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Sample records for catharanthus roseus mesophyll

  1. Patterns of indole alkaloids synthesis in response to heat shock, 5-azacytidine and Na-butyrate treatment of cultured catharanthus roseus mesophyll protoplasts

    SciTech Connect

    Saleem, M.; Cutler, A.J.

    1986-04-01

    Alkaloids of C. roseus are in high demand for therapeutic and other reasons. Cultured Catharanthus cells can produce limited quantities of these alkaloids. The authors have found that cultured mesophyll protoplasts in the presence of /sup 14/C-Tryptamine are capable of synthesizing alkaloids. The pattern of alkaloids synthesis changes when protoplasts are subjected to a heat shock at 37/sup 0/C. The heat shocked protoplasts incorporated 33% more /sup 14/C-Tryptamine and produced 3 new types of alkaloids. Treatment of protoplasts with 5-azacytidine, a DNA hypomethylating agent and Na-butyrate which induces hyperacetylation of histones produced qualitative and quantitative changes in the alkaloid pattern. Four new alkaloids following the above treatments were detected by TLC and HPLC of the extracts. It is suggested that the alkaloid pattern of the cultured protoplasts can be altered by treatment with compounds known as regulators of gene expression. Work is in progress to isolate and identify these new alkaloids.

  2. Monoterpenoid Indole Alkaloids from Catharanthus roseus Cultivated in Yunnan.

    PubMed

    Wang, Bei; Liu, Lu; Chen, Ying-ying; Li, Qiong; Li, Dan; Liu, Va-ping; Luo, Xiao-dong

    2015-12-01

    A new monoterpenoid indole alkaloid, 15,20-dehydro-3α-(2-oxopropyl) coronaridine (1), along with sixteen analogues (2-17) were isolated from the leaves of Catharanthus roseus cultivated in Yunnan. The new alkaloid was elucidated on the basis of extensive spectroscopic analysis, and the known alkaloids were identified by comparison with the reported spectroscopic data. Among them, alkaloid 16 was isolated from Catharanthus for the first time. PMID:26882670

  3. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus

    PubMed Central

    Zhu, Jianhua; Wang, Mingxuan; Wen, Wei; Yu, Rongmin

    2015-01-01

    Catharanthus roseus produces a wide range of terpenoid indole alkaloids (TIA). Many of them, such as vinblastine and vincristine, have significant bioactivity. They are valuable chemotherapy drugs used in combination with other drugs to treat lymphoma and leukemia. The TIA biosynthetic pathway has been investigated for many years, for scientific interest and for their potential in manufacturing applications, to fulfill the market demand. In this review, the progress and perspective of C. roseus TIA biosynthesis and its regulating enzymes are described. In addition, the culture condition, hormones, signaling molecules, precursor feeding on the accumulation of TIA, and gene expression are also evaluated and discussed. PMID:26009689

  4. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus roseus.

    PubMed

    Zhu, Jianhua; Wang, Mingxuan; Wen, Wei; Yu, Rongmin

    2015-01-01

    Catharanthus roseus produces a wide range of terpenoid indole alkaloids (TIA). Many of them, such as vinblastine and vincristine, have significant bioactivity. They are valuable chemotherapy drugs used in combination with other drugs to treat lymphoma and leukemia. The TIA biosynthetic pathway has been investigated for many years, for scientific interest and for their potential in manufacturing applications, to fulfill the market demand. In this review, the progress and perspective of C. roseus TIA biosynthesis and its regulating enzymes are described. In addition, the culture condition, hormones, signaling molecules, precursor feeding on the accumulation of TIA, and gene expression are also evaluated and discussed. PMID:26009689

  5. [Monomeric indole alkaloids from the aerial parts of Catharanthus roseus].

    PubMed

    Zhong, Xiang-Zhang; Wang, Guo-Cai; Wang, Ying; Zhang, Xiao-Qi; Ye, Wen-Cai

    2010-04-01

    Catharanthus roseus (L.) G. Don is a plant of the Catharanthus genus of Apocynaceae which has been reported to have therapeutic effects of detoxication and anticancer. In order to further study the alkaloid constituents of C. roseus, the aerial parts of the plant were extracted with 95% EtOH, and then treated with 2% H2SO4 and NH3H2O to obtain total alkaloids. The total alkaloids were separated and purified by column chromatography over silica gel and prepared by high performance liquid chromatography (HPLC). Their structures were elucidated on the basis of physicochemical properties and spectral data. A new alkaloid together with five known compounds were isolated and identified as vindolinine B (1), lochnericine (2), horhammericine (3), vindorosine (4), vindoline (5), and coronaridine (6). Compound 1 is a new compound and named as vindolinine B. PMID:21355212

  6. Induced Dwarf Mutant in Catharanthus roseus with Enhanced Antibacterial Activity

    PubMed Central

    Verma, A. K.; Singh, R. R.

    2010-01-01

    Evaluation of an ethyl methane sulphonate-induced dwarf mutant of Catharanthus roseus (L.) G. Don revealed that the mutant exhibited marked variation in morphometric parameters. The in vitro antibacterial activity of the aqueous and alcoholic leaf extracts of the mutant and control plants was investigated against medically important bacteria. The mutant leaf extracts showed enhanced antibacterial activity against all the tested bacteria except Bacillus subtilis. PMID:21695004

  7. Functional characterization of amyrin synthase involved in ursolic acid biosynthesis in Catharanthus roseus leaf epidermis.

    PubMed

    Yu, Fang; Thamm, Antje M K; Reed, Darwin; Villa-Ruano, Nemesio; Quesada, Alfonso Lara; Gloria, Edmundo Lozoya; Covello, Patrick; De Luca, Vincenzo

    2013-07-01

    Catharanthus roseus accumulates high levels of the pentacyclic triterpene, ursolic acid, as a component of its wax exudate on the leaf surface. Bioinformatic analyses of transcripts derived from the leaf epidermis provide evidence for the specialized role of this tissue in the biosynthesis of ursolic acid. Cloning and functional expression in yeast of a triterpene synthase derived from this tissue showed it to be predominantly an α-amyrin synthase (CrAS), since the α-amyrin to β-amyrin reaction products accumulated in a 5:1 ratio. Expression analysis of CrAS showed that triterpene biosynthesis occurs predominantly in the youngest leaf tissues and in the earliest stages of seedling development. Further studies using laser capture microdissection to harvest RNA from epidermis, mesophyll, idioblasts, laticifers and vasculature of leaves showed the leaf epidermis to be the preferred sites of CrAS expression and provide conclusive evidence for the involvement of this tissue in the biosynthesis of ursolic acid in C. roseus. PMID:22652241

  8. The seco-iridoid pathway from Catharanthus roseus.

    PubMed

    Miettinen, Karel; Dong, Lemeng; Navrot, Nicolas; Schneider, Thomas; Burlat, Vincent; Pollier, Jacob; Woittiez, Lotte; van der Krol, Sander; Lugan, Raphaël; Ilc, Tina; Verpoorte, Robert; Oksman-Caldentey, Kirsi-Marja; Martinoia, Enrico; Bouwmeester, Harro; Goossens, Alain; Memelink, Johan; Werck-Reichhart, Danièle

    2014-01-01

    The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications. PMID:24710322

  9. The seco-iridoid pathway from Catharanthus roseus

    PubMed Central

    Miettinen, Karel; Dong, Lemeng; Navrot, Nicolas; Schneider, Thomas; Burlat, Vincent; Pollier, Jacob; Woittiez, Lotte; van der Krol, Sander; Lugan, Raphaël; Ilc, Tina; Verpoorte, Robert; Oksman-Caldentey, Kirsi-Marja; Martinoia, Enrico; Bouwmeester, Harro; Goossens, Alain; Memelink, Johan; Werck-Reichhart, Danièle

    2014-01-01

    The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications. PMID:24710322

  10. Hypoglycemic Activity of Aqueous Extracts from Catharanthus roseus

    PubMed Central

    Vega-Ávila, Elisa; Cano-Velasco, José Luis; Alarcón-Aguilar, Francisco J.; Fajardo Ortíz, María del Carmen; Almanza-Pérez, Julio César; Román-Ramos, Rubén

    2012-01-01

    Introduction. Catharanthus roseus (L.) is used in some countries to treat diabetes. The aim of this study was to evaluate the hypoglycemic activity of extracts from the flower, leaf, stem, and root in normal and alloxan-induced diabetic mice. Methods. Roots, leaves, flowers, and stems were separated to obtain organic and aqueous extracts. The blood glucose lowering activity of these extracts was determinate in healthy and alloxan-induced (75 mg/Kg) diabetic mice, after intraperitoneal administration (250 mg/Kg body weight). Blood samples were obtained and blood glucose levels were analyzed employing a glucometer. The data were statistically compared by ANOVA. The most active extract was fractioned. Phytochemical screen and chromatographic studies were also done. Results. The aqueous extracts from C. roseus reduced the blood glucose of both healthy and diabetic mice. The aqueous stem extract (250 mg/Kg) and its alkaloid-free fraction (300 mg/Kg) significantly (P < 0.05) reduced blood glucose in diabetic mice by 52.90 and 51.21%. Their hypoglycemic activity was comparable to tolbutamide (58.1%, P < 0.05). Conclusions. The best hypoglycemic activity was presented for the aqueous extracts and by alkaloid-free stem aqueous fraction. This fraction is formed by three polyphenols compounds. PMID:23056144

  11. Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus.

    PubMed

    Zhu, Xiaoxuan; Zeng, Xinyi; Sun, Chao; Chen, Shilin

    2014-09-01

    Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids (TIAs) in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology. PMID:25159992

  12. Vacuolar Transport of the Medicinal Alkaloids from Catharanthus roseus Is Mediated by a Proton-Driven Antiport1[W

    PubMed Central

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-01-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3′,4′-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4+ and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H+ gradient preestablished across the tonoplast by either vacuolar H+-ATPase or vacuolar H+-pyrophosphatase. The initial rates of H+ gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H+ antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  13. Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

    PubMed

    Carqueijeiro, Inês; Noronha, Henrique; Duarte, Patrícia; Gerós, Hernâni; Sottomayor, Mariana

    2013-07-01

    Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters. PMID:23686419

  14. First report of Tomato chlorotic spot virus on Annual Vinca (Catharanthus roseus) in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tomato chlorotic spot virus was identified in the ornamental crop Catharanthus roseus (commonly known as vinca) in south Florida, the first report of this virus naturally infecting this species. Genetic diversity of the virus was characterized. This report provides an overview of this emerging vir...

  15. 7-O-methylpelargonidin glycosides from the pale red flowers of Catharanthus roseus.

    PubMed

    Tatsuzawa, Fumi

    2013-08-01

    Two new anthocyanidin glycosides were isolated from the pale red flowers of Catharanthus roseus 'Equator Apricot with Red Eye', and identified as 7-O-methylpelargonidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] and 7-O-methylpelargonidin 3-O-(beta-galactopyranoside) by chemical and spectroscopic methods. PMID:24079176

  16. Cytogenetic characterization and genome size of the medicinal plant Catharanthus roseus (L.) G. Don

    PubMed Central

    Guimarães, Guilherme; Cardoso, Luísa; Oliveira, Helena; Santos, Conceição; Duarte, Patrícia; Sottomayor, Mariana

    2012-01-01

    Background and aims Catharanthus roseus is a highly valuable medicinal plant producing several terpenoid indole alkaloids (TIAs) with pharmaceutical applications, including the anticancer agents vinblastine and vincristine. Due to the interest in its TIAs, C. roseus is one of the most extensively studied medicinal plants and has become a model species for the study of plant secondary metabolism. However, very little is known about the cytogenetics and genome size of this species, in spite of their importance for breeding programmes, TIA genetics and emerging genomic research. Therefore, the present paper provides a karyotype description and fluorescence in situ hybridization (FISH) data for C. roseus, as well as a rigorous characterization of its genome size. Methodology The organization of C. roseus chromosomes was characterized using several DNA/chromatin staining techniques and FISH of rDNA. Genome size was investigated by flow cytometry using an optimized methodology. Principal results The C. roseus full chromosome complement of 2n = 16 includes two metacentric, four subtelocentric and two telocentric chromosome pairs, with the presence of a single nucleolus organizer region in chromosome 6. An easy and reliable flow cytometry protocol for nuclear genome analysis of C. roseus was optimized, and the C-value of this species was estimated to be 1C = 0.76 pg, corresponding to 738 Mbp. Conclusions The organization and size of the C. roseus genome were characterized, providing an important basis for future studies of this important medicinal species, including further cytogenetic mapping, genomics, TIA genetics and breeding programmes. PMID:22479673

  17. Enhanced catharanthine and vindoline production in suspension cultures of Catharanthus roseus by ultraviolet-B light

    PubMed Central

    Ramani, Shilpa; Jayabaskaran, Chelliah

    2008-01-01

    Suspension cultures of Catharanthus roseus were used to evaluate ultraviolet-B (UV-B) treatment as an abiotic elicitor of secondary metabolites. A dispersed cell suspension culture from C. roseus leaves in late exponential phase and stationary phase were irradiated with UV-B for 5 min. The stationary phase cultures were more responsive to UV-B irradiation than late exponential phase cultures. Catharanthine and vindoline increased 3-fold and 12-fold, respectively, on treatment with a 5-min UV-B irradiation. PMID:18439256

  18. Catharanthus roseus flower extract has wound-healing activity in Sprague Dawley rats

    PubMed Central

    Nayak, BS; Pinto Pereira, Lexley M

    2006-01-01

    Background Catharanthus roseus L (C. roseus) has been used to treat a wide assortment of diseases including diabetes. The objective of our study was to evaluate the antimicrobial and wound healing activity of the flower extract of Catharanthus in rats. Methods Wound healing activity was determined in rats, after administration (100 mg kg-1 day-1) of the ethanol extract of C. roseus flower, using excision, incision and dead space wounds models. The animals were divided into two groups of 6 each in all the models. In the excision model, group 1 animals were topically treated with carboxymethyl cellulose as placebo control and group 2 received topical application of the ethanol extract of C. roseus at a dose of 100 mg/kg body weight/day. In an incision and dead space model group 1 animals were given normal saline and group 2 received the extract orally at a dose of 100 mg kg-1 day-1. Healing was assessed by the rate of wound contraction, period of epithelization, tensile strength (skin breaking strength), granulation tissue weight, and hydoxyproline content. Antimicrobial activity of the flower extract against four microorganisms was also assessed Results The extract of C. roseus significantly increased the wound breaking strength in the incision wound model compared with controls (P < 0.001). The extract-treated wounds were found to epithelialize faster, and the rate of wound contraction was significantly increased in comparison to control wounds (P < 0.001), Wet and dry granulation tissue weights, and hydroxyproline content in a dead space wound model increased significantly (p < 0.05). Pseudomonas aeruginosa and Staphylococcus aureus demonstrated sensitivity to C. roseus Conclusion Increased wound contraction and tensile strength, augmented hydroxyproline content along with antimicrobial activity support the use of C. roseus in the topical management of wound healing. PMID:17184528

  19. [Identification and expression analysis of WRKY transcription factors in medicinal plant Catharanthus roseus].

    PubMed

    Yang, Zhirong; Wang, Xingchun; Xue, Jin'ai; Meng, Lingzhi; Li, Runzhi

    2013-06-01

    WRKY transcription factors, one of the largest families of transcriptional regulators in plants, involve in multiple life activities including plant growth and development as well as stress responses. However, little is known about the types and functions of WRKY transcription factors in Catharanthus roseus, an important medicinal plant. In this study, we identified 47 CrWRKY transcriptional factors from 26 009 proteins in Catharanthus roseus, and classified them into three distinct groups (G1, G2 and G3) according to the structure of WRKY domain and evolution of the protein family. The expression profiling showed that these CrWRKY genes expressed in a tissue/organ specific manner. The 47 CrWRKY genes were clustered into three types of expression patterns. The first type includes the CrWRKYs highly expressed in flowers and the protoplast treated with methy jasmonate (MeJA) or yeast extraction (YE). The second type contains the CrWRKYs highly expressed in stem and hairy root. The third type represents the CrWRKYs highly expressed in root, stem, leaf, seedling and the hairy root treated by MeJA. Real time quantitative PCR was employed to further identify the expression patterns of the 16 selected CrWRKY genes in various organs, the MeJA-treated protoplasts and hairy roots of Catharanthus roseus, and similar results were obtained. Notably, the expresion of more than 1/3 CrWRKY genes were regulated by MeJA or YE, indicating that these CrWRKYs are likely involed in the signalling webs which modulate the biosynthesis of terpenoid indole alkaloid and plant responses to various stresses. The present results provide a framework for functional identification of the CrWRKYs and understanding of the regulation network of terpenoid indole alkaloid biosynthesis in Catharanthus roseus. PMID:24063238

  20. Transcriptome analysis of Catharanthus roseus for gene discovery and expression profiling.

    PubMed

    Verma, Mohit; Ghangal, Rajesh; Sharma, Raghvendra; Sinha, Alok K; Jain, Mukesh

    2014-01-01

    The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals. PMID:25072156

  1. Transcriptome Analysis of Catharanthus roseus for Gene Discovery and Expression Profiling

    PubMed Central

    Sharma, Raghvendra; Sinha, Alok K.; Jain, Mukesh

    2014-01-01

    The medicinal plant, Catharanthus roseus, accumulates wide range of terpenoid indole alkaloids, which are well documented therapeutic agents. In this study, deep transcriptome sequencing of C. roseus was carried out to identify the pathways and enzymes (genes) involved in biosynthesis of these compounds. About 343 million reads were generated from different tissues (leaf, flower and root) of C. roseus using Illumina platform. Optimization of de novo assembly involving a two-step process resulted in a total of 59,220 unique transcripts with an average length of 1284 bp. Comprehensive functional annotation and gene ontology (GO) analysis revealed the representation of many genes involved in different biological processes and molecular functions. In total, 65% of C. roseus transcripts showed homology with sequences available in various public repositories, while remaining 35% unigenes may be considered as C. roseus specific. In silico analysis revealed presence of 11,620 genic simple sequence repeats (excluding mono-nucleotide repeats) and 1820 transcription factor encoding genes in C. roseus transcriptome. Expression analysis showed roots and leaves to be actively participating in bisindole alkaloid production with clear indication that enzymes involved in pathway of vindoline and vinblastine biosynthesis are restricted to aerial tissues. Such large-scale transcriptome study provides a rich source for understanding plant-specialized metabolism, and is expected to promote research towards production of plant-derived pharmaceuticals. PMID:25072156

  2. Catharanthus roseus: a natural source for the synthesis of silver nanoparticles

    PubMed Central

    Mukunthan, KS; Elumalai, EK; Patel, Trupti N; Murty, V Ramachandra

    2011-01-01

    Objective To develop a simple rapid procedure for bioreduction of silver nanoparticles (AgNPs) using aqueous leaves extracts of Catharanthus roseus (C. roseus). Methods Characterization were determined by using UV-Vis spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray and X-ray diffraction. Results SEM showed the formation of silver nanoparticles with an average size of 67 nm to 48 nm. X-ray diffraction analysis showed that the particles were crystalline in nature with face centered cubic geometry. Conclusions C. roseus demonstrates strong potential for synthesis of silver nanoparticles by rapid reduction of silver ions (Ag+ to Ag0). This study provides evidence for developing large scale commercial production of value-added products for biomedical/nanotechnology-based industries. PMID:23569773

  3. Antihyperglycemic activity of Catharanthus roseus leaf powder in streptozotocin-induced diabetic rats

    PubMed Central

    Rasineni, Karuna; Bellamkonda, Ramesh; Singareddy, Sreenivasa Reddy; Desireddy, Saralakumari

    2010-01-01

    Catharanthus roseus Linn (Apocynaceae), is a traditional medicinal plant used to control diabetes, in various regions of the world. In this study we evaluated the possible antidiabetic and hypolipidemic effect of C. roseus (Catharanthus roseus) leaf powder in diabetic rats. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ, 55 mg/kg body wt) to male Wistar rats. The animals were divided into four groups: Control, control-treated, diabetic, and diabetic-treated group. Diabetic-treated and control-treated rats were treated with C. roseus leaf powder suspension in 2 ml distilled water, orally (100 mg/kg body weight/day/60 days). In diabetic rats (D-group) the plasma glucose was increased and the plasma insulin was decreased gradually. In the diabetic-treated group lowering of plasma glucose and an increase in plasma insulin were observed after 15 days and by the end of the experimental period the plasma glucose had almost reached the normal level, but insulin had not. The significant enhancement in plasma total cholesterol, triglycerides, LDL and VLDL-cholesterol, and the atherogenic index of diabetic rats were normalized in diabetic-treated rats. Decreased hepatic and muscle glycogen content and alterations in the activities of enzymes of glucose metabolism (glycogen phosphorylase, hexokinase, phosphofructokinase, pyruvate kinase, and glucose-6-phosphate dehydrogenase), as observed in the diabetic control rats, were prevented with C. roseus administration. Our results demonstrated that C. roseus with its antidiabetic and hypolipidemic properties could be a potential herbal medicine in treating diabetes. PMID:21808566

  4. Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus.

    PubMed

    Zhu, Wei; Yang, Bingxian; Komatsu, Setsuko; Lu, Xiaoping; Li, Ximin; Tian, Jingkui

    2015-01-01

    Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. PMID:26284098

  5. A virus-induced gene silencing approach to understanding alkaloid metabolism in Catharanthus roseus

    PubMed Central

    Liscombe, David K.; O’Connor, Sarah E.

    2011-01-01

    The anticancer agents vinblastine and vincristine are bisindole alkaloids derived from coupling vindoline and catharanthine, monoterpenoid indole alkaloids produced exclusively by Madagascar periwinkle (Catharanthus roseus) plants. Industrial production of vinblastine and vincristine currently relies on isolation from C. roseus leaves, a process that affords these compounds in 0.0003–0.01% yields. Metabolic engineering efforts to improve alkaloid content or provide alternative sources of the bisindole alkaloids ultimately rely on the isolation and characterization of the genes involved. Several vindoline biosynthetic genes have been isolated, and the cellular and subcellular organization of the corresponding enzymes has been well studied. However, due to the leaf-specific localization of vindoline biosynthesis, and the lack of production of this precursor in cell suspension and hairy root cultures of C. roseus, further elucidation of this pathway demands the development of reverse genetics approaches to assay gene function in planta. The bipartite pTRV vector system is a Tobacco Rattle Virus-based virus-induced gene silencing (VIGS) platform that has provided efficient and effective means to assay gene function in diverse plant systems. We have developed a VIGS method to investigate gene function in C. roseus plants using the pTRV vector system. The utility of this approach in understanding gene function in C. roseus leaves is demonstrated by silencing known vindoline biosynthetic genes previously characterized in vitro. PMID:21802100

  6. Binary stress induces an increase in indole alkaloid biosynthesis in Catharanthus roseus

    PubMed Central

    Zhu, Wei; Yang, Bingxian; Komatsu, Setsuko; Lu, Xiaoping; Li, Ximin; Tian, Jingkui

    2015-01-01

    Catharanthus roseus is an important medicinal plant, which produces a variety of indole alkaloids of significant pharmaceutical relevance. In the present study, we aimed to investigate the potential stress-induced increase of indole alkaloid biosynthesis in C. roseus using proteomic technique. The contents of the detectable alkaloids ajmalicine, vindoline, catharanthine, and strictosidine in C. roseus were significantly increased under binary stress. Proteomic analysis revealed that the abundance of proteins related to tricarboxylic acid cycle and cell wall was largely increased; while, that of proteins related to tetrapyrrole synthesis and photosynthesis was decreased. Of note, 10-hydroxygeraniol oxidoreductase, which is involved in the biosynthesis of indole alkaloid was two-fold more abundant in treated group compared to the control. In addition, mRNA expression levels of genes involved in the indole alkaloid biosynthetic pathway indicated an up-regulation in their transcription in C. roseus under UV-B irradiation. These results suggest that binary stress might negatively affect the process of photosynthesis in C. roseus. In addition, the induction of alkaloid biosynthesis appears to be responsive to binary stress. PMID:26284098

  7. Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus.

    PubMed

    Pollier, Jacob; Vanden Bossche, Robin; Rischer, Heiko; Goossens, Alain

    2014-10-01

    Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis. PMID:25058454

  8. Structural identification of putative USPs in Catharanthus roseus.

    PubMed

    Bahieldin, Ahmed; Atef, Ahmed; Shokry, Ahmed M; Al-Karim, Saleh; Al Attas, Sanaa G; Gadallah, Nour O; Edris, Sherif; Al-Kordy, Magdy A; Omer, Abdulkader M Shaikh; Sabir, Jamal S M; Ramadan, Ahmed M; Al-Hajar, Abdulrahman S M; Makki, Rania M; Hassan, Sabah M; El-Domyati, Fotouh M

    2015-10-01

    Nucleotide sequences of the C. roseus SRA database were assembled and translated in order to detect putative universal stress proteins (USPs). Based on the known conserved USPA domain, 24 Pfam putative USPA proteins in C. roseus were detected and arranged in six architectures. The USPA-like domain was detected in all architectures, while the protein kinase-like (or PK-like), (tyr)PK-like and/or U-box domains are shown downstream it. Three other domains were also shown to coexist with the USPA domain in C. roseus putative USPA sequences. These domains are tetratricopeptide repeat (or TPR), apolipophorin III (or apoLp-III) and Hsp90 co-chaperone Cdc37. Subsequent analysis divided USPA-like domains based on the ability to bind ATP. The multiple sequence alignment indicated the occurrence of eight C. roseus residues of known features of the bacterial 1MJH secondary structure. The data of the phylogenetic tree indicated several distinct groups of USPA-like domains confirming the presence of high level of sequence conservation between the plant and bacterial USPA-like sequences. PMID:26318047

  9. Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus1

    PubMed Central

    De Luca, Vincenzo; Cutler, Adrian J.

    1987-01-01

    The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline. PMID:16665811

  10. Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

    PubMed Central

    Leduc, M.; Tikhomiroff, C.; Cloutier, M.; Perrier, M.

    2006-01-01

    A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate. PMID:16453114

  11. Influence of Some Heavy Metals on Growth, Alkaloid Content and Composition in Catharanthus roseus L.

    PubMed Central

    Srivastava, N. K.; Srivastava, A. K.

    2010-01-01

    Shoot biomass production, alkaloid content and composition as influence by cadmium, manganese, nickel and lead at uniform dose of 5 mM were investigated in Catharanthus roseus plants grown in sand culture. Treatment with Mn, Ni, and Pb significantly enhanced total root alkaloid accumulation. Cd and Ni treatment resulted in two-fold where as Pb treatment resulted in three fold increase in serpentine content of roots. The non-significant affect on biomass suggests that plants can withstand metal stress at the level tested with positive affect on root alkaloid content. PMID:21969751

  12. Computational identification of microRNAs and their targets in Catharanthus roseus expressed sequence tags

    PubMed Central

    Pani, Alok; Mahapatra, Rajani Kanta

    2013-01-01

    No study has been performed on identifying microRNAs (miRNAs) and their targets in the medicinal plant, Catharanthus roseus. In the present study, using the comparative genomics approach, we have predicted two potential C. roseus miRNAs. Furthermore, twelve potential mRNA targets were identified in C. roseus genome based on the characteristics that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences. Among them many of the targets were predicted to encode enzymes that regulate the biosynthesis of terpenoid indole alkaloids (TIA). In addition, most of the predicted targets were the gene coding for transcription factors which are mainly involved in cell growth and development, signaling and metabolism. This is the first in silico study to indicate that miRNA target gene encoding enzymes involved in vinblastine and vincristine biosynthesis, which may help to understand the miRNA-mediated regulation of TIA alkaloid biosynthesis in C. roseus. PMID:26484050

  13. UV-B induced transcript accumulation of DAHP synthase in suspension-cultured Catharanthus roseus cells

    PubMed Central

    2010-01-01

    The enzyme 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase (EC 4.1.2.15) catalyzes the first committed step in the shikimate pathway of tryptophan synthesis, an important precursor for the production of terpenoid indole alkaloids (TIAs). A full-length cDNA encoding nuclear coded chloroplast-specific DAHP synthase transcript was isolated from a Catharanthus roseus cDNA library. This had high sequence similarity with other members of plant DAHP synthase family. This transcript accumulated in suspension cultured C. roseus cells on ultraviolet (UV-B) irradiation. Pretreatment of C.roseus cells with variety of agents such as suramin, N-acetyl cysteine, and inhibitors of calcium fluxes and protein kinases and MAP kinase prevented this effect of UV-B irriadiation. These data further show that the essential components of the signaling pathway involved in accumulation DAHP synthase transcript in C. roseus cells include suramin-sensitive cell surface receptor, staurosporine-sensitive protein kinase and MAP kinase. PMID:20704760

  14. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn. G. Don and their antiplasmodial activities

    PubMed Central

    Ponarulselvam, S; Panneerselvam, C; Murugan, K; Aarthi, N; Kalimuthu, K; Thangamani, S

    2012-01-01

    Objective To develop a novel approach for the green synthesis of silver nanoparticles using aqueous leaves extracts of Catharanthus roseus (C. roseus) Linn. G. Don which has been proven active against malaria parasite Plasmodium falciparum (P. falciparum). Methods Characterizations were determined by using ultraviolet-visible (UV-Vis) spectrophotometry, scanning electron microscopy (SEM), energy dispersive X-ray and X-ray diffraction. Results SEM showed the formation of silver nanoparticles with an average size of 35–55 nm. X-ray diffraction analysis showed that the particles were crystalline in nature with face centred cubic structure of the bulk silver with the broad peaks at 32.4, 46.4 and 28.0. Conclusions It can be concluded that the leaves of C. roseus can be good source for synthesis of silver nanoparticle which shows antiplasmodial activity against P. falciparum. The important outcome of the study will be the development of value added products from medicinal plants C. roseus for biomedical and nanotechnology based industries. PMID:23569974

  15. Computational identification of microRNAs and their targets in Catharanthus roseus expressed sequence tags.

    PubMed

    Pani, Alok; Mahapatra, Rajani Kanta

    2013-12-01

    No study has been performed on identifying microRNAs (miRNAs) and their targets in the medicinal plant, Catharanthus roseus. In the present study, using the comparative genomics approach, we have predicted two potential C. roseus miRNAs. Furthermore, twelve potential mRNA targets were identified in C. roseus genome based on the characteristics that miRNAs exhibit perfect or nearly perfect complementarity with their targeted mRNA sequences. Among them many of the targets were predicted to encode enzymes that regulate the biosynthesis of terpenoid indole alkaloids (TIA). In addition, most of the predicted targets were the gene coding for transcription factors which are mainly involved in cell growth and development, signaling and metabolism. This is the first in silico study to indicate that miRNA target gene encoding enzymes involved in vinblastine and vincristine biosynthesis, which may help to understand the miRNA-mediated regulation of TIA alkaloid biosynthesis in C. roseus. PMID:26484050

  16. ‘Candidatus Phytoplasma hispanicum’, a novel taxon associated with Mexican periwinkle virescence disease of Catharanthus roseus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mexican periwinkle virescence (MPV) phytoplasma was originally discovered in diseased plants of Madagascar periwinkle (Catharanthus roseus) in Yucatán, Mexico. On the basis of results from RFLP analysis of PCR-amplified 16S rRNA gene sequences, strain MPV was previously classified as the first know...

  17. Development of efficient catharanthus roseus regeneration and transformation system using agrobacterium tumefaciens and hypocotyls as explants

    PubMed Central

    2012-01-01

    Background As a valuable medicinal plant, Madagascar periwinkle (Catharanthus roseus) produces many terpenoid indole alkaloids (TIAs), such as vindoline, ajamlicine, serpentine, catharanthine, vinblastine and vincristine et al. Some of them are important components of drugs treating cancer and hypertension. However, the yields of these TIAs are low in wild-type plants, and the total chemical synthesis is impractical in large scale due to high-cost and their complicated structures. The recent development of metabolic engineering strategy offers a promising solution. In order to improve the production of TIAs in C. roseus, the establishment of an efficient genetic transformation method is required. Results To develop a genetic transformation method for C. roseus, Agrobacterium tumefaciens strain EHA105 was employed which harbors a binary vector pCAMBIA2301 containing a report β-glucuronidase (GUS) gene and a selectable marker neomycin phosphotransferase II gene (NTPII). The influential factors were investigated systematically and the optimal transformation condition was achieved using hypocotyls as explants, including the sonication treatment of 10 min with 80 W, A. tumefaciens infection of 30 min and co-cultivation of 2 d in 1/2 MS medium containing 100 μM acetosyringone. With a series of selection in callus, shoot and root inducing kanamycin-containing resistance media, we successfully obtained stable transgenic regeneration plants. The expression of GUS gene was confirmed by histochemistry, polymerase chain reaction, and genomic southern blot analysis. To prove the efficiency of the established genetic transformation system, the rate-limiting gene in TIAs biosynthetic pathway, DAT, which encodes deacetylvindoline-4-O-acetyltransferase, was transferred into C. roseus using this established system and 9 independent transgenic plants were obtained. The results of metabolite analysis using high performance liquid chromatography (HPLC) showed that

  18. Antidiabetic and antioxidant properties of alkaloids from Catharanthus roseus (L.) G. Don.

    PubMed

    Tiong, Soon Huat; Looi, Chung Yeng; Hazni, Hazrina; Arya, Aditya; Paydar, Mohammadjavad; Wong, Won Fen; Cheah, Shiau-Chuen; Mustafa, Mohd Rais; Awang, Khalijah

    2013-01-01

    Catharanthus roseus (L.) G. Don is a herbal plant traditionally used by local populations in India, South Africa, China and Malaysia to treat diabetes. The present study reports the in vitro antioxidant and antidiabetic activities of the major alkaloids isolated from Catharanthus roseus (L.) G. Don leaves extract. Four alkaloids--vindoline I, vindolidine II, vindolicine III and vindolinine IV--were isolated and identified from the dichloromethane extract (DE) of this plant's leaves. DE and compounds I-III were not cytotoxic towards pancreatic β-TC6 cells at the highest dosage tested (25.0 µg/mL). All four alkaloids induced relatively high glucose uptake in pancreatic β-TC6 or myoblast C2C12 cells, with III showing the highest activity. In addition, compounds II-IV demonstrated good protein tyrosine phosphatase-1B (PTP-1B) inhibition activity, implying their therapeutic potential against type 2 diabetes. III showed the highest antioxidant potential in ORAC and DPPH assays and it also alleviated H₂O₂-induced oxidative damage in β-TC6 cells at 12.5 µg/mL and 25.0 µg/mL. PMID:23955322

  19. Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings 1

    PubMed Central

    Fernandez, Jesus Alvarez; Owen, Terence G.; Kurz, Wolfgang G. W.; De Luca, Vincenzo

    1989-01-01

    l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program. Images Figure 3 Figure 5 PMID:16667047

  20. Characterization of an endophytic whorl-forming Streptomyces from Catharanthus roseus stems producing polyene macrolide antibiotic.

    PubMed

    Rakotoniriana, Erick Francisco; Chataigné, Gabrielle; Raoelison, Guy; Rabemanantsoa, Christian; Munaut, Françoise; El Jaziri, Mondher; Urveg-Ratsimamanga, Suzanne; Marchand-Brynaert, Jacqueline; Corbisier, Anne-Marie; Declerck, Stéphane; Quetin-Leclercq, Joëlle

    2012-05-01

    An endophytic whorl-forming Streptomyces sp. designated as TS3RO having antifungal activity against a large number of fungal pathogens, including Sclerotinia sclerotiorum, Rhizoctonia solani, Colletotrichum gloeosporioides, Cryphonectria parasitica, Fusarium oxysporum, Pyrenophora tritici-repentis, Epidermophyton floccosum, and Trichophyton rubrum, was isolated from surface-sterilized Catharanthus roseus stems. Preliminary identification showed that Streptomyces cinnamoneus subsp. sparsus was its closest related species. However, strain TS3RO could readily be distinguished from this species using a combination of phenotypic properties, 16S rDNA sequence similarity, and phylogenetic analyses. Thus, the whorl-forming Streptomyces sp. strain TS3RO is likely a new subspecies within the Streptomyces cinnamoneus group. Direct bioautography on a thin-layer chromatography plate with Cladosporium cucumerinum was conducted throughout the purification steps for bioassay-guided isolation of the active antifungal compounds from the crude extract. Structural elucidation of the isolated bioactive compound was obtained via LC-MS spectrometry, UV-visible spectra, and nuclear magnetic resonance data. It revealed that fungichromin, a known methylpentaene macrolide antibiotic, was the main antifungal component of TS3RO strain, as shown by thin-layer chromatography bioautography. This is the first report of an endophytic whorl-forming Streptomyces isolated from the medically important plant Catharanthus roseus. PMID:22524528

  1. The Leaf Epidermome of Catharanthus roseus Reveals Its Biochemical Specialization[W][OA

    PubMed Central

    Murata, Jun; Roepke, Jonathon; Gordon, Heather; De Luca, Vincenzo

    2008-01-01

    Catharanthus roseus is the sole commercial source of the monoterpenoid indole alkaloids (MIAs), vindoline and catharanthine, components of the commercially important anticancer dimers, vinblastine and vincristine. Carborundum abrasion technique was used to extract leaf epidermis–enriched mRNA, thus sampling the epidermome, or complement, of proteins expressed in the leaf epidermis. Random sequencing of the derived cDNA library established 3655 unique ESTs, composed of 1142 clusters and 2513 singletons. Virtually all known MIA pathway genes were found in this remarkable set of ESTs, while only four known genes were found in the publicly available Catharanthus EST data set. Several novel MIA pathway candidate genes were identified, as demonstrated by the cloning and functional characterization of loganic acid O-methyltransferase involved in secologanin biosynthesis. The pathways for triterpene biosynthesis were also identified, and metabolite analysis showed that oleanane-type triterpenes were localized exclusively to the cuticular wax layer. The pathways for flavonoid and very-long-chain fatty acid biosynthesis were also located in this cell type. The results illuminate the biochemical specialization of Catharanthus leaf epidermis for the production of multiple classes of metabolites. The value and versatility of this EST data set for biochemical and biological analysis of leaf epidermal cells is also discussed. PMID:18326827

  2. Purification and cDNA Cloning of Isochorismate Synthase from Elicited Cell Cultures of Catharanthus roseus

    PubMed Central

    van Tegelen, Léon J.P.; Moreno, Paolo R.H.; Croes, Anton F.; Verpoorte, Robert; Wullems, George J.

    1999-01-01

    Isochorismate is an important metabolite formed at the end of the shikimate pathway, which is involved in the synthesis of both primary and secondary metabolites. It is synthesized from chorismate in a reaction catalyzed by the enzyme isochorismate synthase (ICS; EC 5.4.99.6). We have purified ICS to homogeneity from elicited Catharanthus roseus cell cultures. Two isoforms with an apparent molecular mass of 64 kD were purified and characterized. The Km values for chorismate were 558 and 319 μm for isoforms I and II, respectively. The isoforms were not inhibited by aromatic amino acids and required Mg2+ for enzyme activity. Polymerase chain reaction on a cDNA library from elicited C. roseus cells with a degenerated primer based on the sequence of an internal peptide from isoform II resulted in an amplification product that was used to screen the cDNA library. This led to the first isolation, to our knowledge, of a plant ICS cDNA. The cDNA encodes a protein of 64 kD with an N-terminal chloroplast-targeting signal. The deduced amino acid sequence shares homology with bacterial ICS and also with anthranilate synthases from plants. Southern analysis indicates the existence of only one ICS gene in C. roseus. PMID:9952467

  3. Virus-induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors.

    PubMed

    Carqueijeiro, I; Masini, E; Foureau, E; Sepúlveda, L J; Marais, E; Lanoue, A; Besseau, S; Papon, N; Clastre, M; Dugé de Bernonville, T; Glévarec, G; Atehortùa, L; Oudin, A; Courdavault, V

    2015-11-01

    Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus-based virus-induced gene silencing (VIGS) approach, involving an Agrobacterium-mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic-mediated approach of inoculation of virus-encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down-regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45-90%), similar to results obtained via agro-transformation. Thus, the biolistic-based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium-based approach, e.g. when difficulties arise with agro-inoculations or when Agrobacterium-free procedures are required to avoid plant defence responses. PMID:26284695

  4. Catharanthus roseus mitogen-activated protein kinase 3 confers UV and heat tolerance to Saccharomyces cerevisiae

    PubMed Central

    Raina, Susheel Kumar; Wankhede, Dhammaprakash Pandhari; Sinha, Alok Krishna

    2013-01-01

    Catharanthus roseus is an important source of pharmaceutically important Monoterpenoid Indole Alkaloids (MIAs). Accumulation of many of the MIAs is induced in response to abiotic stresses such as wound, ultra violet (UV) irradiations, etc. Recently, we have demonstrated a possible role of CrMPK3, a C. roseus mitogen-activated protein kinase in stress-induced accumulation of a few MIAs. Here, we extend our findings using Saccharomyces cerevisiae to investigate the role of CrMPK3 in giving tolerance to abiotic stresses. Yeast cells transformed with CrMPK3 was found to show enhanced tolerance to UV and heat stress. Comparison of CrMPK3 and SLT2, a MAPK from yeast shows high-sequence identity particularly at conserved domains. Additionally, heat stress is also shown to activate a 43 kDa MAP kinase, possibly CrMPK3 in C. roseus leaves. These findings indicate the role of CrMPK3 in stress-induced MIA accumulation as well as in stress tolerance. PMID:23221751

  5. Antimicrobial potentials of Catharanthus roseus by disc diffusion assay.

    PubMed

    Bakht, Jehan; Syed, Fatema; Shafi, Mohammad

    2015-05-01

    The present research work investigates the in vitro antimicrobial activity of different solvent extracted samples from the aerial parts (stem, leaf, fruit and flower) of C. roseus against different microbial species using disc diffusion assay at two different concentrations of 1 and 2 mg disc-1. Hexane extracted samples inhibited the growth of all tested microbial strains except S. typhi. Similarly, ethyl acetate extracted samples was effective to control the activity of all the tested microbial strains. E. coli and S. typhi showed resistance to chloroform extracted samples and the remaining eight microbial strains were susceptible to the same extract. Butanol extracted samples did not inhibit the growth of K. pneumonia and S. typhi at low concentration, however, at higher concentration the same extract reduced the growth of different microbes. Methanol extracted samples effectively controlled the growth of all tested microbes at both concentrations except for S. typhi. Water extracted samples did not inhibit the growth at low concentration except E. coli, K. pneumonia and S. aureus and were ineffective against P. aeroginosa at both concentration. C. albicans, showed resistance against chloroform and water extracted samples at low concentration and susceptible to other solvent extracted samples at both concentration. All fractions were effective against plant pathogens i.e. E. carotovora and A. tumefaciens. PMID:26004715

  6. Synthesis and characterization of palladium nanoparticles using Catharanthus roseus leaf extract and its application in the photo-catalytic degradation

    NASA Astrophysics Data System (ADS)

    Kalaiselvi, Aasaithambi; Roopan, Selvaraj Mohana; Madhumitha, Gunabalan; Ramalingam, C.; Elango, Ganesh

    2015-01-01

    The potential effect of Catharanthus roseus leaf extract for the formation of palladium nanoparticles and its application on dye degradation was discussed. The efficiency of C.roseus leaves are used as a bio-material for the first time as reducing agent. Synthesized palladium nanoparticles were supported by UV-vis spectrometry, XRD, FT-IR and TEM analysis. The secondary metabolites which are responsible for the formation of nanoparticles were identified by GC-MS. The results showed that effect of time was directly related to synthesized nanoparticles and functional groups has a critical role in reducing the metal ions and stabilizing the palladium nanoparticles in an eco-friendly process.

  7. Uptake and metabolism of sugars by suspension-cultured catharanthus roseus cells

    SciTech Connect

    Ashihara, Hiroshi; Sagishima, Kyoko; Kubota, Kaoru )

    1989-04-01

    The Uptake and metabolism of sugars by suspension-cultured Catharanthus roseus cells were investigated. Substantially all the sucrose in the culture medium was hydrolyzed to glucose and fructose before being taken up by the cells. The activity of invertase bound to cell walls, determined in situ, was high at the early stage of culture. Glucose was more easily taken up by the cells than was fructose. Tracer experiments using (U-{sup 14}C)glucose and (U-{sup 14}C)fructose indicated that glucose is a better precursor for respiration than fructose, while fructose is preferentially utilized for the synthesis of sucrose, especially in the early phase of cell growth. These results suggest that fructose is utilized for the synthesis of sucrose via the reaction catalyzed by sucrose synthase, prior to the phosphorylation by hexokinase or fructokinase.

  8. Morphogenetic and chemical stability of long-term maintained Agrobacterium-mediated transgenic Catharanthus roseus plants.

    PubMed

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Mathur, Ajay Kumar; Shanker, Karuna

    2015-01-01

    Transgenic Catharanthus roseus plants (transgenic Dhawal [DT] and transgenic Nirmal [NT]) obtained from the Agrobacterium tumefaciens and Agrobacterium rhizognenes-mediated transformations, respectively, have been maintained in vitro for 5 years. Plants were studied at regular intervals for various parameters such as plant height, leaf size, multiplication rate, alkaloid profile and presence of marker genes. DT plant gradually lost the GUS gene expression and it was not detected in the fifth year while NT plant demonstrated the presence of genes rolA, rolB and rolC even in the fifth year, indicating the more stable nature of Ri transgene. Vindoline content in the DT was two times more than in non-transformed control plants. Alkaloid and tryptophan profiles were almost constant during the 5 years. The cluster analysis revealed that the DT plant is more close to the control Nirmal plant followed by NT plant. PMID:25102992

  9. Two new vinblastine-type N-oxide alkaloids from Catharanthus roseus.

    PubMed

    Zhang, Wei-Ku; Xu, Jie-Kun; Tian, Hai-Yan; Wang, Lei; Zhang, Xiao-Qi; Xiao, Xu-Zhi; Li, Ping; Ye, Wen-Cai

    2013-10-01

    Two new vinblastine-type N-oxide alkaloids, 17-desacetoxyvinblastine N'b-oxide (1) and 20'-deoxyvinblastine N'b-oxide (2), were isolated from the leaves of Catharanthus roseus. The structures of 1 and 2 were established by the analysis of their nuclear magnetic resonance and HR-ESI-MS spectroscopic data. All alkaloids were evaluated for their cytotoxic activities against the human hepatocellular carcinoma (HepG2) cell line, human colorectal carcinoma (Lovo) cell line and human breast carcinoma (MCF-7) cell line by the MTT method in vitro, respectively. The results showed that cytotoxic activities of alkaloids 1 and 2 exhibited moderate inhibitory activity on the proliferation of three cancer cells. PMID:23621523

  10. Strictosidine synthase from Catharanthus roseus: purification and characterization of multiple forms.

    PubMed Central

    de Waal, A; Meijer, A H; Verpoorte, R

    1995-01-01

    Multiple (six) forms of strictosidine synthase from Catharanthus roseus cell suspension cultures were purified and characterized. A purification protocol is presented composed of hydrophobic-interaction, gel-permeation and ion-exchange chromatography and chromatofocusing. Four of six isoforms were purified to apparent homogeneity, whereas two others were nearly homogeneous. All strictosidine synthase isoforms were found to be glycoproteins. The isoforms were also found in leaves and roots of the plant, in seedlings and in hairy root cultures. The ratio of the different isoforms differed slightly between these sources. The kinetic parameters of the isoforms showed no significant differences. The maximal velocity (300-400 nkat/mg of protein) is the highest reported so far. It was demonstrated that the apparent Michaelis constant for tryptamine (approx. 9 microM) is much lower than values reported previously. The presence of weak product inhibition (Kp approx. 35 times Km) was established, whereas substrate inhibition was not detected. PMID:7887913

  11. Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus1

    PubMed Central

    De Luca, Vincenzo; Fernandez, Jesus Alvarez; Campbell, Douglas; Kurz, Wolfgang G. W.

    1988-01-01

    Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings. PMID:16665928

  12. Endophytic filamentous fungi from a Catharanthus roseus: Identification and its hydrolytic enzymes.

    PubMed

    Ayob, Farah Wahida; Simarani, Khanom

    2016-05-01

    This paper reported on the various filamentous fungi strains that were isolated from a wild grown Catharanthus roseus. Based on the morphological characteristics and molecular technique through a Polymerase Chain Reaction and DNA sequencing method using internal transcribed spacer (ITS), these fungi had been identified as a Colletotrichum sp., Macrophomina phaseolina, Nigrospora sphaerica and Fusarium solani. The ultrastructures of spores and hyphae were observed under a Scanning Electron Microscope. The hydrolytic enzyme test showed that all strains were positive in secreting cellulase. Colletotrichum sp. and F. solani strains also gave a positive result for amylase while only F. solani was capable to secrete protease. These fungi were putatively classified as endophytic fungi since they produced extracellular enzymes that allow them to penetrate plant cell walls and colonize with symbiotic properties. PMID:27275114

  13. Synthesis of silver nanoparticles using Catharanthus roseus root extract and its larvicidal effects.

    PubMed

    Rajagopal, Thangavel; Jemimah, Irudayaraj Anto Amal; Ponmanickam, Ponnirul; Ayyanar, Muniappan

    2015-11-01

    Phytosynthesis of silver nanoparticles has attracted considerable attention due to their biocompatibility, low toxicity, cost-effectiveness and being a novel method has an eco-friendly approach. Biological activity of root extracts as well as synthesized silver nanoparticles of Catharanthus roseus were evaluated against larvae of Aedes aegyptiand Culex quinquefasciatus. The structure and proportion of the synthesized nanoparticles was defined by exploitation ultraviolet spectrophotometry, X-ray diffraction, fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy and scanning electron microscopy methods. Reduction of silver ions occurred when silver nitrate solution was treated with aqueous root extract at 60°C. Synthesized silver nanoparticles (AgNPs) were confirmed by analyzing the excitation of surface plasmon resonance (SPR) using UV-vis spectrophotometer at 423 nm. FTIR showed aliphatic amines and alkanes corresponding peaks to be presence of responsible compounds to produced nanoparticles in the reaction mixture. Spherical shaped and crystalline nature of particles was recorded under XRD analysis. Presence of silver metal and 35-55nm sized particles were recorded using EDAX and SEM respectively. Larvicidal activitywas observed after24 hrs of exposure to root extracts and synthesized silver nanoparticles. The highest larval mortality was observed in synthesized silver nanopartiucles against Aedes aegypti (LC50= 2.01 ± 0.34; LC90= 5.29 ± 0.07 at 5.0 mg(-1) concentration) and Culex quinquefasciatus (LC50= 1.18 ± 0.15; LC90= 2.55 ± 0.76 at 3.5 to 5.0 mgl(-1) concentration) respectively. The present study provides evidence that synthesized silver nanoparticles of Catharanthus roseus offer potential source for larvicidal activity againstthe larvae of both dengue and filariasis vectors. PMID:26688962

  14. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus.

    PubMed

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  15. Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells

    PubMed Central

    Rischer, Heiko; Orešič, Matej; Seppänen-Laakso, Tuulikki; Katajamaa, Mikko; Lammertyn, Freya; Ardiles-Diaz, Wilson; Van Montagu, Marc C. E.; Inzé, Dirk; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2006-01-01

    Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant. PMID:16565214

  16. A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus*

    PubMed Central

    Giddings, Lesley-Ann; Liscombe, David K.; Hamilton, John P.; Childs, Kevin L.; DellaPenna, Dean; Buell, C. Robin; O'Connor, Sarah E.

    2011-01-01

    Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis of putative cytochrome P450 genes in the Catharanthus roseus transcriptome identified candidate genes with expression profiles similar to known terpene indole alkaloid biosynthetic genes. Screening of these candidate genes by functional expression in Saccharomyces cerevisiae yielded a unique P450-dependent enzyme that stereoselectively hydroxylates the alkaloids tabersonine and lochnericine at the 19-position of the aspidosperma-type alkaloid scaffold. Tabersonine, which can be converted to either vindoline or 19-O-acetylhörhammericine, represents a branch point in alkaloid biosynthesis. The discovery of CYP71BJ1, which forms part of the pathway leading to 19-O-acetylhörhammericine, will help illuminate how this branch point is controlled in C. roseus. PMID:21454651

  17. A vacuolar class III peroxidase and the metabolism of anticancer indole alkaloids in Catharanthus roseus

    PubMed Central

    Duarte, Patrícia; Figueiredo, Raquel; Ros Barceló, Alfonso

    2008-01-01

    Plants possess a unique metabolic diversity commonly designated as secondary metabolism, of which the anticancer alkaloids from Catharanthus roseus are among the most studied. Recently, in a classical function-to-protein-to-gene approach, we have characterized the main class III peroxidase (Prx) expressed in C. roseus leaves, CrPrx1, implicated in a key biosynthetic step of the anticancer alkaloids. We have shown the vacuolar sorting determination of CrPrx1 using GFP fusions and we have obtained further evidence supporting the role of this enzyme in alkaloid biosynthesis, indicating the potential of CrPrx1 as a molecular tool for the manipulation of alkaloid metabolism. Here, we discuss how plant cells may regulate Prx reactions. In fact, Prxs form a large multigenic family whose members accept a broad range of substrates and, in their two subcellular localizations, the cell wall and the vacuole, Prxs co-locate with a large variety of secondary metabolites which can be accepted as substrates. How then, are Prx reactions regulated? Localization data obtained in our lab suggest that arabinogalactan proteins (AGPs) and Prxs may be associated in membrane microdomains, evocative of lipid rafts. Whether plasma membrane and/or tonoplast microcompartmentation involve AGPs and Prxs and whether this enables metabolic channeling determining Prx substrate selection are challenging questions ahead. PMID:19704535

  18. Characterization of 10-hydroxygeraniol dehydrogenase from Catharanthus roseus reveals cascaded enzymatic activity in iridoid biosynthesis.

    PubMed

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)(+) dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP(+) yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  19. Indole alkaloids from Catharanthus roseus: bioproduction and their effect on human health.

    PubMed

    Almagro, Lorena; Fernández-Pérez, Francisco; Pedreño, Maria Angeles

    2015-01-01

    Catharanthus roseus is a medicinal plant belonging to the family Apocynaceae which produces terpenoid indole alkaloids (TIAs) of high medicinal importance. Indeed, a number of activities like antidiabetic, bactericide and antihypertensive are linked to C. roseus. Nevertheless, the high added value of this plant is based on its enormous pharmaceutical interest, producing more than 130 TIAs, some of which exhibit strong pharmacological activities. The most striking biological activity investigated has been the antitumour effect of dimeric alkaloids such as anhydrovinblastine, vinblastine and vincristine which are already in pre-, clinical or in use. The great pharmacological importance of these indole alkaloids, contrasts with the small amounts of them found in this plant, making their extraction a very expensive process. To overcome this problem, researches have looked for alternative sources and strategies to produce them in higher amounts. In this sense, intensive research on the biosynthesis of TIAs and the regulation of their pathways has been developed with the aim to increase by biotechnological approaches, the production of these high added value compounds. This review is focused on the different strategies which improve TIA production, and in the analysis of the beneficial effects that these compounds exert on human health. PMID:25685907

  20. Study of the effect of nickel heavy metals on some physiological parameters of Catharanthus roseus.

    PubMed

    Arefifard, Matin; Mahdieh, Majid; Amirjani, Mohammadreza

    2014-01-01

    Plants, in their life cycle, are usually exposed to various kinds of non-biological stresses including heavy metals. One of these heavy metals is nickel which affects many physiological processes of plants. Studies have shown that the changes in planting conditions can affect the qualitative and quantitative features of Catharanthus roseus; therefore, creating stressful conditions (e.g. NiCl2) can be an effective way to investigate the changes. In this research, we investigated the effect of 0, 2.5, 5, 10, 25 and 50 mM concentrations of NiCl2 on the degree of catalase enzyme activity, amount of proline aggregation and photosynthetic parameters on seeds of pink variety of C. roseus. The results indicated that the degree of catalase enzyme activity and the amount of proline aggregation increased in plants which were exposed to NiCl2 treatments, especially in high concentrations, while the total protein decreased. The stress of Ni also affected photosynthetic parameters, and decreased the amount of pigments, as well as the efficiency of photosystem II. PMID:24870880

  1. Differential Network Analysis Reveals Evolutionary Complexity in Secondary Metabolism of Rauvolfia serpentina over Catharanthus roseus

    PubMed Central

    Pathania, Shivalika; Bagler, Ganesh; Ahuja, Paramvir S.

    2016-01-01

    Comparative co-expression analysis of multiple species using high-throughput data is an integrative approach to determine the uniformity as well as diversification in biological processes. Rauvolfia serpentina and Catharanthus roseus, both members of Apocyanacae family, are reported to have remedial properties against multiple diseases. Despite of sharing upstream of terpenoid indole alkaloid pathway, there is significant diversity in tissue-specific synthesis and accumulation of specialized metabolites in these plants. This led us to implement comparative co-expression network analysis to investigate the modules and genes responsible for differential tissue-specific expression as well as species-specific synthesis of metabolites. Toward these goals differential network analysis was implemented to identify candidate genes responsible for diversification of metabolites profile. Three genes were identified with significant difference in connectivity leading to differential regulatory behavior between these plants. These genes may be responsible for diversification of secondary metabolism, and thereby for species-specific metabolite synthesis. The network robustness of R. serpentina, determined based on topological properties, was also complemented by comparison of gene-metabolite networks of both plants, and may have evolved to have complex metabolic mechanisms as compared to C. roseus under the influence of various stimuli. This study reveals evolution of complexity in secondary metabolism of R. serpentina, and key genes that contribute toward diversification of specific metabolites. PMID:27588023

  2. Characterization of 10-Hydroxygeraniol Dehydrogenase from Catharanthus roseus Reveals Cascaded Enzymatic Activity in Iridoid Biosynthesis

    PubMed Central

    Krithika, Ramakrishnan; Srivastava, Prabhakar Lal; Rani, Bajaj; Kolet, Swati P.; Chopade, Manojkumar; Soniya, Mantri; Thulasiram, Hirekodathakallu V.

    2015-01-01

    Catharanthus roseus [L.] is a major source of the monoterpene indole alkaloids (MIAs), which are of significant interest due to their therapeutic value. These molecules are formed through an intermediate, cis-trans-nepetalactol, a cyclized product of 10-oxogeranial. One of the key enzymes involved in the biosynthesis of MIAs is an NAD(P)+ dependent oxidoreductase system, 10-hydroxygeraniol dehydrogenase (Cr10HGO), which catalyses the formation of 10-oxogeranial from 10-hydroxygeraniol via 10-oxogeraniol or 10-hydroxygeranial. This work describes the cloning and functional characterization of Cr10HGO from C. roseus and its role in the iridoid biosynthesis. Substrate specificity studies indicated that, Cr10HGO has good activity on substrates such as 10-hydroxygeraniol, 10-oxogeraniol or 10-hydroxygeranial over monohydroxy linear terpene derivatives. Further it was observed that incubation of 10-hydroxygeraniol with Cr10HGO and iridoid synthase (CrIDS) in the presence of NADP+ yielded a major metabolite, which was characterized as (1R, 4aS, 7S, 7aR)-nepetalactol by comparing its retention time, mass fragmentation pattern, and co-injection studies with that of the synthesized compound. These results indicate that there is concerted activity of Cr10HGO with iridoid synthase in the formation of (1R, 4aS, 7S, 7aR)-nepetalactol, an important intermediate in iridoid biosynthesis. PMID:25651761

  3. Growth and photosynthetic pigments responses of two varieties of Catharanthus roseus to triadimefon treatment.

    PubMed

    Jaleel, Cheruth Abdul; Gopi, Ragupathi; Panneerselvam, Rajaram

    2008-04-01

    Triadimefon, potential fungicide cum plant-growth retardant was used in this study to investigate its effect on the growth and the photosynthetic pigment contents of two varieties of Catharanthus roseus (L.) G. Don. The plants of both varieties were subjected to 15 mg l(-1) triadimefon treatment by soil drenching 30, 45, 60, and 75 days after planting (DAP). Plants were uprooted on 90 DAP, and morphological parameters, like plant height, number of leaves, leaf area, root length and fresh and dry weights were determined. The photosynthetic pigments, like chlorophylls a and b, total chlorophyll, carotenoids, floral pigment, anthocyanin, were extracted and estimated. It was observed that plant height, number of leaves and leaf area were decreased and that root length, fresh and dry weights were increased under triadimefon treatment. The photosynthetic and floral pigments were increased under triadimefon treatment in both varieties. The results suggest that the application of this plant-growth retardant (triadimefon) has favourable effects on the reduction of plant height; it can thus be used for replacing manual hand pruning and for improving floral and vegetation colour in bedding plants like C. roseus. PMID:18355749

  4. CathaCyc, a metabolic pathway database built from Catharanthus roseus RNA-Seq data.

    PubMed

    Van Moerkercke, Alex; Fabris, Michele; Pollier, Jacob; Baart, Gino J E; Rombauts, Stephane; Hasnain, Ghulam; Rischer, Heiko; Memelink, Johan; Oksman-Caldentey, Kirsi-Marja; Goossens, Alain

    2013-05-01

    The medicinal plant Madagascar periwinkle (Catharanthus roseus) synthesizes numerous terpenoid indole alkaloids (TIAs), such as the anticancer drugs vinblastine and vincristine. The TIA pathway operates in a complex metabolic network that steers plant growth and survival. Pathway databases and metabolic networks reconstructed from 'omics' sequence data can help to discover missing enzymes, study metabolic pathway evolution and, ultimately, engineer metabolic pathways. To date, such databases have mainly been built for model plant species with sequenced genomes. Although genome sequence data are not available for most medicinal plant species, next-generation sequencing is now extensively employed to create comprehensive medicinal plant transcriptome sequence resources. Here we report on the construction of CathaCyc, a detailed metabolic pathway database, from C. roseus RNA-Seq data sets. CathaCyc (version 1.0) contains 390 pathways with 1,347 assigned enzymes and spans primary and secondary metabolism. Curation of the pathways linked with the synthesis of TIAs and triterpenoids, their primary metabolic precursors, and their elicitors, the jasmonate hormones, demonstrated that RNA-Seq resources are suitable for the construction of pathway databases. CathaCyc is accessible online (http://www.cathacyc.org) and offers a range of tools for the visualization and analysis of metabolic networks and 'omics' data. Overlay with expression data from publicly available RNA-Seq resources demonstrated that two well-characterized C. roseus terpenoid pathways, those of TIAs and triterpenoids, are subject to distinct regulation by both developmental and environmental cues. We anticipate that databases such as CathaCyc will become key to the study and exploitation of the metabolism of medicinal plants. PMID:23493402

  5. Alterations in seedling vigour and antioxidant enzyme activities in Catharanthus roseus under seed priming with native diazotrophs

    PubMed Central

    Karthikeyan, B.; Jaleel, C.A.; Gopi, R.; Deiveekasundaram, M.

    2007-01-01

    An experiment was conducted on Catharanthus roseus to study the effect of seed treatments with native diazotrophs on its seedling growth and antioxidant enzyme activities. The treatments had significant influence on various seedling parameters. There is no significant influence on dry matter production with the diazotrophs, Azospirillum and Azotobacter. However, the vital seedling parameters such as germination percentage and vigour index were improved. Azotobacter treatment influenced maximum of 50% germination, whereas Azospirillum and Azotobacter were on par with C. roseus with respect to their vigour index. There was significant difference in the population of total diazotrophs. Azospirillum and Azotobacter between rhizosphere and non-rhizosphere soils of C. roseus had the same trend and were observed at various locations of the study. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased to a significant extent due to the treatment with diazotrophs. PMID:17610323

  6. The juice of fresh leaves of Catharanthus roseus Linn. reduces blood glucose in normal and alloxan diabetic rabbits

    PubMed Central

    Nammi, Srinivas; Boini, Murthy K; Lodagala, Srinivas D; Behara, Ravindra Babu S

    2003-01-01

    Background The leaf juice or water decoction of Catharanthus roseus L. (Apocyanaceae) is used as a folk medicine for the treatment of diabetes all over the world. In the present investigation, the leaf juice of C. roseus has been evaluated for its hypoglycemic activity in normal and alloxan-induced diabetic rabbits. Methods The blood glucose lowering activity of the leaf juice was studied in normal and alloxan-induced (100 mg/kg, i.v.) diabetic rabbits, after oral administration at doses of 0.5, 0.75 and 1.0 ml/kg body weight. Blood samples were collected from the marginal ear vein before and also at 4, 6, 8, 10, 12, 16, 18, 20 & 24 h after drug administration and blood glucose was analyzed by Nelson-Somogyi's method using a visible spectrophotometer. The data was compared statistically by using Student's t-test. Results The leaf juice of C. roseus produced dose-dependent reduction in blood glucose of both normal and diabetic rabbits and comparable with that of the standard drug, glibenclamide. The results indicate a prolonged action in reduction of blood glucose by C. roseus and the mode of action of the active compound(s) of C. roseus is probably mediated through enhance secretion of insulin from the β-cells of Langerhans or through extrapancreatic mechanism. Conclusions The present study clearly indicated a significant antidiabetic activity with the leaf juice of Catharanthus roseus and supports the traditional usage of the fresh leaves by Ayurvedic physicians for the control of diabetes. PMID:12950994

  7. Adaptation of lettuce mosaic virus to Catharanthus roseus involves mutations in the central domain of the VPg.

    PubMed

    Svanella-Dumas, Laurence; Verdin, Eric; Faure, Chantal; German-Retana, Sylvie; Gognalons, Patrick; Danet, Jean Luc; Marais, Armelle; Candresse, Thierry

    2014-05-01

    An isolate of Lettuce mosaic virus (LMV, a Potyvirus) infecting Madagascar periwinckle (Catharanthus roseus) was identified and characterized by Illumina deep sequencing. LMV-Cr has no close affinities to previously sequenced LMV isolates and represents a novel, divergent LMV clade. Inoculation experiments with other representative LMV isolates showed that they are unable to infect C. roseus, which was not known to be a host for LMV. However, three C. roseus variants of one of these isolates, LMV-AF199, could be selected and partially or completely sequenced. These variants are characterized by the accumulation of mutations affecting the C-terminal part of the cylindrical inclusion (CI) helicase and the central part of the VPg. In particular, a serine to proline mutation at amino acid 143 of the VPg was observed in all three independently selected variants and is also present in the LMV-Cr isolate, making it a prime candidate as a host-range determinant. Other mutations at VPg positions 65 and 144 could also contribute to the ability to infect C. roseus. Inoculation experiments involving a recombinant LMV expressing a permissive lettuce eukaryotic translation initiation factor 4E (eIF4E) suggest that eIF4E does not contribute to the interaction of most LMV isolates with C. roseus. PMID:24400938

  8. Metabolic Discrimination of Catharanthus roseus Leaves Infected by Phytoplasma Using 1H-NMR Spectroscopy and Multivariate Data Analysis1

    PubMed Central

    Choi, Young Hae; Tapias, Elisabet Casas; Kim, Hye Kyong; Lefeber, Alfons W.M.; Erkelens, Cornelis; Verhoeven, Jacobus Th.J.; Brzin, Jernej; Zel, Jana; Verpoorte, Robert

    2004-01-01

    A comprehensive metabolomic profiling of Catharanthus roseus L. G. Don infected by 10 types of phytoplasmas was carried out using one-dimensional and two-dimensional NMR spectroscopy followed by principal component analysis (PCA), an unsupervised clustering method requiring no knowledge of the data set and used to reduce the dimensionality of multivariate data while preserving most of the variance within it. With a combination of these techniques, we were able to identify those metabolites that were present in different levels in phytoplasma-infected C. roseus leaves than in healthy ones. The infection by phytoplasma in C. roseus leaves causes an increase of metabolites related to the biosynthetic pathways of phenylpropanoids or terpenoid indole alkaloids: chlorogenic acid, loganic acid, secologanin, and vindoline. Furthermore, higher abundance of Glc, Glu, polyphenols, succinic acid, and Suc were detected in the phytoplasma-infected leaves. The PCA of the 1H-NMR signals of healthy and phytoplasma-infected C. roseus leaves shows that these metabolites are major discriminating factors to characterize the phytoplasma-infected C. roseus leaves from healthy ones. Based on the NMR and PCA analysis, it might be suggested that the biosynthetic pathway of terpenoid indole alkaloids, together with that of phenylpropanoids, is stimulated by the infection of phytoplasma. PMID:15286294

  9. Cell-specific localization of alkaloids in Catharanthus roseus stem tissue measured with Imaging MS and Single-cell MS.

    PubMed

    Yamamoto, Kotaro; Takahashi, Katsutoshi; Mizuno, Hajime; Anegawa, Aya; Ishizaki, Kimitsune; Fukaki, Hidehiro; Ohnishi, Miwa; Yamazaki, Mami; Masujima, Tsutomu; Mimura, Tetsuro

    2016-04-01

    Catharanthus roseus (L.) G. Don is a medicinal plant well known for producing antitumor drugs such as vinblastine and vincristine, which are classified as terpenoid indole alkaloids (TIAs). The TIA metabolic pathway in C. roseus has been extensively studied. However, the localization of TIA intermediates at the cellular level has not been demonstrated directly. In the present study, the metabolic pathway of TIA in C. roseus was studied with two forefront metabolomic techniques, that is, Imaging mass spectrometry (MS) and live Single-cell MS, to elucidate cell-specific TIA localization in the stem tissue. Imaging MS indicated that most TIAs localize in the idioblast and laticifer cells, which emit blue fluorescence under UV excitation. Single-cell MS was applied to four different kinds of cells [idioblast (specialized parenchyma cell), laticifer, parenchyma, and epidermal cells] in the stem longitudinal section. Principal component analysis of Imaging MS and Single-cell MS spectra of these cells showed that similar alkaloids accumulate in both idioblast cell and laticifer cell. From MS/MS analysis of Single-cell MS spectra, catharanthine, ajmalicine, and strictosidine were found in both cell types in C. roseus stem tissue, where serpentine was also accumulated. Based on these data, we discuss the significance of TIA synthesis and accumulation in the idioblast and laticifer cells of C. roseus stem tissue. PMID:27001858

  10. Characterization of Alkaloid Uptake by Catharanthus roseus (L.) G. Don Protoplasts 1

    PubMed Central

    McCaskill, David G.; Martin, DeAndra L.; Scott, A. Ian

    1988-01-01

    The accumulation of alkaloids by protoplasts of Catharanthus roseus (L.) G. Don var. Little Bright Eye was studied to determine the specificity of uptake and the role of ion trapping in the storage of alkaloids. Accumulation of the indole alkaloids vindoline, ajmalicine, tabersonine, and vinblastine was found to be biphasic, with an initial burst of uptake followed by a slow, prolonged phase of accumulation. The concentration and pH dependence of the initial burst of uptake for vindoline suggested that uptake occurred by simple diffusion. Uptake of nicotine was monophasic, with a half life of 5.2 minutes. The accumulation ratio (Ci/Ce) for nicotine at steady state and for the initial burst of uptake for vindoline and ajmalicine suggested that accumulation was driven by the pH gradient between the vacuole and the external assay medium. The second, sustained phase of uptake of vindoline was sensitive to inhibition by either 20 millimolar NaN3 or 0.5 millimolar Cu2+. In azide-treated protoplasts, the uptake for vindoline conformed to the kinetics of simple diffusion, with a half life of 4 minutes. The second phase of uptake for ajmalicine, although sensitive to inhibition by Cu2+, was insensitive to inhibition by NaN3. The biphasic uptake of the indole alkaloids was not due to any significant metabolism. It is concluded that accumulation and storage of the indole alkaloids is due only partly to ion trapping of the alkaloids by the low pH of the vacuole lumen. In the case of vindoline, there appears to be a specific energy-requiring uptake that is not seen with nicotine (which is not endogenous to Catharanthus). Accumulation of ajmalicine appears to involve both ion trapping and an azide-insensitive component, which may be due to complexation with organic counterions and phenolics. PMID:16666154

  11. Abnormalities in carbohydrate and lipid metabolisms in high-fructose dietfed insulin-resistant rats: amelioration by Catharanthus roseus treatments.

    PubMed

    Rasineni, Karuna; Bellamkonda, Ramesh; Singareddy, Sreenivasa Reddy; Desireddy, Saralakumari

    2013-09-01

    High intake of dietary fructose has been shown to exert a number of adverse metabolic effects in humans and experimental animals. The present study was proposed to elucidate the effect of Catharanthus roseus (C. roseus) leaf powder treatment on alterations in carbohydrate and lipid metabolisms in rats fed with high-fructose diet. Male Wistar rats of body weight around 180 g were divided into four groups, two of these groups (groups C and C+CR) were fed with standard pellet diet and the other two groups (groups F and F+CR) were fed with high-fructose (66 %) diet. C. roseus leaf powder suspension in water (100 mg/kg body weight/day) was administered orally to group C+CR and group F+CR. At the end of a 60-day experimental period, biochemical parameters related to carbohydrate and lipid metabolisms were assayed. C. roseus treatment completely prevented the fructose-induced increased body weight, hyperglycemia, and hypertriglyceridemia. Hyperinsulinemia and insulin resistance observed in group F was significantly decreased with C. roseus treatment in group F+CR. The alterations observed in the activities of enzymes of carbohydrate and lipid metabolisms and contents of hepatic tissue lipids in group F rats were significantly restored to near normal values by C. roseus treatment in group F+CR. In conclusion, our study demonstrates that C. roseus treatment is effective in preventing fructose-induced insulin resistance and hypertriglyceridemia while attenuating the fructose-induced alterations in carbohydrate and lipid metabolisms. This study suggests that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it. PMID:23334857

  12. Development of SSR and gene-targeted markers for construction of a framework linkage map of Catharanthus roseus

    PubMed Central

    Shokeen, Bhumika; Choudhary, Shalu; Sethy, Niroj Kumar; Bhatia, Sabhyata

    2011-01-01

    Background and Aims Catharanthus roseus is a plant of great medicinal importance, yet inadequate knowledge of its genome structure and the unavailability of genomic resources have been major impediments in the development of improved varieties. The aims of this study were to develop co-dominant sequence-tagged microsatellite sites (STMS) and gene-targeted markers (GTMs) and utilize them for the construction of a framework intraspecific linkage map of C. roseus. Methods For simple sequence repeat (SSR) isolation, a genomic library enriched for (GA)n repeats was constructed from C. roseus ‘Nirmal’ (CrN1). In addition, GTMs were also designed from 12 genes of the TIA (terpenoid indole alkaloid) pathway – the medicinally most significant pathway in C. roseus. An F2 mapping population was also generated by crossing two diverse accessions of C. roseus CrN1 (Nirmal)×CrN82 (Kew). Key Results A new set of 314 STMS markers and 64 GTMs were developed in this study. A segregating F2 mapping population consisting of 111 F2 individuals was generated. For generating the linkage map, a set of 423 co-dominant markers (378 newly developed and 45 published earlier) were screened for polymorphism between the parental genotypes, of which 134 were identified to be polymorphic. A total of 114 markers were mapped on eight linkage groups that spanned a 632·7 cM region of the genome with an average marker distance of 5·55 cM. Further, the mechanism of hypervariability at the gene-targeted loci was investigated at the sequence level. Conclusions For the first time, a large array of STMS markers and GTMs was generated in the model medicinal plant C. roseus. Moreover, the first microsatellite marker-based linkage map was described in this study. Together, these will serve as a foundation for future genomics studies related to quantitative trait loci analysis and molecular breeding in C. roseus. PMID:21788377

  13. Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture 1

    PubMed Central

    Maki, Hisae; Ando, Satoshi; Kodama, Hiroaki; Komamine, Atsushi

    1991-01-01

    Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G1 phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G1 to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs. PMID:16668290

  14. Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus1

    PubMed Central

    Whitmer, Serap; Canel, Camilo; Hallard, Didier; Gonçalves, Cecilia; Verpoorte, Robert

    1998-01-01

    We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures. PMID:9490777

  15. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus

    PubMed Central

    Yu, Fang; De Luca, Vincenzo

    2013-01-01

    The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine–vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface. PMID:24019465

  16. Vindogentianine, a hypoglycemic alkaloid from Catharanthus roseus (L.) G. Don (Apocynaceae).

    PubMed

    Tiong, Soon Huat; Looi, Chung Yeng; Arya, Aditya; Wong, Won Fen; Hazni, Hazrina; Mustafa, Mohd Rais; Awang, Khalijah

    2015-04-01

    Vindogentianine, a new indole alkaloid together with six known alkaloids, vindoline, vindolidine, vindolicine, vindolinine, perivine and serpentine were isolated from leaf extract (DA) of Catharanthus roseus (L.) G. Don. Their structures were elucidated by spectroscopic methods; NMR, MS, UV and IR. Vindogentianine is a dimer containing a vindoline moiety coupled to a gentianine moiety. After 24h incubation, vindogentianine exhibited no cytotoxic effect in C2C12 mouse myoblast and β-TC6 mouse pancreatic cells (IC50>50μg/mL). Real-time cell proliferation monitoring also indicated vindogentianine had little or no effect on C2C12 mouse myoblast cell growth at the highest dose tested (200μg/mL), without inducing cell death. Vindogentianine exhibited potential hypoglycemic activity in β-TC6 and C2C12 cells by inducing higher glucose uptake and significant in vitro PTP-1B inhibition. However, in vitro α-amylase and α-glucosidase inhibition assay showed low inhibition under treatment of vindogentianine. This suggests that hypoglycemic activity of vindogentianine may be due to the enhancement of glucose uptake and PTP-1B inhibition, implying its therapeutic potential against type 2 diabetes. PMID:25665941

  17. Kinetic Analysis of Phospholipase C from Catharanthus roseus Transformed Roots Using Different Assays1

    PubMed Central

    Hernández-Sotomayor, S.M. Teresa; De Los Santos-Briones, César; Muñoz-Sánchez, J. Armando; Loyola-Vargas, Victor M.

    1999-01-01

    The properties of phospholipase C (PLC) partially purified from Catharanthus roseus transformed roots were analyzed using substrate lipids dispersed in phospholipid vesicles, phospholipid-detergent mixed micelles, and phospholipid monolayers spread at an air-water interface. Using [33P]phosphatidylinositol 4,5-bisphosphate (PIP2) of high specific radioactivity, PLC activity was monitored directly by measuring the loss of radioactivity from monolayers as a result of the release of inositol phosphate and its subsequent dissolution on quenching in the subphase. PLC activity was markedly affected by the surface pressure of the monolayer, with reduced activity at extremes of initial pressure. The optimum surface pressure for PIP2 hydrolysis was 20 mN/m. Depletion of PLC from solution by incubation with sucrose-loaded PIP2 vesicles followed by ultracentrifugation demonstrated stable attachment of PLC to the vesicles. A mixed micellar system was established to assay PLC activity using deoxycholate. Kinetic analyses were performed to determine whether PLC activity was dependent on both bulk PIP2 and PIP2 surface concentrations in the micelles. The interfacial Michaelis constant was calculated to be 0.0518 mol fraction, and the equilibrium dissociation constant of PLC for the lipid was 45.5 μm. These findings will add to our understanding of the mechanisms of regulation of plant PLC. PMID:10444091

  18. Multicellular compartmentation of catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate

    PubMed Central

    St-Pierre, B; Vazquez-Flota, FA; De Luca V

    1999-01-01

    In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed. PMID:10330473

  19. Biochemical and Ultrastructural Changes in Sida cordifolia L. and Catharanthus roseus L. to Auto Pollution.

    PubMed

    Verma, Vijeta; Chandra, Neelam

    2014-01-01

    Auto pollution is the by-product of our mechanized mobility, which adversely affects both plant and human life. However, plants growing in the urban locations provide a great respite to us from the brunt of auto pollution by absorbing the pollutants at their foliar surface. Foliar surface configuration and biochemical changes in plant species, namely, Sida cordifolia L. and Catharanthus roseus L. grown at roadside (polluted site 1, Talkatora; polluted site 2, Charbagh) in Lucknow city and in the garden of the university campus, which has been taken as reference site, were investigated. It was observed that air pollution caused by auto exhaust showed marked alterations in photosynthetic pigments (chlorophyll, carotenoid, and phaeophytin), and relative water content was reduced while antioxidative enzymes like catalase and peroxidase were found to be enhanced. The changes in the foliar configuration reveal marked alteration in epidermal traits, with decreased number of stomata, stomatal indices, and epidermal cells per unit area, while length and breadth of stomata and epidermal cells were found to be increased in leaves samples wich can be used as biomarkers of auto pollution. PMID:27355010

  20. Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker.

    PubMed

    Torres, Adalgisa Ribeiro; Araújo, Welington Luiz; Cursino, Luciana; de Barros Rossetto, Priscilla; Mondin, Mateus; Hungria, Mariangela; Azevedo, João Lúcio

    2013-07-01

    This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant. PMID:23695435

  1. Cytoplasmic Acidification Induced by Inorganic Phosphate Uptake in Suspension Cultured Catharanthus roseus Cells

    PubMed Central

    Sakano, Katsuhiro; Yazaki, Yoshiaki; Mimura, Tetsuro

    1992-01-01

    Cytoplasmic acidification during inorganic phosphate (Pi) absorption by Catharanthus roseus cells were studied by means of a fluorescent pH indicator, 2′,7′-bis-(2-carboxyethyl)-5 carboxyfluorescein (acetomethylester) (BCECF), and 31P-nuclear magnetic resonance spectroscopy. Cytoplasmic acidification measured by decrease in the fluorescence intensity started immediately after Pi application. Within a minute or so, a stable state was attained and no further acidification occurred, whereas Pi absorption was still proceeding. As soon as Pi in the medium was exhausted, cytoplasmic pH started to recover. Coincidentally, the medium pH started to recover toward the original acidic pH. The Pi-induced changes in the cytoplasmic pH were confirmed by 31P-nuclear magnetic resonance study. Maximum acidification of the cytoplasm induced by 1.7 millimolar Pi was 0.2 pH units. Vacuolar pH was also affected by Pi. In some experiments, but not all, pH decreased reversibly by 0.2 to 0.3 pH units during Pi absorption. Results suggest that the cytoplasmic pH is regulated by proton pumps in the plasma membrane and in the tonoplast. In addition, other mechanisms that could consume extra protons in the cytoplasm are suggested. ImagesFigure 1 PMID:16668939

  2. ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus.

    PubMed

    Yu, Fang; De Luca, Vincenzo

    2013-09-24

    The Madagascar periwinkle (Catharanthus roseus) is highly specialized for the biosynthesis of many different monoterpenoid indole alkaloids (MIAs), many of which have powerful biological activities. Such MIAs include the commercially important chemotherapy drugs vinblastine, vincristine, and other synthetic derivatives that are derived from the coupling of catharanthine and vindoline. However, previous studies have shown that biosynthesis of these MIAs involves extensive movement of metabolites between specialized internal leaf cells and the leaf epidermis that require the involvement of unknown secretory processes for mobilizing catharanthine to the leaf surface and vindoline to internal leaf cells. Spatial separation of vindoline and catharanthine provides a clear explanation for the low levels of dimers that accumulate in intact plants. The present work describes the molecular cloning and functional identification of a unique catharanthine transporter (CrTPT2) that is expressed predominantly in the epidermis of young leaves. CrTPT2 gene expression is activated by treatment with catharanthine, and its in planta silencing redistributes catharanthine to increase the levels of catharanthine-vindoline drug dimers in the leaves. Phylogenetic analysis shows that CrTPT2 is closely related to a key transporter involved in cuticle assembly in plants and that may be unique to MIA-producing plant species, where it mediates secretion of alkaloids to the plant surface. PMID:24019465

  3. Negative-pressure cavitation extraction of four main vinca alkaloids from Catharanthus roseus leaves.

    PubMed

    Mu, Fansong; Yang, Liuqing; Wang, Wei; Luo, Meng; Fu, Yujie; Guo, Xiaorui; Zu, Yuangang

    2012-01-01

    In the present study, an improved method termed negative-pressure cavitation extraction (NPCE) followed by reverse phase high-performance liquid chromatography (RP-HPLC) was developed for the extraction and quantification of vindoline (VDL), catharanthine (CTR), vincristine (VCR) and vinblastine (VLB) from Catharanthus roseus leaves. The optimized method employed 60-mesh particles, 80% ethanol, a negative pressure of -0.075 MPa, a solid to liquid ratio of 1:20, 30 min of extraction and three extraction cycles. Under these optimized conditions, the extraction yields of VDL, CTR, VCR and VLB are 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These extraction yields are equivalent to those from the well-known ultrasonic extraction method and higher than the yields from maceration extraction and heating reflux extraction. Our results suggest that NPCE-RP-HPLC represents an excellent alternative for the extraction and quantification of vinca alkaloids for pilot- and industrial-scale applications. PMID:22832876

  4. Biochemical and Ultrastructural Changes in Sida cordifolia L. and Catharanthus roseus L. to Auto Pollution

    PubMed Central

    Verma, Vijeta; Chandra, Neelam

    2014-01-01

    Auto pollution is the by-product of our mechanized mobility, which adversely affects both plant and human life. However, plants growing in the urban locations provide a great respite to us from the brunt of auto pollution by absorbing the pollutants at their foliar surface. Foliar surface configuration and biochemical changes in plant species, namely, Sida cordifolia L. and Catharanthus roseus L. grown at roadside (polluted site 1, Talkatora; polluted site 2, Charbagh) in Lucknow city and in the garden of the university campus, which has been taken as reference site, were investigated. It was observed that air pollution caused by auto exhaust showed marked alterations in photosynthetic pigments (chlorophyll, carotenoid, and phaeophytin), and relative water content was reduced while antioxidative enzymes like catalase and peroxidase were found to be enhanced. The changes in the foliar configuration reveal marked alteration in epidermal traits, with decreased number of stomata, stomatal indices, and epidermal cells per unit area, while length and breadth of stomata and epidermal cells were found to be increased in leaves samples wich can be used as biomarkers of auto pollution. PMID:27355010

  5. Synthesis and characterization of palladium nanoparticles using Catharanthus roseus leaf extract and its application in the photo-catalytic degradation.

    PubMed

    Kalaiselvi, Aasaithambi; Roopan, Selvaraj Mohana; Madhumitha, Gunabalan; Ramalingam, C; Elango, Ganesh

    2015-01-25

    The potential effect of Catharanthus roseus leaf extract for the formation of palladium nanoparticles and its application on dye degradation was discussed. The efficiency of C.roseus leaves are used as a bio-material for the first time as reducing agent. Synthesized palladium nanoparticles were supported by UV-vis spectrometry, XRD, FT-IR and TEM analysis. The secondary metabolites which are responsible for the formation of nanoparticles were identified by GC-MS. The results showed that effect of time was directly related to synthesized nanoparticles and functional groups has a critical role in reducing the metal ions and stabilizing the palladium nanoparticles in an eco-friendly process. PMID:25062057

  6. Subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus and Tabernaemontana divaricata.

    PubMed

    Stevens, L H; Blom, T J; Verpoorte, R

    1993-08-01

    The subcellular localization of tryptophan decarboxylase, strictosidine synthase and strictosidine glucosidase in suspension cultured cells of Catharanthus roseus (L.) G. Don and Tabernaemontana divaricata (L.) R. Br. ex Roem. et Schult, was investigated. It was found that tryptophan decarboxylase is an extra-vacuolar enzyme, whereas strictosidine synthase is active inside the vacuole. Strong indications were obtained for the localization of strictosidine glucosidase on the outside of the tonoplast. The results suggest that tryptamine is transported into the vacuole where it is condensed with secologanin to form strictosidine, and that strictosidine passes the tonoplast and is subsequently hydrolysed outside the vacuole. PMID:24201788

  7. Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs.

    PubMed

    Mahroug, Samira; Courdavault, Vincent; Thiersault, Martine; St-Pierre, Benoit; Burlat, Vincent

    2006-05-01

    Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid-monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid-monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms. PMID:16322983

  8. Evaluation of the nutritive and organoleptic values of food products developed by incorporated Catharanthus roseus (Sadabahar) fresh leaves explore their hypoglycemic potential.

    PubMed

    Bisla, Gita; Choudhary, Shailza; Chaudhary, Vijeta

    2014-01-01

    Diabetes becomes a real problem of public health in developing countries, where its prevalence is increasing steadily. Diabetes mellitus can be found in almost every population in the world. Since the Ayurvedic practice started in India, plants are being used in the cure of diseases. Although the Catharanthus roseus have been used for their alleged health benefits and avail their hypoglycemic effect, used as medicine by diabetics. Medicinal plants have rarely been incorporated in food preparations. To fill these lacunae, food products were prepared by using Catharanthus roseus (Sadabahar) fresh leaves with hypoglycemic properties. Commonly consumed recipes in India are prepared for diabetic patients and were developed at different levels at 3 g, 4 g, and 6 g per serving. Food product development and their acceptability appraisal through organoleptic evaluation were carried out by semitrained panel comprising 15 trained panelists from the department of Food Science and Nutrition, Banasthali University. Seven products were developed by incorporating Catharanthus roseus fresh leaves. Nine point hedonic scale was used as a medium to know about the product acceptability at various variances. All products are moderately acceptable at different concentrations except product fare "6 g" which was more acceptable than the standard. Among the three variations of incorporating the Catharanthus roseus (Sadabahar) Leaves, 3 g variation is more acceptable than other variations. PMID:24790561

  9. Evaluation of the Nutritive and Organoleptic Values of Food Products Developed by Incorporated Catharanthus roseus (Sadabahar) Fresh Leaves Explore Their Hypoglycemic Potential

    PubMed Central

    Bisla, Gita; Choudhary, Shailza; Chaudhary, Vijeta

    2014-01-01

    Diabetes becomes a real problem of public health in developing countries, where its prevalence is increasing steadily. Diabetes mellitus can be found in almost every population in the world. Since the Ayurvedic practice started in India, plants are being used in the cure of diseases. Although the Catharanthus roseus have been used for their alleged health benefits and avail their hypoglycemic effect, used as medicine by diabetics. Medicinal plants have rarely been incorporated in food preparations. To fill these lacunae, food products were prepared by using Catharanthus roseus (Sadabahar) fresh leaves with hypoglycemic properties. Commonly consumed recipes in India are prepared for diabetic patients and were developed at different levels at 3 g, 4 g, and 6 g per serving. Food product development and their acceptability appraisal through organoleptic evaluation were carried out by semitrained panel comprising 15 trained panelists from the department of Food Science and Nutrition, Banasthali University. Seven products were developed by incorporating Catharanthus roseus fresh leaves. Nine point hedonic scale was used as a medium to know about the product acceptability at various variances. All products are moderately acceptable at different concentrations except product fare “6 g” which was more acceptable than the standard. Among the three variations of incorporating the Catharanthus roseus (Sadabahar) Leaves, 3 g variation is more acceptable than other variations. PMID:24790561

  10. Artemisinic Acid Serves as a Novel ORCA3 Inducer to Enhance Biosynthesis of Terpenoid Indole Alkaloids in Catharanthus roseus Cambial Meristematic Cells.

    PubMed

    Wang, Mingxuan; Zi, Jiachen; Zhu, Jianhua; Chen, Shan; Wang, Pu; Song, Liyan; Yu, Rongmin

    2016-06-01

    To investigate the effect of artemisinic acid (AA) on improving the production of terpenoid indole alkaloids (TIAs) of Catharanthus roseus cambial meristematic cells (CMCs), feeding AA to C. roseus CMCs caused 2.35-fold and 2.51-fold increases in the production of vindoline and catharanthine, respectively, compared with those of the untreated CMCs. qRT-PCR experiments showed that AA resulted in a 1.36-8.52 fold increase in the transcript levels of several related genes, including octadecanoid-derivative responsive Catharanthus AP2-domain protein 3 (ORCA3), tryptophan decarboxylase (TDC), strictosidine synthase (STR) and desacetoxyvindoline 4-hydroxylase (D4H). However, no effect was observed on the concentration of either jasmonic acid (JA), or the octadecanoid-pathway inhibitors block TIA accumulation caused by AA. The results indicated that AA might serve as a novel ORCA3 inducer to manipulate biosynthesis of TIAs in C. roseus CMCs via an unknown mechanism. PMID:27534099

  11. Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor) – A comparative study

    PubMed Central

    Mujib, A.; Ali, Muzamil; Isah, Tasiu; Dipti

    2014-01-01

    The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somatic embryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 μM 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 μM) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 μM) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids

  12. Somatic embryo mediated mass production of Catharanthus roseus in culture vessel (bioreactor) - A comparative study.

    PubMed

    Mujib, A; Ali, Muzamil; Isah, Tasiu; Dipti

    2014-11-01

    The purpose of this study was to evaluate and compare the use of liquid and solid Murashige and Skoog (MS) medium in different culture vessels for mass production of Catharanthus roseus, an important source of anticancerous compounds, vincristine and vinblastine. Three media conditions i.e. agar-solidified medium (S), liquid medium in agitated conical flask (L) and growtek bioreactor (B) were used. Rapid propagation was achieved through in vitro somatic embryogenesis pathway. The process of embryogenesis has been categorized into induction, proliferation, maturation and germination stages. All in vitro embryogenesis stages were conducted by withdrawing spent liquid medium and by adding fresh MS medium. In optimized 4.52 μM 2,4-D added MS, the callus biomass growth was low in solid (1.65 g) compared to liquid medium in agitated conical flask (1.95 g) and in bioreactor (2.11 g). The number of normal somatic embryos was more in solid medium (99.75/50 mg of callus mass) compared to liquid medium used in conical flask (83.25/callus mass) and growtek bioreactor (84.88/callus mass). The in vitro raised embryos maturated in GA3 (2.60 μM) added medium; and in bioreactor the embryo growth was high, a maximum length of 9.82 mm was observed at the end of four weeks. These embryos germinated into seedlings in BAP (2.22 μM) added medium and the embryo germination ability was more (59.41%) in bioreactor compared to liquid medium in conical flask (55.5%). Shoot length (11.25 mm) was also high in bioreactor compared to agitated conical flask. The liquid medium used in agitated conical flask and bioreactor increased seedling production efficiency, at the same time it also reduced plant recovery time. The embryo generated plants grew normally in outdoor conditions. The exploitation of medium to large culture vessel or bioreactor may make the process more efficient in getting large number of Catharanthus plant as it is the only source of anti-cancerous alkaloids

  13. Exploiting EST databases for the mining and characterization of short sequence repeat (SSR) markers in Catharanthus roseus L.

    PubMed Central

    Joshi, Raj Kumar; Kar, Basudeba; Nayak, Sanghamitra

    2011-01-01

    Periwinkle (Catharanthus roseus L.) (Family: Apocyanaceae) is a ornamental plants with great medicinal properties. Although it is represented by seven species, little work has been carried out on its genetic characterization due to non-availability of reliable molecular markers. Simple sequence repeats (SSRs) have been widely applied as molecular markers in genetic studies. With the rapid increase in the deposition of nucleotide sequences in the public databases and advent of bioinformatics tools, it has become a cost effective and fast approach to scan for microsatellite repeats and exploit the possibility of converting it into potential genetic markers. Expressed sequence tags (EST's) from Catharanthus roseus were used for the screening of Class I (hyper variable) simple sequence repeats (SSR's). A total of 502 microsatellite repeats were detected from 21730 EST sequences of turmeric after redundancy elimination. The average density of Class I SSRs account to 1 SSR per 10.21 kb of EST. Mononucleotides was the most abundant class of microsatellite motifs. It accounted for 44.02% of the total, followed by the trinucleotide (26.09%) and dinucleotide repeats (14.34%). Among all the repeat motifs, (A/T)n accounted for the highest Proportion (36.25%) followed by (AAG)n. These detected SSRs can be used to design primers that have functional importance and should also facilitate the analysis of genetic diversity, variability, linkage mapping and evolutionary relationships in plants especially medicinal plants. PMID:21383904

  14. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    PubMed Central

    Ramani, Shilpa; Chelliah, Jayabaskaran

    2007-01-01

    Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s), Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. PMID:17988378

  15. Developmental and Light Regulation of Desacetoxyvindoline 4-Hydroxylase in Catharanthus roseus (L.) G. Don.1

    PubMed Central

    Vazquez-Flota, Felipe A.; De Luca, Vincenzo

    1998-01-01

    The expression of desacetoxyvindoline 4-hydroxylase (D4H), which catalyzes the second to the last reaction in vindoline biosynthesis in Catharanthus roseus, appears to be under complex, multilevel developmental and light regulation. Developmental studies with etiolated and light-treated seedlings suggested that although light had variable effects on the levels of d4h transcripts, those of D4H protein and enzyme activity could be increased, depending on seedling development, up to 9- and 8-fold, respectively, compared with etiolated seedlings. However, light treatment of etiolated seedlings could stop and reverse the decline of d4h transcripts at later stages of seedling development. Repeated exposure of seedlings to light was also required to maintain the full spectrum of enzyme activity observed during seedling development. Further studies showed that a photoreversible phytochrome appeared to be involved in the activation of D4H, since red-light treatment of etiolated seedlings increased the detectable levels of d4h transcripts, D4H protein, and D4H enzyme activity, whereas far-red-light treatment completely reversed this process. Additional studies also confirmed that different major isoforms of D4H protein exist in etiolated (isoelectric point, 4.7) and light-grown (isoelectric point, 4.6) seedlings, suggesting that a component of the light-mediated activation of D4H may involve an undetermined posttranslational modification. The biological reasons for this complex control of vindoline biosynthesis may be related to the need to produce structures that could sequester away from cellular activities the cytotoxic vinblastine and vincristine dimers that are derived partially from vindoline. PMID:9701591

  16. Interaction between abscisic acid and nitric oxide in PB90-induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures.

    PubMed

    Chen, Qian; Chen, Zunwei; Lu, Li; Jin, Haihong; Sun, Lina; Yu, Qin; Xu, Hongke; Yang, Fengxia; Fu, Mengna; Li, Shengchao; Wang, Huizhong; Xu, Maojun

    2013-01-01

    Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells. PMID:23554409

  17. A Cytochrome P-450 Monooxygenase Catalyzes the First Step in the Conversion of Tabersonine to Vindoline in Catharanthus roseus.

    PubMed Central

    St-Pierre, B.; De Luca, V.

    1995-01-01

    Hydroxylation at the C-16 position of the indole alkaloid tabersonine has been suggested as the first step toward vindoline biosynthesis in Catharanthus roseus. Tabersonine 16-hydroxylase (16-OH) activity was detected in total protein extracts from young leaves of C. roseus using a novel coupled assay system. Enzyme activity was dependent on NADPH and molecular oxygen and was inhibited by CO, clotrimazole, miconazole, and cytochrome c. 16-OH was localized to the endoplasmic reticulum by linear sucrose density gradient centrifugation. These data suggest that 16-OH is a cytochrome P-450-dependent monooxygenase. The activity of 16-OH reached a maximum in seedlings 9 d postimbibition and was induced by light. The leaf-specific distribution of 16-OH in the mature plant is consistent with the localization of other enzymes in the tabersonine to vindoline pathway. However, in contrast to enzymes that catalyze the last four steps of vindoline biosynthesis, enzymes responsible for the first two steps from tabersonine (16-OH and 16-O-methyltransfersase) were detected in C. roseus cell-suspension cultures. These data complement the complex model of vindoline biosynthesis that has evolved with respect to enzyme compartmentalization, metabolic transport, and control mechanisms. PMID:12228585

  18. Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

    PubMed

    Xing, Shi-Hai; Guo, Xin-Bo; Wang, Quan; Pan, Qi-Fang; Tian, Yue-Sheng; Liu, Pin; Zhao, Jing-Ya; Wang, Guo-Feng; Sun, Xiao-Fen; Tang, Ke-Xuan

    2011-01-01

    The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine. PMID:21660143

  19. Effect of Gloriosa superba and Catharanthus roseus Extracts on IFN-γ-Induced Keratin 17 Expression in HaCaT Human Keratinocytes.

    PubMed

    Pattarachotanant, Nattaporn; Rakkhitawatthana, Varaporn; Tencomnao, Tewin

    2014-01-01

    Gloriosa superba and Catharanthus roseus are useful in traditional medicine for treatment of various skin diseases and cancer. However, their molecular effect on psoriasis has not been investigated. In this study, the effect of ethanol extracts derived from G. superba leaves and C. roseus stems on the expression of psoriatic marker, keratin 17 (K17), was investigated in human keratinocytes using biochemical and molecular experimental approaches. Both extracts could reduce the expression of K17 in a dose-dependent manner through JAK/STAT pathway as demonstrated by an observation of reduced phosphorylation of STAT3 (p-STAT3). The inhibitory activity of G. superba extract was more potent than that of C. roseus. The Pearson's correlation between K17 and cell viability was shown positive. Taken together, the extracts of G. superba and C. roseus may be developed as alternative therapies for psoriasis. PMID:25435888

  20. Effect of Gloriosa superba and Catharanthus roseus Extracts on IFN-γ-Induced Keratin 17 Expression in HaCaT Human Keratinocytes

    PubMed Central

    Pattarachotanant, Nattaporn; Rakkhitawatthana, Varaporn

    2014-01-01

    Gloriosa superba and Catharanthus roseus are useful in traditional medicine for treatment of various skin diseases and cancer. However, their molecular effect on psoriasis has not been investigated. In this study, the effect of ethanol extracts derived from G. superba leaves and C. roseus stems on the expression of psoriatic marker, keratin 17 (K17), was investigated in human keratinocytes using biochemical and molecular experimental approaches. Both extracts could reduce the expression of K17 in a dose-dependent manner through JAK/STAT pathway as demonstrated by an observation of reduced phosphorylation of STAT3 (p-STAT3). The inhibitory activity of G. superba extract was more potent than that of C. roseus. The Pearson's correlation between K17 and cell viability was shown positive. Taken together, the extracts of G. superba and C. roseus may be developed as alternative therapies for psoriasis. PMID:25435888

  1. Induction and Flow Cytometry Identification of Tetraploids from Seed-Derived Explants through Colchicine Treatments in Catharanthus roseus (L.) G. Don

    PubMed Central

    Xing, Shi-Hai; Guo, Xin-Bo; Wang, Quan; Pan, Qi-Fang; Tian, Yue-Sheng; Liu, Pin; Zhao, Jing-Ya; Wang, Guo-Feng; Sun, Xiao-Fen; Tang, Ke-Xuan

    2011-01-01

    The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine. PMID:21660143

  2. A pair of tabersonine 16-hydroxylases initiates the synthesis of vindoline in an organ-dependent manner in Catharanthus roseus.

    PubMed

    Besseau, Sébastien; Kellner, Franziska; Lanoue, Arnaud; Thamm, Antje M K; Salim, Vonny; Schneider, Bernd; Geu-Flores, Fernando; Höfer, René; Guirimand, Grégory; Guihur, Anthony; Oudin, Audrey; Glevarec, Gaëlle; Foureau, Emilien; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Werck-Reichhart, Danièle; Burlat, Vincent; De Luca, Vincenzo; O'Connor, Sarah E; Courdavault, Vincent

    2013-12-01

    Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis. PMID:24108213

  3. Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.) 1

    PubMed Central

    Vetter, Hans-Peter; Mangold, Ursula; Schröder, Gudrun; Marner, Franz-Josef; Werck-Reichhart, Danielle; Schröder, Joachim

    1992-01-01

    We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 μg/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. Images Figure 5 Figure 7 PMID:16653087

  4. CrMPK3, a mitogen activated protein kinase from Catharanthus roseus and its possible role in stress induced biosynthesis of monoterpenoid indole alkaloids

    PubMed Central

    2012-01-01

    Background Mitogen activated protein kinase (MAPK) cascade is an important signaling cascade that operates in stress signal transduction in plants. The biologically active monoterpenoid indole alkaloids (MIA) produced in Catharanthus roseus are known to be induced under several abiotic stress conditions such as wounding, UV-B etc. However involvement of any signaling component in the accumulation of MIAs remains poorly investigated so far. Here we report isolation of a novel abiotic stress inducible Catharanthus roseus MAPK, CrMPK3 that may have role in accumulation of MIAs in response to abiotic stress. Results CrMPK3 expressed in bacterial system is an active kinase as it showed auto-phosphorylation and phosphorylation of Myelin Basic Protein. CrMPK3 though localized in cytoplasm, moves to nucleus upon wounding. Wounding, UV treatment and MeJA application on C. roseus leaves resulted in the transcript accumulation of CrMPK3 as well as activation of MAPK in C. roseus leaves. Immuno-precipitation followed by immunoblot analysis revealed that wounding, UV treatment and methyl jasmonate (MeJA) activate CrMPK3. Transient over-expression of CrMPK3 in C. roseus leaf tissue showed enhanced expression of key MIA biosynthesis pathway genes and also accumulation of specific MIAs. Conclusion Results from our study suggest a possible involvement of CrMPK3 in abiotic stress signal transduction towards regulation of transcripts of key MIA biosynthetic pathway genes, regulators and accumulation of major MIAs. PMID:22871174

  5. 7-deoxyloganetic acid synthase catalyzes a key 3 step oxidation to form 7-deoxyloganetic acid in Catharanthus roseus iridoid biosynthesis.

    PubMed

    Salim, Vonny; Wiens, Brent; Masada-Atsumi, Sayaka; Yu, Fang; De Luca, Vincenzo

    2014-05-01

    Iridoids are key intermediates required for the biosynthesis of monoterpenoid indole alkaloids (MIAs), as well as quinoline alkaloids. Although most iridoid biosynthetic genes have been identified, one remaining three step oxidation required to form the carboxyl group of 7-deoxyloganetic acid has yet to be characterized. Here, it is reported that virus-induced gene silencing of 7-deoxyloganetic acid synthase (7DLS, CYP76A26) in Catharanthus roseus greatly decreased levels of secologanin and the major MIAs, catharanthine and vindoline in silenced leaves. Functional expression of this gene in Saccharomyces cerevisiae confirmed its function as an authentic 7DLS that catalyzes the 3 step oxidation of iridodial-nepetalactol to form 7-deoxyloganetic acid. The identification of CYP76A26 removes a key bottleneck for expression of iridoid and related MIA pathways in various biological backgrounds. PMID:24594312

  6. Screening and kinetic studies of catharanthine and ajmalicine accumulation and their correlation with growth biomass in Catharanthus roseus hairy roots.

    PubMed

    Benyammi, Roukia; Paris, Cédric; Khelifi-Slaoui, Majda; Zaoui, Djamila; Belabbassi, Ouarda; Bakiri, Nouara; Meriem Aci, Myassa; Harfi, Boualem; Malik, Sonia; Makhzoum, Abdullah; Desobry, Stéphane; Khelifi, Lakhdar

    2016-10-01

    Context Catharanthus roseus (L.) G. Don (Apocynaceae) is still one of the most important sources of terpene indole alkaloids including anticancer and hypertensive drugs as vincristine and vinblastine. These final compounds have complex pathway and many enzymes are involved in their biosynthesis. Indeed, ajmalicine and catharanthine are important precursors their increase can lead to enhance levels of molecules of interest. Objective This study aims at selecting the highest yield of hairy root line(s) and at identifying best times for further treatments. We study kinetics growth and alkaloids (ajmalicine and catharanthine) accumulation of three selected hairy root lines during the culture cycle in order to determine the relationship between biomass production and alkaloids accumulation. Materials and methods Comparative analysis has been carried out on three selected lines of Catharanthus roseus hairy roots (LP10, LP21 and L54) for their kinetics of growth and the accumulation of ajamalicine and catharanthine, throughout a 35-day culture cycle. The methanolic extract for each line in different times during culture cycle is analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Results Maximum accumulation of the alkaloids is recorded for LP10 line in which the peak of ajmalicine and catharanthine accumulation reached to 3.8 and 4.3 mg/g dry weight (DW), respectively. This increase coincides with an exponential growth phase. Discussion and conclusion Our results suggest that the evolution of accumulation of ajmalicine and catharanthine are positively correlated with the development of the biomass growth. Significantly, for LP10 line the most promising line to continue optimizing the production of TIAs. Additionally, the end of exponential phase remains the best period for elicitor stimuli. PMID:26983347

  7. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis

    PubMed Central

    Pandey, Shiv S.; Singh, Sucheta; Babu, C. S. Vivek; Shanker, Karuna; Srivastava, N. K.; Shukla, Ashutosh K.; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229–403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  8. Fungal endophytes of Catharanthus roseus enhance vindoline content by modulating structural and regulatory genes related to terpenoid indole alkaloid biosynthesis.

    PubMed

    Pandey, Shiv S; Singh, Sucheta; Babu, C S Vivek; Shanker, Karuna; Srivastava, N K; Shukla, Ashutosh K; Kalra, Alok

    2016-01-01

    Not much is known about the mechanism of endophyte-mediated induction of secondary metabolite production in Catharanthus roseus. In the present study two fungal endophytes, Curvularia sp. CATDLF5 and Choanephora infundibulifera CATDLF6 were isolated from the leaves of the plant that were found to enhance vindoline content by 229-403%. The isolated endophytes did not affect the primary metabolism of the plant as the maximum quantum efficiency of PSII, net CO2 assimilation, plant biomass and starch content of endophyte-inoculated plants was similar to endophyte-free control plants. Expression of terpenoid indole alkaloid (TIA) pathway genes, geraniol 10-hydroxylase (G10H), tryptophan decarboxylase (TDC), strictosidine synthase (STR), 16-hydoxytabersonine-O-methyltransferase (16OMT), desacetoxyvindoline-4-hydroxylase (D4H), deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in endophyte-inoculated plants. Endophyte inoculation upregulated the expression of the gene for transcriptional activator octadecanoid-responsive Catharanthus AP2-domain protein (ORCA3) and downregulated the expression of Cys2/His2-type zinc finger protein family transcriptional repressors (ZCTs). The gene for the vacuolar class III peroxidase (PRX1), responsible for coupling vindoline and catharanthine, was upregulated in endophyte-inoculated plants. These endophytes may enhance vindoline production by modulating the expression of key structural and regulatory genes of vindoline biosynthesis without affecting the primary metabolism of the host plant. PMID:27220774

  9. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots

    PubMed Central

    Rizvi, Noreen F.; Weaver, Jessica D.; Cram, Erin J.; Lee-Parsons, Carolyn W. T.

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  10. Effect of Chromium on Antioxidant Potential of Catharanthus roseus Varieties and Production of Their Anticancer Alkaloids: Vincristine and Vinblastine

    PubMed Central

    Tandon, Pramod Kumar; Khatoon, Sayyada

    2014-01-01

    Catharanthus roseus (L.) G. Don, a medicinal plant, has a very important place in the traditional as well as modern pharmaceutical industry. Two common varieties of this plant rosea and alba are named so because of pink and white coloured flowers, respectively. This plant comprises of about 130 terpenoid indole alkaloids and two of them, vincristine and vinblastine, are common anticancer drugs. The effect of chromium (Cr) on enzymatic and non-enzymatic antioxidant components and on secondary metabolites vincristine and vinblastine was studied under pot culture conditions of both varieties of C. roseus. Antioxidant responses of these varieties were analyzed under 0, 10, 50, and 100 μM chromium (Cr) level in order to investigate the plant's protective mechanisms against Cr induced oxidative stress. The results indicated that Cr affects all the studied parameters and decreases growth performance. However, vincristine and vinblastine contents were increased under Cr stress. Results are quite encouraging, as this plant shows good antioxidant potential and increased the level of active constituents under Cr stress. PMID:24734252

  11. Identification and quantification of active alkaloids in Catharanthus roseus by liquid chromatography-ion trap mass spectrometry.

    PubMed

    Chen, Qinhua; Zhang, Wenpeng; Zhang, Yulin; Chen, Jing; Chen, Zilin

    2013-08-15

    Catharanthus roseus is an important dicotyledonous medicinal plant that produces anticancer compounds. The active alkaloids vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine were identified by direct-injection ion trap-mass spectrometry (IT-MS) for collecting MS(1-2) spectra. The determinations of five alkaloids were accomplished by liquid chromatography (LC) with UV and MS detections. The analytes provided good signals corresponding to the protonated molecular ions [M+H](+) and product ions. The precursor ions and product ions for quantification of vinblastine, vindoline, ajmalicine, catharanthine, and vinleurosine were m/z 825→807, 457→397, 353→144, 337→144 and 809→748 by LC-IT-MS, respectively. Two methods were used to evaluate a number of validation characteristics (repeatability, LOD, calibration range, and recovery). MS provided a high selectivity and sensitivity for determination of five alkaloids in positive mode. After optimisation of the methods, separation, identification and quantification of the five components in C. roseus were comprehensively accomplished by HPLC with UV and MS detection. PMID:23561180

  12. Effect of chromium on antioxidant potential of Catharanthus roseus varieties and production of their anticancer alkaloids: vincristine and vinblastine.

    PubMed

    Rai, Vartika; Tandon, Pramod Kumar; Khatoon, Sayyada

    2014-01-01

    Catharanthus roseus (L.) G. Don, a medicinal plant, has a very important place in the traditional as well as modern pharmaceutical industry. Two common varieties of this plant rosea and alba are named so because of pink and white coloured flowers, respectively. This plant comprises of about 130 terpenoid indole alkaloids and two of them, vincristine and vinblastine, are common anticancer drugs. The effect of chromium (Cr) on enzymatic and non-enzymatic antioxidant components and on secondary metabolites vincristine and vinblastine was studied under pot culture conditions of both varieties of C. roseus. Antioxidant responses of these varieties were analyzed under 0, 10, 50, and 100  μM chromium (Cr) level in order to investigate the plant's protective mechanisms against Cr induced oxidative stress. The results indicated that Cr affects all the studied parameters and decreases growth performance. However, vincristine and vinblastine contents were increased under Cr stress. Results are quite encouraging, as this plant shows good antioxidant potential and increased the level of active constituents under Cr stress. PMID:24734252

  13. Investigation of a substrate-specifying residue within Papaver somniferum and Catharanthus roseus aromatic amino acid decarboxylases.

    PubMed

    Torrens-Spence, Michael P; Lazear, Michael; von Guggenberg, Renee; Ding, Haizhen; Li, Jianyong

    2014-10-01

    Plant aromatic amino acid decarboxylases (AAADs) catalyze the decarboxylation of aromatic amino acids with either benzene or indole rings. Because the substrate selectivity of AAADs is intimately related to their physiological functions, primary sequence data and their differentiation could provide significant physiological insights. However, due to general high sequence identity, plant AAAD substrate specificities have been difficult to identify through primary sequence comparison. In this study, bioinformatic approaches were utilized to identify several active site residues within plant AAAD enzymes that may impact substrate specificity. Next a Papaver somniferum tyrosine decarboxylase (TyDC) was selected as a model to verify our putative substrate-dictating residues through mutation. Results indicated that mutagenesis of serine 372 to glycine enables the P. somniferum TyDC to use 5-hydroxytryptophan as a substrate, and reduces the enzyme activity toward 3,4-dihydroxy-L-phenylalanine (dopa). Additionally, the reverse mutation in a Catharanthus roseus tryptophan decarboxylase (TDC) enables the mutant enzyme to utilize tyrosine and dopa as substrates with a reduced affinity toward tryptophan. Molecular modeling and molecular docking of the P. somniferum TyDC and the C. roseus TDC enzymes provided a structural basis to explain alterations in substrate specificity. Identification of an active site residue that impacts substrate selectivity produces a primary sequence identifier that may help differentiate the indolic and phenolic substrate specificities of individual plant AAADs. PMID:25107664

  14. Correspondence between flowers and leaves in terpenoid indole alkaloid metabolism of the phytoplasma-infected Catharanthus roseus plants.

    PubMed

    Srivastava, Suchi; Pandey, Richa; Kumar, Sushil; Nautiyal, Chandra Shekhar

    2014-11-01

    Several plants of Catharanthus roseus cv 'leafless inflorescence (lli)' showing phenotype of phytoplasma infection were observed for symptoms of early flowering, virescence, phyllody, and apical clustering of branches. Symptomatic plants were studied for the presence/absence and identity of phytoplasma in flowers. Transcription levels of several genes involved in plants' metabolism and development, accumulation of pharmaceutically important terpenoid indole alkaloids in flowers and leaves and variation in the root-associated microbial flora were examined. The expression profile of 12 genes studied was semi-quantitatively similar in control leaves and phytoplasma-infected leaves and flowers, in agreement with the symptoms of virescence and phyllody in phytoplasma-infected plants. The flowers of phytoplasma-infected plants possessed the TIA profile of leaves and accumulated catharanthine, vindoline, and vincristine and vinblastine in higher concentrations than leaves. The roots of the infected plants displayed lower microbial diversity than those of normal plants. In conclusion, phytoplasma affected the biology of C. roseus lli plants multifariously, it reduced the differences between the metabolite accumulates of the leaves and flowers and restrict the microbial diversity of rhizosphere. PMID:24658891

  15. The effects of UV-B stress on the production of terpenoid indole alkaloids in Catharanthus roseus hairy roots.

    PubMed

    Binder, Bernard Y K; Peebles, Christie A M; Shanks, Jacqueline V; San, Ka-Yiu

    2009-01-01

    In nature, plants generate protective secondary metabolites in response to environmental stresses. Such metabolites include terpenoid indole alkaloids (TIAs), which absorb UV-B light and serve putatively to protect the plant from harmful radiation. Catharanthus roseus plants, multiple shoot cultures, and cell suspension cultures exposed to UV-B light show significant increases in the production of TIAs, including precursors to vinblastine and vincristine, which have proven effective in the treatment of leukemia and lymphoma. Here, the effect of UV-B light on C. roseus hairy roots was examined. Analysis of alkaloid concentrations up to 168 h after UV-B exposure shows significant increases in the concentrations of lochnericine and significant decreases in the concentration of hörhammericine over time (ANOVA, P < 0.05). Our results also indicate that increasing UV-B exposure time up to 20 min caused significant increases in lochnericine, serpentine, and ajmalicine and a decrease in hörhammericine (t-test, p < 0.05). PMID:19479674

  16. Silencing the Transcriptional Repressor, ZCT1, Illustrates the Tight Regulation of Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus Hairy Roots.

    PubMed

    Rizvi, Noreen F; Weaver, Jessica D; Cram, Erin J; Lee-Parsons, Carolyn W T

    2016-01-01

    The Catharanthus roseus plant is the source of many valuable terpenoid indole alkaloids (TIAs), including the anticancer compounds vinblastine and vincristine. Transcription factors (TFs) are promising metabolic engineering targets due to their ability to regulate multiple biosynthetic pathway genes. To increase TIA biosynthesis, we elicited the TIA transcriptional activators (ORCAs and other unidentified TFs) with the plant hormone, methyl jasmonate (MJ), while simultaneously silencing the expression of the transcriptional repressor ZCT1. To silence ZCT1, we developed transgenic hairy root cultures of C. roseus that expressed an estrogen-inducible Zct1 hairpin for activating RNA interference. The presence of 17β-estradiol (5μM) effectively depleted Zct1 in hairy root cultures elicited with MJ dosages that either optimize or inhibit TIA production (250 or 1000μM). However, silencing Zct1 was not sufficient to increase TIA production or the expression of the TIA biosynthetic genes (G10h, Tdc, and Str), illustrating the tight regulation of TIA biosynthesis. The repression of the TIA biosynthetic genes at the inhibitory MJ dosage does not appear to be solely regulated by ZCT1. For instance, while Zct1 and Zct2 levels decreased through activating the Zct1 hairpin, Zct3 levels remained elevated. Since ZCT repressors have redundant yet distinct functions, silencing all three ZCTs may be necessary to relieve their repression of alkaloid biosynthesis. PMID:27467510

  17. Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.

    PubMed

    Rai, Avanish; Smita, Shachi S; Singh, Anup Kumar; Shanker, Karuna; Nagegowda, Dinesh A

    2013-09-01

    Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis. PMID:23543438

  18. Cytosine hypomethylation at CHG and CHH sites in the pleiotropic mutants of Mendelian inheritance in Catharanthus roseus.

    PubMed

    Kumari, Renu; Yadav, Gitanjali; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

    2013-12-01

    The 5S and 18S rDNA sequences of Catharanthus roseus cv 'Nirmal' (wild type) and its leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill) single mutants and lli egd, lli ill and egd ill double mutants were characterized. The lli, egd and ill mutants of Mendelian inheritance bore the names after their most conspicuous morphological feature(s). They had been chemically induced and isolated for their salt tolerance. The double mutants were isolated as morphological segregants from crosses between single mutants. The morphological features of the two parents accompanied salt tolerance in the double mutants. All the six mutants were hypomethylated at repeat sequences, upregulated and downregulated for many genes and carried pleiotropic alterations for several traits. Here the 5S and 18S rDNAs of C. roseus were found to be relatively low in cytosine content. Cytosines were preponderantly in CG context (53%) and almost all of them were methylated (97%). The cytosines in CHH and CHG (where H = A, T or C) contexts were largely demethylated (92%) in mutants. The demethylation was attributable to reduced expression of RDR2 and DRM2 led RNA dependant DNA methylation and CMT3 led maintenance methylation pathways. Mutants had gained some cytosines by substitution of C at T sites. These perhaps arose on account of errors in DNA replication, mediated by widespread cytosine demethylation at CHG and CHH sites. It was concluded that the regulation of cytosine ethylation mechanisms was disturbed in the mutants. ILL, EGD and LLI genes were identified as the positive regulators of other genes mediating the RdDM and CMT3 pathways, for establishment and maintenance of cytosine methylation in C. roseus. PMID:24371171

  19. Overexpression of ORCA3 and G10H in Catharanthus roseus Plants Regulated Alkaloid Biosynthesis and Metabolism Revealed by NMR-Metabolomics

    PubMed Central

    Pan, Qifang; Wang, Quan; Yuan, Fang; Xing, Shihai; Zhao, Jingya; Choi, Young Hae; Verpoorte, Robert; Tian, Yuesheng; Wang, Guofeng; Tang, Kexuan

    2012-01-01

    In order to improve the production of the anticancer dimeric indole alkaloids in Catharanthuse roseus, much research has been dedicated to culturing cell lines, hairy roots, and efforts to elucidate the regulation of the monoterpenoid indole alkaloid (MIA) biosynthesis. In this study, the ORCA3 (Octadecanoid-derivative Responsive Catharanthus AP2-domain) gene alone or integrated with the G10H (geraniol 10-hydroxylase) gene were first introduced into C. roseus plants. Transgenic C. roseus plants overexpressing ORCA3 alone (OR lines), or co-overexpressing G10H and ORCA3 (GO lines) were obtained by genetic modification. ORCA3 overexpression induced an increase of AS, TDC, STR and D4H transcripts but did not affect CRMYC2 and G10H transcription. G10H transcripts showed a significant increase under G10H and ORCA3 co-overexpression. ORCA3 and G10H overexpression significantly increased the accumulation of strictosidine, vindoline, catharanthine and ajmalicine but had limited effects on anhydrovinblastine and vinblastine levels. NMR-based metabolomics confirmed the higher accumulation of monomeric indole alkaloids in OR and GO lines. Multivariate data analysis of 1H NMR spectra showed change of amino acid, organic acid, sugar and phenylpropanoid levels in both OR and GO lines compared to the controls. The result indicated that enhancement of MIA biosynthesis by ORCA3 and G10H overexpression might affect other metabolic pathways in the plant metabolism of C. roseus. PMID:22916202

  20. Large scale in-silico identification and characterization of simple sequence repeats (SSRs) from de novo assembled transcriptome of Catharanthus roseus (L.) G. Don.

    PubMed

    Kumar, Santosh; Shah, Niraj; Garg, Vanika; Bhatia, Sabhyata

    2014-06-01

    Transcriptomic data of C. roseus offering ample sequence resources for providing better insights into gene diversity: large resource of genic SSR markers to accelerate genomic studies and breeding in Catharanthus . Next-generation sequencing is an efficient system for generating high-throughput complete transcripts/genes and developing molecular markers. We present here the transcriptome sequencing of a 26-day-old Catharanthus roseus seedling tissue using Illumina GAIIX platform that resulted in a total of 3.37 Gb of nucleotide sequence data comprising 29,964,104 reads which were de novo assembled into 26,581 unigenes. Based on similarity searches 58 % of the unigenes were annotated of which 13,580 unique transcripts were assigned 5016 gene ontology terms. Further, 7,687 of the unigenes were found to have Cluster of Orthologous Group classifications, and 4,006 were assigned to 289 Kyoto Encyclopedia of Genes and Genome pathways. Also, 5,221 (19.64 %) of transcripts were distributed to 81 known transcription factor (TF) families. In-silico analysis of the transcriptome resulted in identification of 11,004 SSRs in 26.62 % transcripts from which 2,520 SSR markers were designed which exhibited a non-random pattern of distribution. The most abundant was the trinucleotide repeats (AAG/CTT) followed by the dinucleotide repeats (AG/CT). Location specific analysis of SSRs revealed that SSRs were preferentially associated with the 5'-UTRs with a predicted role in regulation of gene expression. A PCR validation of a set of 48 primers revealed 97.9 % successful amplification, and 76.6 % of them showed polymorphism across different Catharanthus species as well as accessions of C. roseus. In summary, this study will provide an insight into understanding the seedling development and resources for novel gene discovery and SSR development for utilization in marker-assisted selective breeding in C. roseus. PMID:24482265

  1. Precursor feeding studies and molecular characterization of geraniol synthase establish the limiting role of geraniol in monoterpene indole alkaloid biosynthesis in Catharanthus roseus leaves.

    PubMed

    Kumar, Krishna; Kumar, Sarma Rajeev; Dwivedi, Varun; Rai, Avanish; Shukla, Ashutosh K; Shanker, Karuna; Nagegowda, Dinesh A

    2015-10-01

    The monoterpene indole alkaloids (MIAs) are generally derived from strictosidine, which is formed by condensation of the terpene moiety secologanin and the indole moiety tryptamine. There are conflicting reports on the limitation of either terpene or indole moiety in the production of MIAs in Catharanthus roseus cell cultures. Formation of geraniol by geraniol synthase (GES) is the first step in secologanin biosynthesis. In this study, feeding of C. roseus leaves with geraniol, but not tryptophan (precursor for tryptamine), increased the accumulation of the MIAs catharanthine and vindoline, indicating the limitation of geraniol in MIA biosynthesis. This was further validated by molecular and in planta characterization of C. roseus GES (CrGES). CrGES transcripts exhibited leaf and shoot specific expression and were induced by methyl jasmonate. Virus-induced gene silencing (VIGS) of CrGES significantly reduced the MIA content, which was restored to near-WT levels upon geraniol feeding. Moreover, over-expression of CrGES in C. roseus leaves increased MIA content. Further, CrGES exhibited correlation with MIA levels in leaves of different C. roseus cultivars and has significantly lower expression relative to other pathway genes. These results demonstrated that the transcriptional regulation of CrGES and thus, the in planta geraniol availability plays crucial role in MIA biosynthesis. PMID:26398791

  2. A simple and rapid HPLC-DAD method for simultaneously monitoring the accumulation of alkaloids and precursors in different parts and different developmental stages of Catharanthus roseus plants.

    PubMed

    Pan, Qifang; Saiman, Mohd Zuwairi; Mustafa, Natali Rianika; Verpoorte, Robert; Tang, Kexuan

    2016-03-01

    A rapid and simple reversed phase liquid chromatographic system has been developed for simultaneous analysis of terpenoid indole alkaloids (TIAs) and their precursors. This method allowed separation of 11 compounds consisting of eight TIAs (ajmalicine, serpentine, catharanthine, vindoline, vindolinine, vincristine, vinblastine, and anhydrovinblastine) and three related precursors i.e., tryptophan, tryptamine and loganin. The system has been applied for screening the TIAs and precursors in Catharanthus roseus plant extracts. In this study, different organs i.e., flowers, leaves, stems, and roots of C. roseus were investigated. The results indicate that TIAs and precursor accumulation varies qualitatively and quantitatively in different organs of C. roseus. The precursors showed much lower levels than TIAs in all organs. Leaves and flowers accumulate higher level of vindoline, catharanthine and anhydrovinblastine while roots have higher level of ajmalicine, vindolinine and serpentine. Moreover, the alkaloid profiles of leaves harvested at different ages and different growth stages were studied. The results show that the levels of monoindole alkaloids decreased while bisindole alkaloids increased with leaf aging and upon plant growth. The HPLC method has been successfully applied to detect TIAs and precursors in different types of C. roseus samples to facilitate further study of the TIA pathway and its regulation in C. roseus plants. PMID:26854826

  3. Effects of β-cyclodextrin and methyl jasmonate on the production of vindoline, catharanthine, and ajmalicine in Catharanthus roseus cambial meristematic cell cultures.

    PubMed

    Zhou, Pengfei; Yang, Jiazeng; Zhu, Jianhua; He, Shuijie; Zhang, Wenjin; Yu, Rongmin; Zi, Jiachen; Song, Liyan; Huang, Xuesong

    2015-09-01

    Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus, and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids. PMID:25981997

  4. An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death.

    PubMed

    Palem, Padmini P C; Kuriakose, Gini C; Jayabaskaran, Chelliah

    2015-01-01

    Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp--CrP14, obtained from stem tissues, and Talaromyces radicus--CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 μg/ml and 20 μg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus--CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus--CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 μg/l) in modified M2 medium and of vinblastine (70 μg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns. PMID:26697875

  5. Vindoline Formation in Shoot Cultures of Catharanthus roseus is Synchronously Activated with Morphogenesis Through the Last Biosynthetic Step

    PubMed Central

    Campos-Tamayo, Freddy; Hernández-Domínguez, Elizabeta; Vázquez-Flota, Felipe

    2008-01-01

    Background and Aims The Madagascar periwinkle (Catharanthus roseus) produces the monoterpenoid alkaloid vindoline, which requires a specialized cell organization present only in the aerial tissues. Vindoline content can be affected by photoperiod and this effect seems to be associated with the morphogenetic capacity of branches; this association formed the basis of the study reported here. Methods Vindoline-producing in vitro shoot cultures were exposed either to continuous light or a 16-h photoperiod regime. New plantlet formation and alkaloid biosynthesis were analysed throughout a culture cycle. Key Results In cultures under the photoperiod, the formation of new plantlets occurred in a more synchronized fashion as compared to those under continuous light. The accumulation of vindoline in cultures under the photoperiod occurred in co-ordination with plantlet formation, in constrast to cultures under continuous light, and coincided with a peak of activity of deacetylvindoline acetyl CoA acetyltransferase (DAT), the enzyme that catalyses the last step in vindoline biosynthesis. When new plantlet formation was blocked in cultures under the photoperiod by treatment with phytoregulators, vindoline synthesis was also reduced via an effect on DAT activity. No association between plantlet formation and other biosynthetic enzymes, such as tryptophan decarboxylase (TDC) and deacetoxyvindoline 4-hydroxylase (D4H), was found. Effects of light treatment on vindoline synthesis were not mediated by ORCA-3 proteins (which are involved in the induction of alkaloid synthesis in response to elicitation), suggesting that the presence of a different set of regulatory proteins. Conclusions The data suggest that vindoline biosynthesis is associated with morphogenesis in shoot cultures of C. roseus. PMID:18587132

  6. Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

    PubMed Central

    Han, Mei; Heppel, Simon C.; Su, Tao; Bogs, Jochen; Zu, Yuangang; An, Zhigang; Rausch, Thomas

    2013-01-01

    In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation. PMID:23650515

  7. An Endophytic Fungus, Talaromyces radicus, Isolated from Catharanthus roseus, Produces Vincristine and Vinblastine, Which Induce Apoptotic Cell Death

    PubMed Central

    Jayabaskaran, Chelliah

    2015-01-01

    Endophytic fungi isolated from Catharanthus roseus were screened for the production of vincristine and vinblastine. Twenty-two endophytic fungi isolated from various tissues of C. roseus were characterized taxonomically by sequence analysis of the internal transcribed spacer (ITS) region of rDNA and grouped into 10 genera: Alternaria, Aspergillus, Chaetomium, Colletotrichum, Dothideomycetes, Eutypella, Eutypa, Flavodon, Fusarium and Talaromyces. The antiproliferative activity of these fungi was assayed in HeLa cells using the MTT assay. The fungal isolates Eutypella sp—CrP14, obtained from stem tissues, and Talaromyces radicus—CrP20, obtained from leaf tissues, showed the strongest antiproliferative activity, with IC50 values of 13.5 μg/ml and 20 μg/ml, respectively. All 22 endophytic fungi were screened for the presence of the gene encoding tryptophan decarboxylase (TDC), the key enzyme in the terpenoid indole alkaloid biosynthetic pathway, though this gene could only be amplified from T. radicus—CrP20 (NCBI GenBank accession number KC920846). The production of vincristine and vinblastine by T. radicus—CrP20 was confirmed and optimized in nine different liquid media. Good yields of vincristine (670 μg/l) in modified M2 medium and of vinblastine (70 μg/l) in potato dextrose broth medium were obtained. The cytotoxic activity of partially purified fungal vincristine was evaluated in different human cancer cell lines, with HeLa cells showing maximum susceptibility. The apoptosis-inducing activity of vincristine derived from this fungus was established through cell cycle analysis, loss of mitochondrial membrane potential and DNA fragmentation patterns. PMID:26697875

  8. The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus.

    PubMed

    Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O'Connor, Sarah E; Rischer, Heiko; Memelink, Johan; Goossens, Alain

    2015-06-30

    Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix-loop-helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures. PMID:26080427

  9. The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

    PubMed Central

    Van Moerkercke, Alex; Steensma, Priscille; Schweizer, Fabian; Pollier, Jacob; Gariboldi, Ivo; Payne, Richard; Vanden Bossche, Robin; Miettinen, Karel; Espoz, Javiera; Purnama, Purin Candra; Kellner, Franziska; Seppänen-Laakso, Tuulikki; O’Connor, Sarah E.; Rischer, Heiko; Memelink, Johan; Goossens, Alain

    2015-01-01

    Plants make specialized bioactive metabolites to defend themselves against attackers. The conserved control mechanisms are based on transcriptional activation of the respective plant species-specific biosynthetic pathways by the phytohormone jasmonate. Knowledge of the transcription factors involved, particularly in terpenoid biosynthesis, remains fragmentary. By transcriptome analysis and functional screens in the medicinal plant Catharanthus roseus (Madagascar periwinkle), the unique source of the monoterpenoid indole alkaloid (MIA)-type anticancer drugs vincristine and vinblastine, we identified a jasmonate-regulated basic helix–loop–helix (bHLH) transcription factor from clade IVa inducing the monoterpenoid branch of the MIA pathway. The bHLH iridoid synthesis 1 (BIS1) transcription factor transactivated the expression of all of the genes encoding the enzymes that catalyze the sequential conversion of the ubiquitous terpenoid precursor geranyl diphosphate to the iridoid loganic acid. BIS1 acted in a complementary manner to the previously characterized ethylene response factor Octadecanoid derivative-Responsive Catharanthus APETALA2-domain 3 (ORCA3) that transactivates the expression of several genes encoding the enzymes catalyzing the conversion of loganic acid to the downstream MIAs. In contrast to ORCA3, overexpression of BIS1 was sufficient to boost production of high-value iridoids and MIAs in C. roseus suspension cell cultures. Hence, BIS1 might be a metabolic engineering tool to produce sustainably high-value MIAs in C. roseus plants or cultures. PMID:26080427

  10. Volatile composition of Catharanthus roseus (L.) G. Don using solid-phase microextraction and gas chromatography/mass spectrometry.

    PubMed

    De Pinho, P Guedes; Gonçalves, Rui F; Valentão, Patrícia; Pereira, David M; Seabra, Rosa M; Andrade, Paula B; Sottomayor, Mariana

    2009-04-01

    A total of 88 volatile and semi-volatile components were formally or tentatively identified in flowers, leaves and stems of Catharanthus roseus (L.) G. Don (cv. Little Bright Eye), by headspace solid-phase microextraction (HS-SPME) and by dichloromethane extraction, combined with gas chromatography-mass spectrometry (GC-MS). These include some diterpenic compounds (manool and manoyl oxides), a sesquiterpen (alpha-bisabolol), and some pyridine, pyrazine, indol and carotenoid derivatives. Applying multivariate analysis (principal component analysis and agglomerative hierarchic cluster analysis) to the HS-SPME-GC-MS data, it was possible to characterize each part of the vegetal material using a relative small number of compounds. Hence, flowers were richer in terpenic molecules (including limonene), alpha-bisabolol, methyljasmonate, cis-jasmone, 2-phenylethanol, phenylacetaldehyde, trans-2-octenal, benzylic alcohol and 2-isobutyl-3-methoxypyrazine. Leaves can be characterized by the methyl and propyl esters of fatty acids, mono- and disaturated, trans-phytol, carotenoid derivative compounds, hydrofarnesylacetone, methylanthranilate, manool and epi-manool oxide, while stems have high levels of volatile aldehydes, such as hexanal, octanal, cis-2-nonenal, cis-2-decenal, cis, trans-2,6-nonadienal, trans, trans-2,4-decadienal and cis, trans-2,4-decadienal. Dichloromethane extraction allowed also the identification of some alkaloid-like compounds that were not detected by HS-SPME. PMID:19186019

  11. Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells).

    PubMed Central

    Wissing, J. B.; Kornak, B.; Funke, A.; Riedel, B.

    1994-01-01

    Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %. PMID:12232252

  12. Isolation, Purification and Characterization of Vinblastine and Vincristine from Endophytic Fungus Fusarium oxysporum Isolated from Catharanthus roseus

    PubMed Central

    Kumar, Ashutosh; Patil, Deepak; Rajamohanan, Pattuparambil Ramanpillai; Ahmad, Absar

    2013-01-01

    Endophytic fungi reside in a symbiotic fashion inside their host plants, mimic their chemistry and interestingly, produce the same natural products as their hosts and are thus being screened for the production of valuable compounds like taxol, camptothecin, podophyllotoxin, etc. Vinblastine and vincristine are excellent anti-cancer drugs but their current production using plants is non-abundant and expensive. In order to make these drugs readily available to the patients at affordable prices, we isolated the endophytic fungi from Catharanthus roseus plant and found a fungus AA-CRL-6 which produces vinblastine and vincristine in appreciable amounts. These drugs were purified by TLC and HPLC and characterized using UV-Vis spectroscopy, ESI-MS, MS/MS and 1H NMR. One liter of culture filtrate yielded 76 µg and 67 µg of vinblastine and vincristine respectively. This endophytic fungal strain was identified as Fusarium oxysporum based upon its cultural and morphological characteristics and internal transcribed spacer (ITS) sequence analysis. PMID:24066024

  13. Salicylic acid restrains nickel toxicity, improves antioxidant defence system and enhances the production of anticancer alkaloids in Catharanthus roseus (L.).

    PubMed

    Idrees, Mohd; Naeem, M; Aftab, Tariq; Khan, M Masroor A; Moinuddin

    2013-05-15

    Salicylic acid (SA) has been reported to ameliorate various stresses in plants. In order to explore the role of SA under nickel (Ni) stress, thirty-days old plants of periwinkle (Catharanthus roseus L.) were supplied with eight treatments comprising basal application of Ni (0, 50, 100 and 150 mg kg(-1)) and foliar application of SA (0 and 10(-5)M) under net house conditions. Ni application significantly reduced the growth attributes including plant height, leaf-area index and fresh and dry weights of shoot and root. Increasing Ni concentration led to a gradual decrease in photosynthetic parameters and activities of nitrate reductase and carbonic anhydrase. The plants, undergoing Ni stress, exhibited a significant increase in the activity of superoxide dismutase, catalase and peroxidase together with an increase in electrolyte leakage and proline content. Total alkaloid content was also declined in Ni-treated plants. Foliar application of SA (10(-5)M) reduced the deleterious effects of Ni on plant growth, accelerating the restoration of growth processes. SA also improved the total alkaloid content under normal as well as adverse conditions. Foliar spray of SA significantly improved the content of anticancer alkaloids vincristine (by 22.2%) and vinblastine (by 50.0%) in plants treated with 150 mg kg(-1) of Ni. PMID:23597961

  14. Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

    PubMed Central

    Sakano, Katsuhiro

    1990-01-01

    Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H+ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H+/H2PO4− stoichiometry of the cotransport is most likely to be 4. PMID:16667491

  15. Ornamental exterior versus therapeutic interior of Madagascar periwinkle (Catharanthus roseus): the two faces of a versatile herb.

    PubMed

    Nejat, Naghmeh; Valdiani, Alireza; Cahill, David; Tan, Yee-How; Maziah, Mahmood; Abiri, Rambod

    2015-01-01

    Catharanthus roseus (L.) known as Madagascar periwinkle (MP) is a legendary medicinal plant mostly because of possessing two invaluable antitumor terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The plant has also high aesthetic value as an evergreen ornamental that yields prolific blooms of splendid colors. The plant possesses yet another unique characteristic as an amiable experimental host for the maintenance of the smallest bacteria found on earth, the phytoplasmas and spiroplasmas, and serves as a model for their study. Botanical information with respect to synonyms, vernacular names, cultivars, floral morphology, and reproduction adds to understanding of the plant while the geography and ecology of periwinkle illustrate the organism's ubiquity. Good agronomic practices ensure generous propagation of healthy plants that serve as a source of bioactive compounds and multitudinous horticultural applications. The correlation between genetic diversity, variants, and TIA production exists. MP is afflicted with a whole range of diseases that have to be properly managed. The ethnobotanical significance of MP is exemplified by its international usage as a traditional remedy for abundant ailments and not only for cancer. TIAs are present only in micro quantities in the plant and are highly poisonous per se rendering a challenge for researchers to increase yield and reduce toxicity. PMID:25667940

  16. Ornamental Exterior versus Therapeutic Interior of Madagascar Periwinkle (Catharanthus roseus): The Two Faces of a Versatile Herb

    PubMed Central

    Valdiani, Alireza; Cahill, David; Tan, Yee-How; Maziah, Mahmood; Abiri, Rambod

    2015-01-01

    Catharanthus roseus (L.) known as Madagascar periwinkle (MP) is a legendary medicinal plant mostly because of possessing two invaluable antitumor terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The plant has also high aesthetic value as an evergreen ornamental that yields prolific blooms of splendid colors. The plant possesses yet another unique characteristic as an amiable experimental host for the maintenance of the smallest bacteria found on earth, the phytoplasmas and spiroplasmas, and serves as a model for their study. Botanical information with respect to synonyms, vernacular names, cultivars, floral morphology, and reproduction adds to understanding of the plant while the geography and ecology of periwinkle illustrate the organism's ubiquity. Good agronomic practices ensure generous propagation of healthy plants that serve as a source of bioactive compounds and multitudinous horticultural applications. The correlation between genetic diversity, variants, and TIA production exists. MP is afflicted with a whole range of diseases that have to be properly managed. The ethnobotanical significance of MP is exemplified by its international usage as a traditional remedy for abundant ailments and not only for cancer. TIAs are present only in micro quantities in the plant and are highly poisonous per se rendering a challenge for researchers to increase yield and reduce toxicity. PMID:25667940

  17. A polymorphic (GA/CT)n- SSR influences promoter activity of Tryptophan decarboxylase gene in Catharanthus roseus L. Don.

    PubMed

    Kumar, Santosh; Bhatia, Sabhyata

    2016-01-01

    Simple Sequence Repeats (SSRs) of polypurine-polypyrimidine type motifs occur very frequently in the 5' flanks of genes in plants and have recently been implicated to have a role in regulation of gene expression. In this study, 2 accessions of Catharanthus roseus having (CT)8 and (CT)21 varying motifs in the 5'UTR of Tryptophan decarboxylase (Tdc) gene, were investigated for its role in regulation of gene expression. Extensive Tdc gene expression analysis in the 2 accessions was carried out both at the level of transcription and translation. Transcript abundance was estimated using Northern analysis and qRT-PCR, whereas the rate of Tdc gene transcription was assessed using in-situ nuclear run-on transcription assay. Translation status of Tdc gene was monitored by quantification of polysome associated Tdc mRNA using qRT-PCR. These observations were validated through transient expression analysis using the fusion construct [CaM35S:(CT)8-21:GUS]. Our study demonstrated that not only does the length of (CT)n -SSRs influences the promoter activity, but the presence of SSRs per se in the 5'-UTR significantly enhances the level of gene expression. We termed this phenomenon as "microsatellite mediated enhancement" (MME) of gene expression. Results presented here will provide leads for engineering plants with enhanced amounts of medicinally important alkaloids. PMID:27623355

  18. The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus1[W][OA

    PubMed Central

    Suttipanta, Nitima; Pattanaik, Sitakanta; Kulshrestha, Manish; Patra, Barunava; Singh, Sanjay K.; Yuan, Ling

    2011-01-01

    Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the down-regulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Compared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. PMID:21988879

  19. Differential induction of meristematic stem cells of Catharanthus roseus and their characterization.

    PubMed

    Moon, So Hyun; Venkatesh, Jelli; Yu, Jae-Woong; Park, Se Won

    2015-11-01

    Plant cell culture technology has been introduced for the mass production of the many useful components. A variety of plant-derived compounds is being used in various fields, such as pharmaceuticals, foods, and cosmetics. Plant cell cultures are believed to be derived from the dedifferentiation process. In the present study, an undifferentiated cambial meristematic cell (CMCs) of Catharanthus is isolated using histological and genetic methods, and compared with dedifferentiation-derived callus (DDCs) cultures. Furthermore, differential culture conditions for both DDCs- and CMCs-derived cell lines were established. A suitable media for the increased accumulation of terpenoid indole alkaloids (TIAs) was also standardized. Compared with DDCs, CMCs showed marked accumulation of TIAs in cell lines grown on media with 1.5 mg·mL(-1) of NAA and 0.5 mg·mL(-1) of kinetin. CMCs-derived cultures of Catharanthus, as a source of key anticancer drugs (viblastine and vincristine), would overcome the obstacles usually associated with the production of natural metabolites through the use of DDCs. Cell culture systems that are derived from CMCs may also provide a cost-effective and eco-friendly basis for the sustainable production of a number of important plant natural products. PMID:26298518

  20. The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution, Molecular Marker Identification, and Phylogenetic Implications in Asterids

    PubMed Central

    Ku, Chuan; Chung, Wan-Chia; Chen, Ling-Ling; Kuo, Chih-Horng

    2013-01-01

    The Madagascar periwinkle (Catharanthusroseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C. roseus, which could be applied to other C. roseus-related studies. The C. roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffeaarabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepiassyriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C. roseus-specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C. roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported. PMID:23825699

  1. The Complete Plastid Genome Sequence of Madagascar Periwinkle Catharanthus roseus (L.) G. Don: Plastid Genome Evolution, Molecular Marker Identification, and Phylogenetic Implications in Asterids.

    PubMed

    Ku, Chuan; Chung, Wan-Chia; Chen, Ling-Ling; Kuo, Chih-Horng

    2013-01-01

    The Madagascar periwinkle (Catharanthusroseus in the family Apocynaceae) is an important medicinal plant and is the source of several widely marketed chemotherapeutic drugs. It is also commonly grown for its ornamental values and, due to ease of infection and distinctiveness of symptoms, is often used as the host for studies on phytoplasmas, an important group of uncultivated plant pathogens. To gain insights into the characteristics of apocynaceous plastid genomes (plastomes), we used a reference-assisted approach to assemble the complete plastome of C. roseus, which could be applied to other C. roseus-related studies. The C. roseus plastome is the second completely sequenced plastome in the asterid order Gentianales. We performed comparative analyses with two other representative sequences in the same order, including the complete plastome of Coffeaarabica (from the basal Gentianales family Rubiaceae) and the nearly complete plastome of Asclepiassyriaca (Apocynaceae). The results demonstrated considerable variations in gene content and plastome organization within Apocynaceae, including the presence/absence of three essential genes (i.e., accD, clpP, and ycf1) and large size changes in non-coding regions (e.g., rps2-rpoC2 and IRb-ndhF). To find plastome markers of potential utility for Catharanthus breeding and phylogenetic analyses, we identified 41 C. roseus-specific simple sequence repeats. Furthermore, five intergenic regions with high divergence between C. roseus and three other euasterids I taxa were identified as candidate markers. To resolve the euasterids I interordinal relationships, 82 plastome genes were used for phylogenetic inference. With the addition of representatives from Apocynaceae and sampling of most other asterid orders, a sister relationship between Gentianales and Solanales is supported. PMID:23825699

  2. Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells 1

    PubMed Central

    Bouzayen, Mondher; Latché, Alain; Pech, Jean-Claude; Marigo, Gérard

    1989-01-01

    The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a Km of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30°C. Several effectors known to inhibit ATPase (N,N′-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H+ electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN− and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH—, OH—, and NH2— groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions. PMID:16667182

  3. Jasmonic acid effect on the fatty acid and terpenoid indole alkaloid accumulation in cell suspension cultures of Catharanthus roseus.

    PubMed

    Goldhaber-Pasillas, Guitele Dalia; Mustafa, Natali Rianika; Verpoorte, Robert

    2014-01-01

    The stress response after jasmonic acid (JA) treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA) and terpenoid indole alkaloids (TIA). According to multivariate data analyses (MVDA), three major time events were observed and characterized according to the variations of specific FA and TIA: after 0-30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90-360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system. PMID:25029072

  4. The improved resistance to high salinity induced by trehalose is associated with ionic regulation and osmotic adjustment in Catharanthus roseus.

    PubMed

    Chang, Bowen; Yang, Lei; Cong, Weiwei; Zu, Yuangang; Tang, Zhonghua

    2014-04-01

    The effects of exogenous trehalose (Tre) on salt tolerance of pharmaceutical plant Catharanthus roseus and the physiological mechanisms were both investigated in this study. The results showed that the supplement of Tre in saline condition (250 mM NaCl) largely alleviated the inhibitory effects of salinity on plant growth, namely biomass accumulation and total leaf area per plant. In this saline condition, the decreased level of relative water content (RWC) and photosynthetic rate were also greatly rescued by exogenous Tre. This improved performance of plants under high salinity induced by Tre could be partly ascribed to its ability to decrease accumulation of sodium, and increase potassium in leaves. The exogenous Tre led to high levels of fructose, glucose, sucrose and Tre inside the salt-stressed plants during whole the three-week treatment. The major free amino acids such as proline, arginine, threonine and glutamate were also largely elevated in the first two-week course of treatment with Tre in saline solution. It was proposed here that Tre might act as signal to make the salt-stressed plants actively increase internal compatible solutes, including soluble sugars and free amino acids, to control water loss, leaf gas exchange and ionic flow at the onset of salt stress. The application of Tre in saline condition also promoted the accumulation of alkaloids. The regulatory role of Tre in improving salt tolerance was optimal with an exogenous concentration of 10 mM Tre. Larger concentrations of Tre were supra-optimum and adversely affected plant growth. PMID:24589477

  5. Evaluation of Catharanthus roseus leaf extract-mediated biosynthesis of titanium dioxide nanoparticles against Hippobosca maculata and Bovicola ovis.

    PubMed

    Velayutham, Kanayairam; Rahuman, Abdul Abdul; Rajakumar, Govindasamy; Santhoshkumar, Thirunavukkarasu; Marimuthu, Sampath; Jayaseelan, Chidambaram; Bagavan, Asokan; Kirthi, Arivarasan Vishnu; Kamaraj, Chinnaperumal; Zahir, Abdul Abduz; Elango, Gandhi

    2012-12-01

    The purpose of the present study was based on assessments of the antiparasitic activities of synthesized titanium dioxide nanoparticles (TiO(2) NPs) utilizing leaf aqueous extract of Catharanthus roseus against the adults of hematophagous fly, Hippobosca maculata Leach (Diptera: Hippoboscidae), and sheep-biting louse, Bovicola ovis Schrank (Phthiraptera: Trichodectidae). The synthesized TiO(2) NPs were analyzed by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The formation of the TiO(2) NPs synthesized from the XRD spectrum compared with the standard confirmed spectrum of titanium particles formed in the present experiments were in the form of nanocrystals, as evidenced by the peaks at 2θ values of 27.43°, 36.03°, and 54.32°. The FTIR spectra of TiO(2) NPs exhibited prominent peaks at 714 (Ti-O-O bond), 1,076 (C-N stretch aliphatic amines), 1,172 (C-O stretching vibrations in alcoholic groups), 1,642 (N-H bend bond), and 3,426 (O-H stretching due to alcoholic group). SEM analysis of the synthesized TiO(2) NPs clearly showed the clustered and irregular shapes, mostly aggregated and having the size of 25-110 nm. By Bragg's law and Scherrer's constant, it is proved that the mean size of synthesized TiO(2) NPs was 65 nm. The AFM obviously depicts the formation of the rutile and anatase forms in the TiO(2) NPs and also, the surface morphology of the particles is uneven due to the presence of some of the aggregates and individual particles. Adulticidal parasitic activity was observed in varying concentrations of aqueous leaf extract of C. roseus, TiO(2) solution, and synthesized TiO(2) NPs for 24 h. The maximum parasitic activity was observed in aqueous crude leaf extracts of C. roseus against the adults of H. maculata and B. ovis with LD(50) values of 36.17 and 30.35 mg/L, and r (2) values of 0.948 and 0.908, respectively. The highest efficacy was reported in 5 mM TiO(2

  6. Isolation of Catharanthus roseus (L.) G. Don Nuclei and Measurement of Rate of Tryptophan decarboxylase Gene Transcription Using Nuclear Run-On Transcription Assay

    PubMed Central

    Kumar, Santosh; Bhatia, Sabhyata

    2015-01-01

    Background An accurate assessment of transcription ‘rate’ is often desired to describe the promoter activity. In plants, isolation of transcriptionally active nuclei and their subsequent use in nuclear run-on assays has been challenging and therefore limit an accurate measurement of gene transcription ‘rate’. Catharanthus roseus has emerged as a model medicinal plant as it exhibits an unsurpassed spectrum of chemodiversity, producing over 130 alkaloids through the terpenoid indole alkaloid (TIA) pathway and therefore serves as a ‘molecular hub’ to understand gene expression profiles. Results The protocols presented here streamline, adapt and optimize the existing methods of nuclear run-on assay for use in C. roseus. Here, we fully describe all the steps to isolate transcriptionally active nuclei from C. roseus leaves and utilize them to perform nuclear run-on transcription assay. Nuclei isolated by this method transcribed at a level consistent with their response to external stimuli, as transcription rate of TDC gene was found to be higher in response to external stimuli i.e. when seedlings were subjected to UV-B light or to methyl jasmonate (MeJA). However, the relative transcript abundance measured parallel through qRT-PCR was found to be inconsistent with the synthesis rate indicating that some post transcriptional events might have a role in transcript stability in response to stimuli. Conclusions Our study provides an optimized, efficient and inexpensive method of isolation of intact nuclei and nuclear ‘run-on’ transcription assay to carry out in-situ measurement of gene transcription rate in Catharanthus roseus. This would be valuable in investigating the transcriptional and post transcriptional response of other TIA pathway genes in C. roseus. Isolated nuclei may also provide a resource that could be used for performing the chip assay as well as serve as the source of nuclear proteins for in-vitro EMSA studies. Moreover, nascent nuclear run

  7. High-throughput transcriptome analysis of the leafy flower transition of Catharanthus roseus induced by peanut witches'-broom phytoplasma infection.

    PubMed

    Liu, Li-Yu Daisy; Tseng, Hsin-I; Lin, Chan-Pin; Lin, Yen-Yu; Huang, Yuan-Hung; Huang, Chien-Kang; Chang, Tean-Hsu; Lin, Shih-Shun

    2014-05-01

    Peanut witches'-broom (PnWB) phytoplasma are obligate bacteria that cause leafy flower symptoms in Catharanthus roseus. The PnWB-mediated leafy flower transitions were studied to understand the mechanisms underlying the pathogen-host interaction; however, our understanding is limited because of the lack of information on the C. roseus genome. In this study, the whole-transcriptome profiles from healthy flowers (HFs) and stage 4 (S4) PnWB-infected leafy flowers of C. roseus were investigated using next-generation sequencing (NGS). More than 60,000 contigs were generated using a de novo assembly approach, and 34.2% of the contigs (20,711 genes) were annotated as putative genes through name-calling, open reading frame determination and gene ontology analyses. Furthermore, a customized microarray based on this sequence information was designed and used to analyze samples further at various stages of PnWB infection. In the NGS profile, 87.8% of the genes showed expression levels that were consistent with those in the microarray profiles, suggesting that accurate gene expression levels can be detected using NGS. The data revealed that defense-related and flowering gene expression levels were altered in S4 PnWB-infected leafy flowers, indicating that the immunity and reproductive stages of C. roseus were compromised. The network analysis suggested that the expression levels of >1,000 candidate genes were highly associated with CrSVP1/2 and CrFT expression, which might be crucial in the leafy flower transition. In conclusion, this study provides a new perspective for understanding plant pathology and the mechanisms underlying the leafy flowering transition caused by host-pathogen interactions through analyzing bioinformatics data obtained using a powerful, rapid high-throughput technique. PMID:24492256

  8. A Pair of Tabersonine 16-Hydroxylases Initiates the Synthesis of Vindoline in an Organ-Dependent Manner in Catharanthus roseus1[C][W

    PubMed Central

    Besseau, Sébastien; Kellner, Franziska; Lanoue, Arnaud; Thamm, Antje M.K.; Salim, Vonny; Schneider, Bernd; Geu-Flores, Fernando; Höfer, René; Guirimand, Grégory; Guihur, Anthony; Oudin, Audrey; Glevarec, Gaëlle; Foureau, Emilien; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc’h, Nathalie; St-Pierre, Benoit; Werck-Reichhart, Danièle; Burlat, Vincent; De Luca, Vincenzo; O’Connor, Sarah E.; Courdavault, Vincent

    2013-01-01

    Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis. PMID:24108213

  9. Rapid and simultaneous determination of five vinca alkaloids in Catharanthus roseus and human serum using trilinear component modeling of liquid chromatography-diode array detection data.

    PubMed

    Liu, Zhi; Wu, Hai-Long; Li, Yong; Gu, Hui-Wen; Yin, Xiao-Li; Xie, Li-Xia; Yu, Ru-Qin

    2016-07-15

    A novel chemometrics-assisted high performance liquid chromatography method coupled with diode array detector (HPLC-DAD) was proposed for the simultaneous determination of vincristine (VCR), vinblastine (VLB), vindoline (VDL), catharanthine (CAT) and yohimbine (YHB) in Catharanthus roseus (C. roseus) and human serum samples. With the second-order advantage of the alternating trilinear decomposition (ATLD) method, the resolution and rapid determination of five components of interest in complex matrices were performed, even in the present of heavy overlaps and unknown interferences. Therefore, multi-step purification was omitted and five components could be fast eluted out within 7.5min under simple isocratic elution condition (acetonitrile/0.2% formic acid water, 37:63, v/v). Statistical parameters, such as the linear correlation coefficient (R(2)), root-mean-square error of prediction (RMSEP), limit of detection (LOD) and limit of quantitation (LOQ) had been calculated to investigate the accuracy and reliability of the method. The average recoveries of five vinca alkaloids ranged from 97.1% to 101.9% and 98.8% to 103.0% in C. roseus and human serum samples, respectively. The five vinca alkaloids were adequately determined with limits of detection (LODs) of 29.5-49.3ngmL(-1) in C. roseus and 12.4-27.2ngmL(-1) in human serum samples, respectively. The obtained results demonstrated that the analytical strategy provided a feasible alternative for synchronously monitoring the quality of raw herb and the concentration of blood drugs. PMID:26321366

  10. Effects of Adding Vindoline and MeJA on Production of Vincristine and Vinblastine, and Transcription of their Biosynthetic Genes in the Cultured CMCs of Catharanthus roseus.

    PubMed

    Zhang, Wenjin; Yang, Jiazeng; Zi, Jiachen; Zhu, Jianhua; Song, Liyan; Yu, Rongmin

    2015-12-01

    Vincristine and vinblastine were found by Liquid Chromatography-Mass Spectrometry (LC-MS) in Catharanthus roseuscambial meristem cells (CMCs) jointly treated with 0.25 mM vindoline and methyl jasmonate (MeJA), suggesting that C. roseus CMCs contain a complete set of the enzymes which are in response to convert vindoline into vincristine and vinblastine. Based on the facts that the transcript levels of vindoline-biosynthetic genes (STR, SGD and D4H) were up-regulated instead of being down-regulated by adding itself to the culture, and that the transcriptional factor ORCA3 was up-regulated simultaneously, we further confirmed that the transcription of STR, SGD, D4H was manipulated by ORCA3. PMID:26882673

  11. Lack of Control in Inorganic Phosphate Uptake by Catharanthus roseus (L.) G. Don Cells (Cytoplasmic Inorganic Phosphate Homeostasis Depends on the Tonoplast Inorganic Phosphate Transport System?).

    PubMed Central

    Sakano, K.; Yazaki, Y.; Okihara, K.; Mimura, T.; Kiyota, S.

    1995-01-01

    Inorganic phosphate (Pi) uptake by Catharanthus roseus (L.) G. Don cells was studied in relation to its apparent uncontrolled uptake using 31P-nuclear magnetic resonance spectroscopy. Kinetics of Pi uptake by the cells indicated that apparent Km and Vm were about 7 [mu]M and 20 [mu]mol g-1 fresh weight h-1, respectively. Pi uptake in Murashige-Skoog medium under different Pi concentrations and different initial cell densities followed basically the same kinetics. When supplied with abundant Pi, cells absorbed Pi at a constant rate (Vm) for the first hours and accumulated it in the vacuole. As the endogenous pool expanded, the rate of Pi uptake gradually decreased to nil. Maximum Pi accumulation was 100 to 120 [mu]mol g-1 fresh weight if cell swelling during Pi uptake (about 2-fold in cell volume) was not considered. Results indicated that (a) the rate of Pi uptake by Catharanthus cells was independent of initial cell density and was constant over a wide range of Pi concentrations (2 mM to about 10 [mu]M) unless the cells were preloaded with excess Pi, and (b) there was no apparent feedback control over the Pi uptake process in the plasma membrane to avoid Pi toxicity. The importance of the tonoplast Pi transport system in cytoplasmic Pi homeostasis is discussed. PMID:12228474

  12. Promoter analysis reveals cis-regulatory motifs associated with the expression of the WRKY transcription factor CrWRKY1 in Catharanthus roseus.

    PubMed

    Yang, Zhirong; Patra, Barunava; Li, Runzhi; Pattanaik, Sitakanta; Yuan, Ling

    2013-12-01

    WRKY transcription factors (TFs) are emerging as an important group of regulators of plant secondary metabolism. However, the cis-regulatory elements associated with their regulation have not been well characterized. We have previously demonstrated that CrWRKY1, a member of subgroup III of the WRKY TF family, regulates biosynthesis of terpenoid indole alkaloids in the ornamental and medicinal plant, Catharanthus roseus. Here, we report the isolation and functional characterization of the CrWRKY1 promoter. In silico analysis of the promoter sequence reveals the presence of several potential TF binding motifs, indicating the involvement of additional TFs in the regulation of the TIA pathway. The CrWRKY1 promoter can drive the expression of a β-glucuronidase (GUS) reporter gene in native (C. roseus protoplasts and transgenic hairy roots) and heterologous (transgenic tobacco seedlings) systems. Analysis of 5'- or 3'-end deletions indicates that the sequence located between positions -140 to -93 bp and -3 to +113 bp, relative to the transcription start site, is critical for promoter activity. Mutation analysis shows that two overlapping as-1 elements and a CT-rich motif contribute significantly to promoter activity. The CrWRKY1 promoter is induced in response to methyl jasmonate (MJ) treatment and the promoter region between -230 and -93 bp contains a putative MJ-responsive element. The CrWRKY1 promoter can potentially be used as a tool to isolate novel TFs involved in the regulation of the TIA pathway. PMID:23979312

  13. Catharanthus roseus Aqueous Extract is Cytotoxic to Jurkat Leukaemic T-cells but Induces the Proliferation of Normal Peripheral Blood Mononuclear Cells

    PubMed Central

    Ahmad, Nor Hazwani; Rahim, Rohanizah Abdul; Mat, Ishak

    2010-01-01

    Research on natural products has been widely used as a strategy to discover new drugs with potential for applications in complementary medicines because they have fewer side effects than conventional drugs. The aim of the present study was to evaluate the in vitro cytotoxic effects of crude aqueous Catharanthus roseus extract on Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). The aqueous extract was standardised to vinblastine by high performance liquid chromatography (HPLC) and was used to determine cytotoxicity by the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. DNA fragmentation assay was employed to determine if cell death was due to apoptosis. The results showed that the aqueous extract induced cell death of Jurkat cells at 24, 48 and 72 hours post-treatment in a time- and dose-dependent manner. However, cells treated at 48 and 72 hours produced higher cytotoxic effects with half maximal inhibitory concentration (IC50) values of 2.55 μg/ml and 2.38 μg/ml, respectively. In contrast, the extract induced normal PBMC proliferation, especially after 24 hours treatment with 1000 μg/ml. This result indicates that the C. roseus crude aqueous extract showed differential effects of inhibiting the proliferation of the Jurkat cell line and promoting the growth of PBMCs. These data suggest that the extract may be applicable for modulating the normal and transformed immune cells in leukaemia patients. PMID:24575203

  14. Molecular Cloning and Characterization of a Vacuolar Class III Peroxidase Involved in the Metabolism of Anticancer Alkaloids in Catharanthus roseus1[C

    PubMed Central

    Costa, Maria Manuela R.; Hilliou, Frederique; Duarte, Patrícia; Pereira, Luís Gustavo; Almeida, Iolanda; Leech, Mark; Memelink, Johan; Barceló, Alfonso Ros; Sottomayor, Mariana

    2008-01-01

    Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to α-3′,4′-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in α-3′,4′-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant. PMID:18065566

  15. Identification of conserved and novel microRNAs in Catharanthus roseus by deep sequencing and computational prediction of their potential targets.

    PubMed

    Prakash, Pravin; Ghosliya, Dolly; Gupta, Vikrant

    2015-01-10

    MicroRNAs are small endogenous non-coding RNAs of ~19-24 nucleotides and perform regulatory roles in many plant processes. To identify miRNAs involved in regulatory networks controlling diverse biological processes including secondary metabolism in Catharanthus roseus, an important medicinal plant, we employed deep sequencing of small RNA from leaf tissue. A total of 88 potential miRNAs comprising of 81 conserved miRNAs belonging to 35 families and seven novel miRNAs were identified. Precursors for 16 conserved and seven novel cro-miRNAs were identified, and their stem-loop hairpin structures were predicted. Selected cro-miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in different vegetative tissues of C. roseus. Targets were predicted for conserved and novel cro-miRNAs, which were found to be involved in diverse biological role(s) including secondary metabolism. Our study enriches available resources and information regarding miRNAs and their potential targets for better understanding of miRNA-mediated gene regulation in plants. PMID:25445288

  16. The Combined Effects of Ethylene and MeJA on Metabolic Profiling of Phenolic Compounds in Catharanthus roseus Revealed by Metabolomics Analysis.

    PubMed

    Liu, Jia; Liu, Yang; Wang, Yu; Zhang, Zhong-Hua; Zu, Yuan-Gang; Efferth, Thomas; Tang, Zhong-Hua

    2016-01-01

    Phenolic compounds belong to a class of secondary metabolites and are implicated in a wide range of responsive mechanisms in plants triggered by both biotic and abiotic elicitors. In this study, we approached the combinational effects of ethylene and MeJA (methyl jasmonate) on phenolic compounds profiles and gene expressions in the medicinal plant Catharanthus roseus. In virtue of a widely non-targeted metabolomics method, we identified a total of 34 kinds of phenolic compounds in the leaves, composed by 7 C6C1-, 11 C6C3-, and 16 C6C3C6 compounds. In addition, 7 kinds of intermediates critical for the biosynthesis of phenolic compounds and alkaloids were identified and discussed with phenolic metabolism. The combinational actions of ethylene and MeJA effectively promoted the total phenolic compounds, especially the C6C1 compounds (such as salicylic acid, benzoic acid) and C6C3 ones (such as cinnamic acid, sinapic acid). In contrast, the C6C3C6 compounds displayed a notably inhibitory trend in this case. Subsequently, the gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 5 gene tags and the accumulation profiles of 41 metabolite peaks. Generally, we provide an insight into the controlling mode of ethylene-MeJA combination on phenolic metabolism in C. roseus leaves. PMID:27375495

  17. Involvement of the Octadecanoid Pathway and Protein Phosphorylation in Fungal Elicitor-Induced Expression of Terpenoid Indole Alkaloid Biosynthetic Genes in Catharanthus roseus

    PubMed Central

    Menke, Frank L.H.; Parchmann, Stefanie; Mueller, Martin J.; Kijne, Jan W.; Memelink, Johan

    1999-01-01

    Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor α-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously . Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates. PMID:10198087

  18. The Combined Effects of Ethylene and MeJA on Metabolic Profiling of Phenolic Compounds in Catharanthus roseus Revealed by Metabolomics Analysis

    PubMed Central

    Liu, Jia; Liu, Yang; Wang, Yu; Zhang, Zhong-Hua; Zu, Yuan-Gang; Efferth, Thomas; Tang, Zhong-Hua

    2016-01-01

    Phenolic compounds belong to a class of secondary metabolites and are implicated in a wide range of responsive mechanisms in plants triggered by both biotic and abiotic elicitors. In this study, we approached the combinational effects of ethylene and MeJA (methyl jasmonate) on phenolic compounds profiles and gene expressions in the medicinal plant Catharanthus roseus. In virtue of a widely non-targeted metabolomics method, we identified a total of 34 kinds of phenolic compounds in the leaves, composed by 7 C6C1-, 11 C6C3-, and 16 C6C3C6 compounds. In addition, 7 kinds of intermediates critical for the biosynthesis of phenolic compounds and alkaloids were identified and discussed with phenolic metabolism. The combinational actions of ethylene and MeJA effectively promoted the total phenolic compounds, especially the C6C1 compounds (such as salicylic acid, benzoic acid) and C6C3 ones (such as cinnamic acid, sinapic acid). In contrast, the C6C3C6 compounds displayed a notably inhibitory trend in this case. Subsequently, the gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 5 gene tags and the accumulation profiles of 41 metabolite peaks. Generally, we provide an insight into the controlling mode of ethylene-MeJA combination on phenolic metabolism in C. roseus leaves. PMID:27375495

  19. Jasmonate-dependent alkaloid biosynthesis in Catharanthus Roseus hairy root cultures is correlated with the relative expression of Orca and Zct transcription factors.

    PubMed

    Goklany, Sheba; Rizvi, Noreen F; Loring, Ralph H; Cram, Erin J; Lee-Parsons, Carolyn W T

    2013-01-01

    The effects of methyl jasmonate (MJ) dosage on terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus are correlated with the relative levels of specific MJ-responsive transcription factors. In this study, the expression of transcription factors (Orca, Zct, Gbf, Myc2, At-hook, and Wrky1), TIA pathway genes (G10h, Tdc, Str, and Sgd), and TIA metabolites (secologanin, strictosidine, and tabersonine) were investigated in C. roseus hairy root cultures elicited with a range of MJ dosages (0-1,000 µM) during mid-exponential growth. The highest production of TIA metabolites occurs at 250 μM MJ, increasing by 150-370% compared with untreated controls. At this MJ dosage, the expression of the transcriptional activators (Orca) is dramatically increased (29-40 fold) while the levels of the transcriptional repressors (Zct) remain low (2-7 fold). Simultaneously, the expression of genes coding for key enzymes involved in TIA biosynthesis increases by 8-15 fold. In contrast, high MJ dosages (1,000 µM) inhibit the production of TIA metabolites. This dosage is correlated with elevated expression levels of Zct (up to 40-fold) relative to Orca (13-19-fold) and minimal induction of the TIA biosynthetic genes (0-6 fold). The significant changes in the expression of Orca and Zct with MJ dosage do not correspond to changes in the expression of the early-response transcription factors (AT-hook, Myc2, and Wrky1) believed to regulate Orca and Zct. In summary, these observations suggest that the dependence of alkaloid production on MJ dosage in C. roseus may be partly mediated through the relative levels of Orca and Zct family transcription factors. PMID:23970483

  20. Suppression of aggressive strains of 'Candidatus phytoplasma mali' by mild strains in Catharanthus roseus and Nicotiana occidentalis and indication of similar action in apple trees.

    PubMed

    Schneider, Bernd; Sule, Sandor; Jelkmann, Wilhelm; Seemüller, Erich

    2014-05-01

    To study antagonistic interactions of 'Candidatus Phytoplasma mali' strains, graft inoculation of Catharanthus roseus and Nicotiana occidentalis was performed with mild strains 1/93Vin and 1/93Tab as suppressors and three aggressive strains as challengers. Inoculation of the suppressors was carried out in either the cross-protection modus prior to grafting of the challengers or by co-inoculating suppressors and challengers. Monitoring using multiplex real-time polymerase chain reaction assays revealed that, in long-term cross-protection trials with C. roseus, suppressor 1/93Vin was present in all root and randomly collected stem samples over the entire observation period. In contrast, the challengers were never detected in such stem samples and rarely in the roots. Following simultaneous inoculation, the suppressor successively colonized all stem and root regions whereas detection of challenger AT steadily decreased. However, this strain remained detectable in up to 13 and 27% of stem and root samples, respectively. The cross-protection trials with N. occidentalis yielded results similar to that of the cross-protection experiments with C. roseus. Comparison of the symptomatology of infected apple trees with the presence of putatively suppressive strains indicated that suppression of severe strains also occurs in apple. Phylogenetic analysis using a variable fragment of AAA+ ATPase gene AP460 of 'Ca. P. mali' revealed that suppressors 1/93Vin and 1/93Tab, together with several other mild strains maintained in apple, cluster distantly from obviously nonsuppressive strains that were predominantly highly virulent. PMID:24724815

  1. Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

    PubMed

    Pan, Ya-Jie; Liu, Jia; Guo, Xiao-Rui; Zu, Yuan-Gang; Tang, Zhong-Hua

    2015-05-01

    Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the

  2. Antagonism of Ca2+ influx via L-type Ca2+ channels mediates the vasorelaxant effect of Catharanthus roseus-derived vindorosine in rat renal artery.

    PubMed

    Wu, Xiao-Lin; Cheang, Wai San; Zhang, Dong-Mei; Li, Yong; Lau, Chi-Wai; Wang, Guo-Cai; Huang, Yu; Ye, Wen-Cai

    2014-12-01

    Catharanthus roseus is a traditional herbal medicine used in Asian and African countries for the treatment of various diseases including hypertension. The present study examined possible cellular mechanisms for the relaxation of rat renal arteries induced by vindorosine extracted from C. roseus. Intrarenal arteries were isolated from 200-300 g male Sprague-Dawley rats and treated with different pharmacological blockers and inhibitors for the measurement of vascular reactivity on a Multi Myograph System. Fluorescence imaging by laser scanning confocal microscopy was utilized to determine the intracellular Ca(2+) level in the vascular smooth muscles of the renal arteries. Vindorosine in micromolar concentrations relaxes renal arteries precontracted by KCl, phenylephrine, 11-dideoxy-9α,11α-epoxymethanoprostaglandin F2α, and serotonin. Vindorosine-induced relaxations were unaffected by endothelium denudation or by treatment with the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester hydrochloride, the guanylyl cyclase inhibitor 1H-[1, 2, 4]oxadiazolo[4,3-a]quinoxalin-1-one, the cyclooxygenase inhibitor indomethacin, or K(+) channel blockers such as tetraethylammonium ions, glibenclamide, and BaCl2. Vindorosine-induced relaxations were attenuated in the presence of 0.1 µM nifedipine (an L-type Ca(2+) channel blocker). Vindorosine also concentration-dependently suppressed contractions induced by CaCl2 (0.01-5 mM) in Ca-free 60 mM KCl solution. Furthermore, fluorescence imaging using fluo-4 demonstrated that 30 min incubation with 100 µM vindorosine reduced the 60 mM KCl-stimulated Ca(2+) influx in the smooth muscles of rat renal arteries. The present study is probably the first report of blood vessel relaxation by vindorosine and the possible underlying mechanisms involving the inhibition of Ca(2+) entry via L-type Ca(2+) channels in vascular smooth muscles. PMID:25340466

  3. Pleiotropic phenotypes of the salt-tolerant and cytosine hypomethylated leafless inflorescence, evergreen dwarf and irregular leaf lamina mutants of Catharanthus roseus possessing Mendelian inheritance.

    PubMed

    Kumari, Renu; Sharma, Vishakha; Sharma, Vinay; Kumar, Sushil

    2013-12-01

    In Catharanthus roseus, three morphological cum salt-tolerant chemically induced mutants of Mendelian inheritance and their wild-type parent cv Nirmal were characterized for overall cytosine methylation at DNA repeats, expression of 119 protein coding and seven miRNA-coding genes and 50 quantitative traits. The mutants, named after their principal morphological feature(s), were leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill). The Southern-blot analysis of MspI digested DNAs of mutants probed with centromeric and 5S and 18S rDNA probes indicated that, in comparison to wild type, the mutants were extensively demethylated at cytosine sites. Among the 126 genes investigated for transcriptional expression, 85 were upregulated and 41 were downregulated in mutants. All of the five genes known to be stress responsive had increased expression in mutants. Several miRNA genes showed either increased or decreased expression in mutants. The C. roseus counterparts of CMT3, DRM2 and RDR2 were downregulated in mutants. Among the cell, organ and plant size, photosynthesis and metabolism related traits studied, 28 traits were similarly affected in mutants as compared to wild type. Each of the mutants also expressed some traits distinctively. The egd mutant possessed superior photosynthesis and water retention abilities. Biomass was hyperaccumulated in roots, stems, leaves and seeds of the lli mutant. The ill mutant was richest in the pharmaceutical alkaloids catharanthine, vindoline, vincristine and vinblastine. The nature of mutations, origins of mutant phenotypes and evolutionary importance of these mutants are discussed. PMID:24371160

  4. Application of natural deep eutectic solvents to the extraction of anthocyanins from Catharanthus roseus with high extractability and stability replacing conventional organic solvents.

    PubMed

    Dai, Yuntao; Rozema, Evelien; Verpoorte, Robert; Choi, Young Hae

    2016-02-19

    Natural deep eutectic solvents (NADES) have attracted a great deal of attention in recent times as promising green media. They are generally composed of neutral, acidic or basic compounds that form liquids of high viscosity when mixed in certain molar ratio. Despite their potential, viscosity and acid or basic nature of some ingredients may affect the extraction capacity and stabilizing ability of the target compounds. To investigate these effects, extraction with a series of NADES was employed for the analysis of anthocyanins in flower petals of Catharanthus roseus in combination with HPLC-DAD-based metabolic profiling. Along with the extraction yields of anthocyanins their stability in NADES was also studied. Multivariate data analysis indicates that the lactic acid-glucose (LGH), and 1,2-propanediol-choline chloride (PCH) NADES present a similar extraction power for anthocyanins as conventional organic solvents. Furthermore, among the NADES employed, LGH exhibits an at least three times higher stabilizing capacity for cyanidins than acidified ethanol, which facilitates their extraction and analysis process. Comparing NADES to the conventional organic solvents, in addition to their reduced environmental impact, they proved to provide higher stability for anthocyanins, and therefore have a great potential as possible alternatives to those organic solvents in health related areas such as food, pharmaceuticals and cosmetics. PMID:26822320

  5. Phase-Specific Polypeptides and Poly(A)+ RNAs during the Cell Cycle in Synchronous Cultures of Catharanthus roseus Cells 1

    PubMed Central

    Kodama, Hiroaki; Kawakami, Naoto; Watanabe, Akira; Komamine, Atsushi

    1989-01-01

    This study shows an overall analysis of gene expression during the cell cycle in synchronous suspension cultures of Catharanthus roseus cells. First, the cellular cytoplasmic proteins were fractionated by two-dimensional gel electrophoresis and visualized by staining with silver. Seventeen polypeptides showed qualitative or quantitative changes during the cell cycle. Second, the rates of synthesis of cytoplasmic proteins were also investigated by autoradiography by labeling cells with [35S]methionine at each phase of the cell cycle. The rates of synthesis of 13 polypeptides were found to vary during the cell cycle. The silverstained electrophoretic pattern of proteins in the G2 phase in particular showed characteristic changes in levels of polypeptides, while the rates of synthesis of polypeptides synthesized during the G2 phase did not show such phase-specific changes. This result suggests that posttranslational processing of polypeptides occurs during or prior to the G2 phase. In the G1 and S phases and during cytokinesis, several other polypeptides were specifically synthesized. Finally, the variation of mRNAs was analyzed from the autoradiograms of in vitro translation products of poly(A)+ RNA isolated at each phase. Three poly(A)+ RNAs increased in amount from the G1 to the S phase and one poly (A)+ RNA increased preferentially from the G2 phase to cytokinesis. Images Figure 1 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 Figure 10 Figure 11 Figure 12 PMID:16666641

  6. Virus-induced gene silencing identifies Catharanthus roseus 7-deoxyloganic acid-7-hydroxylase, a step in iridoid and monoterpene indole alkaloid biosynthesis.

    PubMed

    Salim, Vonny; Yu, Fang; Altarejos, Joaquín; De Luca, Vincenzo

    2013-12-01

    Iridoids are a major group of biologically active molecules that are present in thousands of plant species, and one versatile iridoid, secologanin, is a precursor for the assembly of thousands of monoterpenoid indole alkaloids (MIAs) as well as a number of quinoline alkaloids. This study uses bioinformatics to screen large databases of annotated transcripts from various MIA-producing plant species to select candidate genes that may be involved in iridoid biosynthesis. Virus-induced gene silencing of the selected genes combined with metabolite analyses of silenced plants was then used to identify the 7-deoxyloganic acid 7-hydroxylase (CrDL7H) that is involved in the 3rd to last step in secologanin biosynthesis. Silencing of CrDL7H reduced secologanin levels by at least 70%, and increased the levels of 7-deoxyloganic acid to over 4 mg g(-1) fresh leaf weight compared to control plants in which this iridoid is not detected. Functional expression of this CrDL7H in yeast confirmed its biochemical activity, and substrate specificity studies showed its preference for 7-deoxyloganic acid over other closely related substrates. Together, these results suggest that hydroxylation precedes carboxy-O-methylation in the secologanin pathway in Catharanthus roseus. PMID:24103035

  7. Molecular characterization of recombinant T1, a non-allergenic periwinkle (Catharanthus roseus) protein, with sequence similarity to the Bet v 1 plant allergen family.

    PubMed Central

    Laffer, Sylvia; Hamdi, Said; Lupinek, Christian; Sperr, Wolfgang R; Valent, Peter; Verdino, Petra; Keller, Walter; Grote, Monika; Hoffmann-Sommergruber, Karin; Scheiner, Otto; Kraft, Dietrich; Rideau, Marc; Valenta, Rudolf

    2003-01-01

    More than 25% of the population suffer from Type I allergy, an IgE-mediated hypersensitivity disease. Allergens with homology to the major birch ( Betula verrucosa ) pollen allergen, Bet v 1, belong to the most potent elicitors of IgE-mediated allergies. T1, a cytokinin-inducible cytoplasmic periwinkle ( Catharanthus roseus ) protein, with significant sequence similarity to members of the Bet v 1 plant allergen family, was expressed in Escherichia coli. Recombinant T1 (rT1) did not react with IgE antibodies from allergic patients, and failed to induce basophil histamine release and immediate-type skin reactions in Bet v 1-allergic patients. Antibodies raised against purified rT1 could be used for in situ localization of natural T1 by immunogold electron microscopy, but did not cross-react with most of the Bet v 1-related allergens. CD analysis showed significant differences regarding secondary structure and thermal denaturation behaviour between rT1 and recombinant Bet v 1, suggesting that these structural differences are responsible for the different allergenicity of the proteins. T1 represents a non-allergenic member of the Bet v 1 family that may be used to study structural requirements of allergenicity and to engineer hypo-allergenic plants by replacing Bet v 1-related allergens for primary prevention of allergy. PMID:12656672

  8. Identification of a human ABCC10 orthologue in Catharanthus roseus reveals a U12-type intron determinant for the N-terminal domain feature.

    PubMed

    El-Guizani, Taissir; Guibert, Clotilde; Triki, Saida; St-Pierre, Benoit; Ducos, Eric

    2014-04-01

    ABC (ATP-binding cassette) transporters are members of a large superfamily of proteins that utilize ATP hydrolysis to translocate a wide range of substrates across biological membranes. In general, members of C subfamily (ABCC) are structurally characterized by an additional (N-terminal) transmembrane domain (TMD0). Phylogenetic analysis of plant ABCCs separates their protein sequences into three distinct clusters: I and II are plant specific whereas cluster III contains both human and plant ABCCs. Screening of the Plant Medicinal Genomics Resource database allowed us to identify 16 ABCCs partial sequences in Catharanthus roseus; two of which belong to the unique CrABCC1 transcript that we identified in cluster III. Genomic organization of CrABCC1 TMD0 coding sequence displays an AT-AC U12-type intron that is conserved in higher plant orthologues. We showed that CrABCC1, like its human orthologue ABCC10, produces alternative transcripts that encode protein sequences with a truncated form of TMD0 without the first transmembrane span (TM1). Subcellular localization of CrABCC1 TMD0 variants using yellow fluorescent protein fusions reveals that the TM1 is required for a correct routing of the TMD0 to the tonoplast. Finally, the specific repartition of CrABCC1 orthologues in some species suggests that this gene was lost several times during evolution and that its physiological function may, rely on a common feature of multicellular eukaryotes. PMID:24840820

  9. Isolation of Genes that Are Preferentially Expressed at the G1/S Boundary during the Cell Cycle in Synchronized Cultures of Catharanthus roseus Cells 1

    PubMed Central

    Kodama, Hiroaki; Ito, Masaki; Hattori, Tsukaho; Nakamura, Kenzo; Komamine, Atsushi

    1991-01-01

    A cDNA library was screened for genes that may be involved in the progression of the cell cycle of cells of higher plants. The Catharanthus roseus L. (G) Don. cells were synchronized by the double phosphate starvation method, and a λgt11 cDNA library was prepared using poly(A)+ RNA from cells in the S phase of the cell cycle. Two independent sequences, cyc02 and cyc07, were identified by differential screening. The levels of cyc02 and cyc07 mRNAs increased dramatically, but transiently, at the G1/S boundary of the cell cycle. High levels of cyc02 mRNA, but not of cyc07 mRNA, were also present in cells arrested at the G1 phase by phosphate starvation. In an asynchronous batch culture, cyc02 and cyc07 mRNAs accumulated transiently at different stages of the growth cycle, cyc02 mRNA early in the stationary phase, and cyc07 mRNA in the midlogarithmic phase. When the proliferation of cells was arrested by nutrient starvation, i.e. by sucrose or nitrogen starvation, the relative amounts of the cyc02 and cyc07 mRNAs decreased. These results indicate that cyc02 and cyc07 contain nucleotide sequences from growth-related genes. The analysis of nucleotide sequence of cyc02 shows that the predicted product of this gene is basic and is composed of 101 amino acids. No significant homology to other known proteins was detected. Images Figure 1 Figure 4 Figure 5 PMID:16667998

  10. Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

    PubMed Central

    2010-01-01

    Background CrMYC2 is an early jasmonate-responsive bHLH transcription factor involved in the regulation of the expression of the genes of the terpenic indole alkaloid biosynthesis pathway in Catharanthus roseus. In this paper, we identified the amino acid domains necessary for the nuclear targeting of CrMYC2. Findings We examined the intracellular localization of whole CrMYC2 and of various deletion mutants, all fused with GFP, using a transient expression assay in onion epidermal cells. Sequence analysis of this protein revealed the presence of four putative basic nuclear localization signals (NLS). Assays showed that none of the predicted NLS is active alone. Further functional dissection of CrMYC2 showed that the nuclear targeting of this transcription factor involves the cooperation of three domains located in the C-terminal region of the protein. The first two domains are located at amino acid residues 454-510 and 510-562 and contain basic classical monopartite NLSs; these regions are referred to as NLS3 (KRPRKR) and NLS4 (EAERQRREK), respectively. The third domain, between residues 617 and 652, is rich in basic amino acids that are well conserved in other phylogenetically related bHLH transcription factors. Our data revealed that these three domains are inactive when isolated but act cooperatively to target CrMYC2 to the nucleus. Conclusions This study identified three amino acid domains that act in cooperation to target the CrMYC2 transcription factor to the nucleus. Further fine structure/function analysis of these amino acid domains will allow the identification of new NLS domains and will allow the investigation of the related molecular mechanisms involved in the nuclear targeting of the CrMYC2 bHLH transcription factor. PMID:21073696

  11. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus.

    PubMed

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  12. Ethylene-Induced Vinblastine Accumulation Is Related to Activated Expression of Downstream TIA Pathway Genes in Catharanthus roseus

    PubMed Central

    Wang, Xi; Pan, Ya-Jie; Chang, Bo-Wen; Hu, Yan-Bo; Guo, Xiao-Rui; Tang, Zhong-Hua

    2016-01-01

    We selected different concentrations of ethephon, to stress C. roseus. We used qRT-PCR and HPLC followed by PCA to obtain comprehensive profiling of the vinblastine biosynthesis in response to ethephon. Based on our findings, the results showed that the high concentration of ethephon had a positive effect at both transcriptional and metabolite level. Meanwhile, there was a remarkable decrease of hydrogen peroxide content and a promoted peroxidase activity in leaves. The loading plot combination with correlation analysis suggested that CrPrx1 could be regarded as a positive regulator and interacts with ethylene response factor (ERF) to play a key role in vinblastine content and peroxidase (POD) activity. This study provides the foundation for a better understanding of the regulation and accumulation of vinblastine in response to ethephon. PMID:27314017

  13. Over-expression of Catharanthus roseus tryptophan decarboxylase and strictosidine synthase in rol gene integrated transgenic cell suspensions of Vinca minor.

    PubMed

    Verma, Priyanka; Sharma, Abhishek; Khan, Shamshad Ahmad; Shanker, Karuna; Mathur, Ajay K

    2015-01-01

    Tryptophan decarboxylase (TDC) and strictosidine synthase (STR) genes from Catharanthus roseus have been successfully over-expressed in the rol gene integrated cell suspensions of V. minor. Thirty seconds SAAT (sonication-assisted Agrobacterium transformation) treatment of plant cell suspension with LBA1119 having construct () generated three stable TDC + STR over-expressing cell lines--PVG1, PVG2, and PVG3. The transgenes were confirmed by β-glucuronidase GUS histochemical assay and PCR amplification of rol genes/GUS gene. All the three cell suspension lines were found to be slow growing. In comparison to the control cell suspensions (GI = 241.0 ± 5.8), PVG3 cell line registered a growth index (GI) of 208.0 ± 10.0 followed by PVG1 (GI = 140.0 ± 14.2) and PVG2 (GI = 85.0 ± 9.6). The PVG3 cell line was also up-scaled in the 5-l stirred tank bioreactor with GI of 745.6 ± 35.3 under optimized parameters. Only PVG3 line registered a twofold increase in total alkaloid content (2.1 ± 0.1% dry wt.) and showed vincamine presence (0.003 ± 0.001% dry wt.) which was further enhanced at the bioreactor level (2.7 ± 0.3 and 0.005 ± 0.001% dry wt., respectively). Real-time (RT) qPCR analysis of PVG3 showed more than sevenfold to eightfold increase in TDC and STR expression [relative quantity value (RQ) = 7.6 ± 0.8 (TDC); RQ = 8.5 ± 0.9 (STR)]. PMID:25106473

  14. Identification of a Bipartite Jasmonate-Responsive Promoter Element in the Catharanthus roseus ORCA3 Transcription Factor Gene That Interacts Specifically with AT-Hook DNA-Binding Proteins1[W

    PubMed Central

    Vom Endt, Débora; Soares e Silva, Marina; Kijne, Jan W.; Pasquali, Giancarlo; Memelink, Johan

    2007-01-01

    Jasmonates are plant signaling molecules that play key roles in defense against certain pathogens and insects, among others, by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the APETALA2-domain transcription factor ORCA3 is involved in the jasmonate-responsive activation of terpenoid indole alkaloid biosynthetic genes. ORCA3 gene expression is itself induced by jasmonate. By loss- and gain-of-function experiments, we located a 74-bp region within the ORCA3 promoter, which contains an autonomous jasmonate-responsive element (JRE). The ORCA3 JRE is composed of two important sequences: a quantitative sequence responsible for a high level of expression and a qualitative sequence that appears to act as an on/off switch in response to methyl jasmonate. We isolated 12 different DNA-binding proteins having one of four different types of DNA-binding domains, using the ORCA3 JRE as bait in a yeast (Saccharomyces cerevisiae) one-hybrid transcription factor screening. The binding of one class of proteins bearing a single AT-hook DNA-binding motif was affected by mutations in the quantitative sequence within the JRE. Two of the AT-hook proteins tested had a weak activating effect on JRE-mediated reporter gene expression, suggesting that AT-hook family members may be involved in determining the level of expression of ORCA3 in response to jasmonate. PMID:17496112

  15. Effects of ambient and elevated CO2 on growth, chlorophyll fluorescence, photosynthetic pigments, antioxidants, and secondary metabolites of Catharanthus roseus (L.) G Don. grown under three different soil N levels.

    PubMed

    Singh, Aradhana; Agrawal, Madhoolika

    2015-03-01

    Catharanthus roseus L. plants were grown under ambient (375 ± 30 ppm) and elevated (560 ± 25 ppm) concentrations of atmospheric CO2 at different rates of N supply (without supplemental N, 0 kg N ha(-1); recommended N, 50 kg N ha(-1); and double recommended N, 100 kg N ha(-1)) in open top chambers under field condition. Elevated CO2 significantly increased photosynthetic pigments, photosynthetic efficiency, and organic carbon content in leaves at recommended (RN) and double recommended N (DRN), while significantly decreased total nitrogen content in without supplemental N (WSN). Activities of superoxide dismutase, catalase, and ascorbate peroxidase were declined, while glutathione reductase, peroxidase, and phenylalanine-ammonia lyase were stimulated under elevated CO2. However, the responses of the above enzymes were modified with different rates of N supply. Elevated CO2 significantly reduced superoxide production rate, hydrogen peroxide, and malondialdehyde contents in RN and DRN. Compared with ambient, total alkaloids content increased maximally at recommended level of N, while total phenolics in WSN under elevated CO2. Elevated CO2 stimulated growth of plants by increasing plant height and numbers of branches and leaves, and the magnitude of increment were maximum in DRN. The study suggests that elevated CO2 has positively affected plants by increasing growth and alkaloids production and reducing the level of oxidative stress. However, the positive effects of elevated CO2 were comparatively lesser in plants grown under limited N availability than in moderate and higher N availability. Furthermore, the excess N supply in DRN has stimulated the growth but not the alkaloids production under elevated CO2. PMID:25304238

  16. Phytic Acid Synthesis and Vacuolar Accumulation in Suspension-Cultured Cells of Catharanthus roseus Induced by High Concentration of Inorganic Phosphate and Cations1[w

    PubMed Central

    Mitsuhashi, Naoto; Ohnishi, Miwa; Sekiguchi, Yoko; Kwon, Yong-Uk; Chang, Young-Tae; Chung, Sung-Kee; Inoue, Yoshinori; Reid, Robert J.; Yagisawa, Hitoshi; Mimura, Tetsuro

    2005-01-01

    We have established a new system for studying phytic acid, myo-inositol hexakisphosphate (InsP6) synthesis in suspension-cultured cells of Catharanthus. InsP6 and other intermediates of myo-inositol (Ins) phosphate metabolism were measured using an ion chromatography method. The detection limit for InsP6 was less than 50 nm, which was sufficient to analyze Ins phosphates in living cells. Synthesis of Ins phosphates was induced by incubation in high inorganic phosphate medium. InsP6 was mainly accumulated in vacuoles and was enhanced when cells were grown in high concentration of inorganic phosphates with the cations K+, Ca2+, or Zn2+. However, there was a strong tendency for InsP6 to accumulate in the vacuole in the presence of Ca2+ and in nonvacuolar compartments when supplied with Zn2+, possibly due to precipitation of InsP6 with Zn2+ in the cytosol. A vesicle transport inhibitor, brefeldin A, stimulated InsP6 accumulation. The amounts of both Ins(3)P1 myo-inositol monophosphate synthase, a key enzyme for InsP6 synthesis, and Ins(1,4,5)P3 kinase were unrelated to the level of accumulation of InsP6. The mechanisms for InsP6 synthesis and localization into vacuoles in plant cells are discussed. PMID:15965017

  17. Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: detection of a phenolic compound with antioxidant potential.

    PubMed

    Verma, Priyanka; Mathur, Ajay K; Masood, Nusrat; Luqman, Suaib; Shanker, Karuna

    2013-02-01

    Five cell suspension lines of Catharanthus roseus resistant to 5-methyl tryptophan (5-MT; an analogue of tryptophan) were selected and characterized for growth, free tryptophan content and terpenoid indole alkaloid accumulation. These lines showed differential tolerance to analogue-induced growth inhibition by 30 to 70 mg/l 5-MT supplementation (LD(50) = 7-15 mg/l). Lines P40, D40, N30, D50 and P70 recorded growth indices (i.e. percent increment over the initial inoculum weight) of 840.9, 765.0, 643.9, 585.7 and 356.5 in the absence and, 656.7, 573.9, 705.8, 489.0 and 236.0 in the presence of 5-MT after 40 days of culture, respectively. A corresponding increment in the free tryptophan level ranging from 46.7 to 160.0 μg/g dry weight in the absence and 168.0 to 468.0 μg/g dry weight in the presence was noted in the variant lines. Higher tryptophan accumulation of 368.0 and 468.0 g/g dry weight in lines N30 and P40 in 5-MT presence also resulted in higher alkaloid accumulation (0.65 to 0.90 % dry weight) in them. High-performance liquid chromatography (HPLC) analysis of the crude alkaloid extracts of the selected lines did not show the presence of any pharmaceutically important monomeric or dimeric alkaloids except catharanthine in traces in the N30 line that was also unique in terms of a chlorophyllous green phenotype. The N30 line under optimized up-scaling conditions in a 7-l stirred tank bioreactor using Murashige and Skoog medium containing 2 mg/l α-naphthalene acetic acid and 0.2 mg/l kinetin attained 18-folds biomass accumulation within 8 weeks. Interestingly, the cell biomass yield was enhanced to 30-folds if 30 mg/l 5-MT was added in the bioreactor vessel one week prior to harvest. Crude alkaloid extract of the cells grown in shake flask and this bioreactor batch also showed the formation of yellow-coloured crystals which upon (1)HNMR and ESI-MS analysis indicated a phenolic identity. This crude alkaloid extract of bioreactor-harvested cells containing

  18. Phytochrome Is Involved in the Light-Regulation of Vindoline Biosynthesis in Catharanthus1

    PubMed Central

    Aerts, Rob J.; De Luca, Vincenzo

    1992-01-01

    The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyl-transferase (DAT) catalyzes the final step in the biosynthesis of the monoterpenoid indole alkaloid, vindoline. Previous studies have shown that the appearance of DAT activity in etiolated seedlings of Catharanthus roseus is induced by exposure of seedlings to light and that enzyme activity is restricted principally to the cotyledons. Evidence is now presented that phytochrome is involved in the light-mediated induction of DAT activity in Catharanthus cotyledons. PMID:16653011

  19. A look inside an alkaloid multisite plant: the Catharanthus logistics.

    PubMed

    Courdavault, Vincent; Papon, Nicolas; Clastre, Marc; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Burlat, Vincent

    2014-06-01

    Environmental pressures forced plants to diversify specialized metabolisms to accumulate noxious molecules such as alkaloids constituting one of the largest classes of defense metabolites. Catharanthus roseus produces monoterpene indole alkaloids via a highly elaborated biosynthetic pathway whose characterization greatly progressed with the recent expansion of transcriptomic resources. The complex architecture of this pathway, sequentially distributed in at least four cell types and further compartmentalized into several organelles, involves partially identified inter-cellular and intra-cellular translocation events acting as potential key-regulators of metabolic fluxes. The description of this spatial organization and the inherent secretion and sequestration of metabolites not only provide new insight into alkaloid cell biology and its involvement in plant defense processes but also present new biotechnological challenges for synthetic biology. PMID:24727073

  20. Estimating Mesophyll Conductance in the Tropical Rainforest

    NASA Astrophysics Data System (ADS)

    Coughlin, I.

    2015-12-01

    In the current research modeling the carbon cycle, some of the biggest setbacks are methodological barriers to calculating the gross primary production (GPP) in the terrestrial biosphere. However, recent developments in high precision gas measurements now allow the use of COS as a potential tracer for determination of GPP, independently of CO2 .Since the tropics are implicated as being the source of the most significant reduction of carbon uptake by the majority of models, making accurate GPP measurements in the tropics is particularly important for carbon modeling. In order to constrain measurements of GPP in the tropics, carbonyl sulfide fluxes on a leaf chamber scale and a canopy-wide scale will be analyzed in a field site in the central Amazon. Accompanying this experiment, I am measuring the resistance of CO2 passing through the intercellular airspaces in the leaf to the sites of carboxylation, known as mesophyll conductance. Mesophyll conductance is poorly documented in the tropics, and remains a centrally limiting factor in plant uptake of COS and CO2 - with upward estimates of 40% of the CO2 diffusional limitation of photosynthesis hinging on mesophyll conductance (Warren, 2008). This makes mesophyll conductance comparable in magnitude to that of the stomatal conductance, suggesting that mesophyll conductance is one of the most fundamental measurements necessary for developing the predictive capacity of plants' response to ecosystem changes. Accurate measurements of the mesophyll conductance also lead to better informed models that can upscale assimilation measurements from leaf chambers, by providing quantitative constraints for modeling the uptake of carbonyl sulfide and carbon dioxide by the leaf. Additionally, since mesophyll conductance reacts to environmental variation, it can be used as an indicator for leaf stress. Measurements are taken using the 'variable J' technique, involving the use of combined fluorescence measurements and gas exchange data

  1. Auxins Induce Tryptophan Decarboxylase Activity in Radicles of Catharanthus Seedlings 1

    PubMed Central

    Aerts, Rob J.; Alarco, Anne-Marie; De Luca, Vincenzo

    1992-01-01

    Germinating seedlings of Catharanthus roseus produce monoterpenoid indole alkaloids as a result of a transient increase of tryptophan decarboxylase (TDC) activity. The influence of auxins on this transient rise of TDC activity was studied. External application of indolebutyric acid or 2,4-dichlorophenoxyacetic acid at a concentration of 20 to 40 μm enhanced and prolonged the rise in TDC activity in developing seedlings. Auxin treatment also influenced the morphology of the seedlings; it induced a shortening and thickening of the hypocotyl and the radicle and promoted the initiation of lateral roots in the radicle. During development, the radicles of auxin-treated seedlings displayed a gradual increase in TDC activity that was absent in the radicles of untreated controls. Examination of immunoblots revealed anti-TDC reactive proteins in extracts from radicles of auxin-treated seedlings, but none in extracts from radicles of control seedlings. In contrast, TDC activity and immunoreactive protein levels in the aerial parts of controls and auxin-treated seedlings were comparable. Our results indicate that externally applied auxins induce both abnormal development and TDC activity in the radicles of Catharanthus seedlings. Although auxins slightly delayed the light-mediated induction of the cotyledon-specific last step in vindoline biosynthesis (i.e. acetylcoenzyme A: deacetylvindolin-O-acetyltransferase activity), seedlings still synthesized vindoline, one of the major alkaloid end products. Images Figure 2 PMID:16653009

  2. Molecular pharmacokinetics of catharanthus (vinca) alkaloids.

    PubMed

    Levêque, Dominique; Jehl, François

    2007-05-01

    This review focuses on the published data regarding the molecular determinants (enzymes, transporters, orphan nuclear receptors) of Catharanthus (vinca) alkaloids pharmacokinetics in humans. The clinical impact of these determinants (drug disposition, drug-drug interactions) is also discussed. PMID:17442684

  3. Production of Annual Vinca (Catharanthus roseus) in WholeTree Substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate the potential use of container substrates composed of processed whole pine trees (WholeTree). Three species [loblolly pine (Pinus taeda), slash pine (Pinus elliottii) and longleaf pine (Pinus palustris)] of 8-10 year old pine trees were harvested at groun...

  4. 'Candidatus Phytoplasma malaysianum', a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus).

    PubMed

    Nejat, Naghmeh; Vadamalai, Ganesan; Davis, Robert E; Harrison, Nigel A; Sijam, Kamaruzaman; Dickinson, Matthew; Abdullah, Siti Nor Akmar; Zhao, Yan

    2013-02-01

    This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described 'Candidatus Phytoplasma' species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rRNA gene shared 96.5 % or less sequence similarity with that of previously described 'Ca. Phytoplasma' species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, 'Candidatus Phytoplasma malaysianum'. The 16S rRNA gene F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated a member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and the new group included diverse strains infecting periwinkle, coconut palm and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B and 16SrXXXII-C, respectively. PMID:22523165

  5. Unlocking the diversity of alkaloids in Catharanthus roseus: nuclear localization suggests metabolic channeling in secondary metabolism.

    PubMed

    Stavrinides, Anna; Tatsis, Evangelos C; Foureau, Emilien; Caputi, Lorenzo; Kellner, Franziska; Courdavault, Vincent; O'Connor, Sarah E

    2015-03-19

    The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway. PMID:25772467

  6. Unlocking the Diversity of Alkaloids in Catharanthus roseus: Nuclear Localization Suggests Metabolic Channeling in Secondary Metabolism

    PubMed Central

    Stavrinides, Anna; Tatsis, Evangelos C.; Foureau, Emilien; Caputi, Lorenzo; Kellner, Franziska; Courdavault, Vincent; O’Connor, Sarah E.

    2015-01-01

    Summary The extraordinary chemical diversity of the plant-derived monoterpene indole alkaloids, which include vinblastine, quinine, and strychnine, originates from a single biosynthetic intermediate, strictosidine aglycone. Here we report for the first time the cloning of a biosynthetic gene and characterization of the corresponding enzyme that acts at this crucial branchpoint. This enzyme, an alcohol dehydrogenase homolog, converts strictosidine aglycone to the heteroyohimbine-type alkaloid tetrahydroalstonine. We also demonstrate how this enzyme, which uses a highly reactive substrate, may interact with the upstream enzyme of the pathway. PMID:25772467

  7. Candidatus Phytoplasma malaysianum, a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytopla...

  8. Phytoremediation of TNT: C. roseus hairy roots as a model system

    SciTech Connect

    Lauritzen, J.R.; Hughes, J.B.; Shanks, J.V.

    1996-12-31

    Widespread contamination by 2,4,6-trinitrotoluene (TNT) of Soil exists at former munitions production and handling facilities. Phytoremediation may be an effective alternative to existing methods of TNT remediation: incineration is highly expensive and recalcitrant reduction products are formed in composting. Recently, the intrinsic ability of plants to transform TNT has been demonstrated using hairy root cultures of Catharanthus roseus as a model system. Kinetic studies were performed at concentrations of 30 and 50 mg/L TNT in growth medium. The pseudo-first order rate constants for disappearance ranged from 0.0103 to 0.0161 (L/g-day); TNT disappears completely within seven to ten days of exposure. The fate of the TNT molecule in plants is also currently under study, mass balance studies were performed with 1-{sup 14}C TNT. After a seven day exposure period, 72% of the label was associated with the roots and 30% was associated with the medium. However, HPLC analysis shows that less than 5% (wt%) of the TNT added is recoverable from both the plants and the media in the form of reduction products. 11 refs., 2 figs.

  9. TEMPRANILLO Reveals the Mesophyll as Crucial for Epidermal Trichome Formation.

    PubMed

    Matías-Hernández, Luis; Aguilar-Jaramillo, Andrea E; Osnato, Michela; Weinstain, Roy; Shani, Eilon; Suárez-López, Paula; Pelaz, Soraya

    2016-03-01

    Plant trichomes are defensive specialized epidermal cells. In all accepted models, the epidermis is the layer involved in trichome formation, a process controlled by gibberellins (GAs) in Arabidopsis rosette leaves. Indeed, GA activates a genetic cascade in the epidermis for trichome initiation. Here we report that TEMPRANILLO (TEM) genes negatively control trichome initiation not only from the epidermis but also from the leaf layer underneath the epidermis, the mesophyll. Plants over-expressing or reducing TEM specifically in the mesophyll, display lower or higher trichome numbers, respectively. We surprisingly found that fluorescently labeled GA3 accumulates exclusively in the mesophyll of leaves, but not in the epidermis, and that TEM reduces its accumulation and the expression of several newly identified GA transporters. This strongly suggests that TEM plays an essential role, not only in GA biosynthesis, but also in regulating GA distribution in the mesophyll, which in turn directs epidermal trichome formation. Moreover, we show that TEM also acts as a link between GA and cytokinin signaling in the epidermis by negatively regulating downstream genes of both trichome formation pathways. Overall, these results call for a re-evaluation of the present theories of trichome formation as they reveal mesophyll essential during epidermal trichome initiation. PMID:26802039

  10. Variable rsponses of mesophyll conductance to substomatal carbon dioxide concentration in common bean and soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some reports indicate that mesophyll conductance to carbon dioxide varies greatly with the sub-stomatal carbon dioxide concentration during the measurement, while other reports indicate little or no change. I used the oxygen sensitivity of photosynthesis to determine the response of mesophyll condu...

  11. Evaluation of antimicrobial activity of extracts of in vivo and in vitro grown Vinca rosea L. (Catharanthus roseus) against pathogens.

    PubMed

    Naz, Shagufta; Haq, Rukhama; Aslam, Farah; Ilyas, Saiqa

    2015-05-01

    The antimicrobial activity of Vinca rosea was evaluated against pathogenic bacterial strains (Bacillus subtilis, B. licheniformis and Azotobacter sp.) and fungal strains (Asprgillus niger, Alternaria solani and Rhizopus oryzae) using agar well diffusion method. Methanolic extracts of in vivo leaf, in vitro leaf, in vitro calluses of leaf, nodal and fruit explants were used and exhibited antimicrobial activity as indicated by minimum inhibitory concentration (MIC). In vitro extracts showed better results as compared to the in vivo extracts for both the antibacterial as well as the antifungal activity. Among all the extracts, maximum zone of inhibition (30.3 mm ± 0.58(a)) was formed by in vitro leaf callus extract concentration of 2.0mg/ml against B. licheniformis. Similarly in case of antifungal activity, maximum zone of inhibition (34.6mm ± 0.57(a)) was formed by in vitro leaf callus extract and MIC value is 6.0mg/ml against A. niger. Hence these results clearly depicts that V. rosea possess a great strength to fight against the microbial activity and can be used against various infections. PMID:26004716

  12. Enhancement of vindoline and vinblastine production in suspension-cultured cells of Catharanthus roseus by artemisinic acid elicitation.

    PubMed

    Liu, Jinwei; Zhu, Jianhua; Tang, Le; Wen, Wei; Lv, Shuangshuang; Yu, Rongmin

    2014-01-01

    Elicitation is an important strategy to improve production of secondary metabolites in vitro. Artemisinic acid was studied as a novel elicitor to enhance the yield of terpenoid indole alkaloids in the present paper. Our results demonstrated that the concentrations of vindoline and vinblastine were increased by sixfold and twofold, respectively, compared to those of the control group after treatment with artemisinic acid. To elucidate the underlying mechanism, we investigated the gene expression of four enzymes involved in the biosynthetic pathway of vinblastine in the suspension-cultured cells of Catharanthu sroseus. RT-PCR experiment showed that artemisinic acid was able to up-regulate the transcriptions of tryptophan decarboxylase, geraniol 10-hydroxylase, tabersonine 16-hydroxylase and deacetoxyvindoline 4-hydroxylase. PMID:23864440

  13. Expression Patterns of Genes Involved in the Defense and Stress Response of Spiroplasma citri Infected Madagascar Periwinkle Catharanthus roseus

    PubMed Central

    Nejat, Naghmeh; Vadamalai, Ganesan; Dickinson, Matthew

    2012-01-01

    Madagascar periwinkle is an ornamental and a medicinal plant, and is also an indicator plant that is highly susceptible to phytoplasma and spiroplasma infections from different crops. Periwinkle lethal yellows, caused by Spiroplasma citri, is one of the most devastating diseases of periwinkle. The response of plants to S. citri infection is very little known at the transcriptome level. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate the expression levels of four selected genes involved in defense and stress responses in naturally and experimentally Spiroplasma citri infected periwinkles. Strictosidine β-glucosidase involved in terpenoid indole alkaloids (TIAs) biosynthesis pathway showed significant upregulation in experimentally and naturally infected periwinkles. The transcript level of extensin increased in leaves of periwinkles experimentally infected by S. citri in comparison to healthy ones. A similar level of heat shock protein 90 and metallothionein expression was observed in healthy, naturally and experimentally spiroplasma-diseased periwinkles. Overexpression of Strictosidine β-glucosidase demonstrates the potential utility of this gene as a host biomarker to increase the fidelity of S. citri detection and can also be used in breeding programs to develop stable disease-resistance varieties. PMID:22408455

  14. Genotypically Identifying Wheat Mesophyll Conductance Regulation under Progressive Drought Stress

    PubMed Central

    Olsovska, Katarina; Kovar, Marek; Brestic, Marian; Zivcak, Marek; Slamka, Pavol; Shao, Hong Bo

    2016-01-01

    Photosynthesis limitation by CO2 flow constraints from sub-stomatal cavities to carboxylation sites in chloroplasts under drought stress conditions is, at least in some plant species or crops not fully understood, yet. Leaf mesophyll conductance for CO2 (gm) may considerably affect both photosynthesis and water use efficiency (WUE) in plants under drought conditions. The aim of our study was to detect the responses of gm in leaves of four winter wheat (Triticum aestivum L.) genotypes from different origins under long-term progressive drought. Based on the measurement of gas-exchange parameters the variability of genotypic responses was analyzed at stomatal (stomata closure) and non-stomatal (diffusional and biochemical) limits of net CO2 assimilation rate (AN). In general, progressive drought caused an increasing leaf diffusion resistance against CO2 flow leading to the decrease of AN, gm and stomatal conductance (gs), respectively. Reduction of gm also led to inhibition of carboxylation efficiency (Vcmax). On the basis of achieved results a strong positive relationship between gm and gs was found out indicating a co-regulation and mutual independence of the relationship under the drought conditions. In severely stressed plants, the stomatal limitation of the CO2 assimilation rate was progressively increased, but to a less extent in comparison to gm, while a non-stomatal limitation became more dominant due to the prolonged drought. Mesophyll conductance (gm) seems to be a suitable mechanism and parameter for selection of improved diffusional properties and photosynthetic carbon assimilation in C3 plants, thus explaining their better photosynthetic performance at a whole plant level during periods of drought. PMID:27551283

  15. Genotypically Identifying Wheat Mesophyll Conductance Regulation under Progressive Drought Stress.

    PubMed

    Olsovska, Katarina; Kovar, Marek; Brestic, Marian; Zivcak, Marek; Slamka, Pavol; Shao, Hong Bo

    2016-01-01

    Photosynthesis limitation by CO2 flow constraints from sub-stomatal cavities to carboxylation sites in chloroplasts under drought stress conditions is, at least in some plant species or crops not fully understood, yet. Leaf mesophyll conductance for CO2 (gm) may considerably affect both photosynthesis and water use efficiency (WUE) in plants under drought conditions. The aim of our study was to detect the responses of gm in leaves of four winter wheat (Triticum aestivum L.) genotypes from different origins under long-term progressive drought. Based on the measurement of gas-exchange parameters the variability of genotypic responses was analyzed at stomatal (stomata closure) and non-stomatal (diffusional and biochemical) limits of net CO2 assimilation rate (AN). In general, progressive drought caused an increasing leaf diffusion resistance against CO2 flow leading to the decrease of AN, gm and stomatal conductance (gs), respectively. Reduction of gm also led to inhibition of carboxylation efficiency (Vcmax). On the basis of achieved results a strong positive relationship between gm and gs was found out indicating a co-regulation and mutual independence of the relationship under the drought conditions. In severely stressed plants, the stomatal limitation of the CO2 assimilation rate was progressively increased, but to a less extent in comparison to gm, while a non-stomatal limitation became more dominant due to the prolonged drought. Mesophyll conductance (gm) seems to be a suitable mechanism and parameter for selection of improved diffusional properties and photosynthetic carbon assimilation in C3 plants, thus explaining their better photosynthetic performance at a whole plant level during periods of drought. PMID:27551283

  16. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals.

    PubMed

    Mochizuki-Kawai, Hiroko; Niki, Tomoko; Shibuya, Kenichi; Ichimura, Kazuo

    2015-01-01

    In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals. PMID:26605547

  17. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

    PubMed Central

    Mochizuki-Kawai, Hiroko; Niki, Tomoko; Shibuya, Kenichi; Ichimura, Kazuo

    2015-01-01

    In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals. PMID:26605547

  18. Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

    PubMed Central

    Kubínová, Zuzana

    2014-01-01

    Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use. PMID:24336344

  19. Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate.

    PubMed

    Misra, Biswapriya B; de Armas, Evaldo; Tong, Zhaohui; Chen, Sixue

    2015-01-01

    Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3-). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3- responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3-. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage. PMID:26641455

  20. Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate

    PubMed Central

    Misra, Biswapriya B.; de Armas, Evaldo; Tong, Zhaohui; Chen, Sixue

    2015-01-01

    Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3-). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3- responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3-. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage. PMID:26641455

  1. Hymenobacter roseus sp. nov., isolated from sand.

    PubMed

    Subhash, Y; Sasikala, Ch; Ramana, Ch V

    2014-12-01

    Strain JC245(T) was isolated from a sand sample, and appeared as dark pink colonies on agar plates with cells staining Gram-negative. Catalase and oxidase activities were positive. Casein was hydrolysed while chitin, gelatin and starch were not. Major (>5 %) fatty acids were iso-C15 : 0, iso-C15 : 0 3-OH, C16 : 1ω5c, C16 : 1ω6c/C16 : 1ω7c, anteiso-C17 : 1 B/iso-C17 : 1 I and iso-C17 : 0 3-OH. Strain JC245(T) contained phosphatidylethanolamine and two unidentified lipids as the major polar lipids, with minor amounts of four unidentified lipids and an unidentified amino lipid. Bacterial hopane derivatives and adenosylhopane were the major hopanoids. Hydroxyflexixanthin was identified as one of the major carotenoids of strain JC245(T) along with five unidentified carotenoids. The genomic DNA G+C content was 52.5 mol%. 16S rRNA gene sequence comparisons indicated that strain JC245(T) represents a member of the genus Hymenobacter within the family Cytophagaceae of the phylum Bacteroidetes. Strain JC245(T) shared the highest 16S rRNA gene sequence similarity with Hymenobacter roseosalivarius AA-718(T) (98.3 %) and other members of the genus Hymenobacter (<95.1 %). However, strain JC245(T) showed 21±2 % relatedness (based on DNA-DNA hybridization) with H. roseosalivarius DSM 11622(T). Distinct morphological, physiological and genotypic differences from previously described taxa support the classification of strain JC245(T) as a representative of a novel species in the genus Hymenobacter, for which the name Hymenobacter roseus sp. nov. is proposed. The type strain is JC245(T) ( = KCTC 42090(T) = LMG 28260(T)). PMID:25242537

  2. Biodegradation of a synthetic lubricant by Micrococcus roseus

    SciTech Connect

    Wright, M.A.; Taylor, F.; Brown, D.E.; Higgins, I.J. ); Randles, S.J. )

    1993-04-01

    The loss of large quantities of lubricants, both synthetic and mineral oil based, is causing increasing concern because they are not only an unquantified hazard to the environment, but also a potential hazard to the long-term health of people. This study examines the metabolic pathways and eventual fate of synthetic lubricants in micoorganisms involved in biodegradation. The synthetic ester Emkarate 1550 (E1550), which includes a tertiary alcohol (TMP), and the bacterium, Micrococcus roseus were used in the experiments. The results indicate that M. roseus cleaves the E1550 ester by the action of esterases bound to the surface of the cell, with the products released into the surrounding medium. The organic acids, octaoate and decanoate, are taken up and metabolized, whereas the TMP (1,1,1-tri(hydroxymethyl)propane) component is not metabolized and accumulates in the medium. 16 refs., 5 figs., 1 tab.

  3. TEMPRANILLO Reveals the Mesophyll as Crucial for Epidermal Trichome Formation1[OPEN

    PubMed Central

    Aguilar-Jaramillo, Andrea E.; Osnato, Michela; Shani, Eilon

    2016-01-01

    Plant trichomes are defensive specialized epidermal cells. In all accepted models, the epidermis is the layer involved in trichome formation, a process controlled by gibberellins (GAs) in Arabidopsis rosette leaves. Indeed, GA activates a genetic cascade in the epidermis for trichome initiation. Here we report that TEMPRANILLO (TEM) genes negatively control trichome initiation not only from the epidermis but also from the leaf layer underneath the epidermis, the mesophyll. Plants over-expressing or reducing TEM specifically in the mesophyll, display lower or higher trichome numbers, respectively. We surprisingly found that fluorescently labeled GA3 accumulates exclusively in the mesophyll of leaves, but not in the epidermis, and that TEM reduces its accumulation and the expression of several newly identified GA transporters. This strongly suggests that TEM plays an essential role, not only in GA biosynthesis, but also in regulating GA distribution in the mesophyll, which in turn directs epidermal trichome formation. Moreover, we show that TEM also acts as a link between GA and cytokinin signaling in the epidermis by negatively regulating downstream genes of both trichome formation pathways. Overall, these results call for a re-evaluation of the present theories of trichome formation as they reveal mesophyll essential during epidermal trichome initiation. PMID:26802039

  4. Apoplastic mesophyll signals induce rapid stomatal responses to CO2 in Commelina communis.

    PubMed

    Fujita, Takashi; Noguchi, Ko; Terashima, Ichiro

    2013-07-01

    Previous studies have suggested that the mesophyll contributes to stomatal CO(2) responses. The effects of changes in CO(2) concentration (100 or 700 ppm) on stomatal responses in red or white light were examined microscopically in a leaf segment, an epidermal strip and an epidermal strip placed on a mesophyll segment of Commelina communis, all mounted on a buffer-containing gel. In both red and white light, stomata of the leaf segment opened/closed rapidly at low/high CO(2). In red light, epidermal strip stomata barely responded to CO(2). In white light, they opened at low CO(2), but hardly closed at high CO(2). Stomata of the epidermal strip placed on the mesophyll responded in the same manner as those on the leaf segment. Insertion of a doughnut-shaped cellophane spacer (but not polyethylene spacer) between the epidermal strip and the mesophyll hardly altered these responses. Stomata in leaf segments treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthesis inhibitor, did not open in red light, but opened/closed at low/high CO(2) in white light. These results indicate that the apoplast transfer of 'mesophyll signals' and the stomatal opening at low CO(2) are dependent on photosynthesis, whereas the stomatal closure at high CO(2) is independent of photosynthesis. PMID:23560389

  5. Photorespiratory Properties of Mesophyll Protoplasts of Nicotiana plumbaginifolia

    PubMed Central

    Rey, Pascal; Peltier, Gilles

    1989-01-01

    The photorespiratory activity of mesophyll protoplasts of Nicotiana plumbaginifolia has been clearly demonstrated by the presence of a Warburg-effect, the occurrence of an important CO2-sensitive O2 uptake and the effect of some photorespiratory inhibitors on photosynthetic activity. At a nonsaturating dissolved inorganic carbon (DIC) concentration (0.1 millimolar), we observed that the rate of CO2 fixation was 60% lower at 50% O2 compared to that measured at 2% O2. Using 18O2 and mass spectrometry, we measured O2 exchange as a function of light intensity and of DIC concentration. Oxygen uptake measured at the CO2 compensation point (47.4 micromoles O2 per hour per milligram chlorophyll) was three-fold higher than that measured at a saturating CO2 concentration. Cyanide or iodoacetamide, inhibitors of the Calvin cycle, were found to reduce the O2 uptake to the same extent as CO2 saturation. We conclude from these results that the major part of the CO2-sensitive O2 uptake is due to photorespiration. Further, we investigated the effect on net photosynthesis of some inhibitors of the glycolate pathway. At CO2 saturation (10 millimolar DIC), 5 millimolar aminoacetonitrile (AAN), and 1 millimolar aminooxyacetate (AOA) did not cause any significant decrease in net photosynthesis. However, when these two inhibitors were added under a period of active photorespiration (10 minutes at the CO2 compensation point at 20% O2), we observed a decrease in the rate of net photosynthesis at 10 millimolar DIC measured afterward (respectively, 18 and 29%). This inhibition did not appear at 2% O2, but was stronger at 50% O2 (40% for AAN and 47% for AOA). With 0.05 millimolar butyl 2-hydroxy-3-butynoate (BHB) or 0.5 millimolar l-methionine-dl-sulfoximine (l-MSO), rates of net photosynthesis at 10 millimolar DIC were decreased by 10 to 15%. Additional decreases were observed after a period at the CO2 compensation point at 20% O2 (30% for BHB and 20% for l-MSO). From the sites of action of

  6. Leaf light reflectance, transmittance, absorptance, and optical and geometrical parameters for eleven plant genera with different leaf mesophyll arrangements.

    NASA Technical Reports Server (NTRS)

    Gausman, H. W.; Allen, W. A.; Wiegand, C. L.; Escobar, D. E.; Rodriguez, R. R.

    1971-01-01

    Review of research on radiation interactions within plant canopies and communities and interactions of various leaf structures (mesophyll arrangements) with electromagnetic radiation involved in the interpretation of data sensed from air or spacecraft. The hypothesis underlying the research reported is that leaf mesophyll arrangements influence spectral energy measurements of leaves.

  7. Isolation of Cells Specialized in Anticancer Alkaloid Metabolism by Fluorescence-Activated Cell Sorting.

    PubMed

    Carqueijeiro, Inês; Guimarães, Ana Luísa; Bettencourt, Sara; Martínez-Cortés, Teresa; Guedes, Joana G; Gardner, Rui; Lopes, Telma; Andrade, Cláudia; Bispo, Cláudia; Martins, Nuno Pimpão; Andrade, Paula; Valentão, Patrícia; Valente, Inês M; Rodrigues, José A; Duarte, Patrícia; Sottomayor, Mariana

    2016-08-01

    Plant specialized metabolism often presents a complex cell-specific compartmentation essential to accomplish the biosynthesis of valuable plant natural products. Hence, the disclosure and potential manipulation of such pathways may depend on the capacity to isolate and characterize specific cell types. Catharanthus roseus is the source of several medicinal terpenoid indole alkaloids, including the low-level anticancer vinblastine and vincristine, for which the late biosynthetic steps occur in specialized mesophyll cells called idioblasts. Here, the optical, fluorescence, and alkaloid-accumulating properties of C. roseus leaf idioblasts are characterized, and a methodology for the isolation of idioblast protoplasts by fluorescence-activated cell sorting is established, taking advantage of the distinctive autofluorescence of these cells. This achievement represents a crucial step for the development of differential omic strategies leading to the identification of candidate genes putatively involved in the biosynthesis, pathway regulation, and transmembrane transport leading to the anticancer alkaloids from C. roseus. PMID:27356972

  8. Catharanthus mosaic virus: A potyvirus from a gymnosperm, Welwitschia mirabilis.

    PubMed

    Koh, Shu Hui; Li, Hua; Admiraal, Ryan; Jones, Michael G K; Wylie, Stephen J

    2015-05-01

    A virus from a symptomatic plant of the gymnosperm Welwitschia mirabilis Hook. growing as an ornamental plant in a domestic garden in Western Australia was inoculated to a plant of Nicotiana benthamiana where it established a systemic infection. The complete genome sequence of 9636 nucleotides was determined using high-throughput and Sanger sequencing technologies. The genome sequence shared greatest identity (83% nucleotides and 91% amino acids) with available partial sequences of catharanthus mosaic virus, indicating that the new isolate belonged to that taxon. Analysis of the phylogeny of the complete virus sequence placed it in a monotypic group in the genus Potyvirus. This is the first record of a virus from W. mirabilis, the first complete genome sequence of catharanthus mosaic virus determined, and the first record from Australia. This finding illustrates the risk to natural and managed systems posed by the international trade in live plants and propagules, which enables viruses to establish in new regions and infect new hosts. PMID:25804761

  9. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    PubMed Central

    2011-01-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  10. Factors affecting polyhydroxybutyrate accumulation in mesophyll cells of sugarcane and switchgrass

    PubMed Central

    2014-01-01

    Background Polyhydroxyalkanoates are linear biodegradable polyesters produced by bacteria as a carbon store and used to produce a range of bioplastics. Widespread polyhydroxyalkanoate production in C4 crops would decrease petroleum dependency by producing a renewable supply of biodegradable plastics along with residual biomass that could be converted into biofuels or energy. Increasing yields to commercial levels in biomass crops however remains a challenge. Previously, lower accumulation levels of the short side chain polyhydroxyalkanoate, polyhydroxybutyrate (PHB), were observed in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells in transgenic maize (Zea mays), sugarcane (Saccharum sp.), and switchgrass (Panicum virgatum L.) leading to a significant decrease in the theoretical yield potential. Here we explore various factors which might affect polymer accumulation in mesophyll cells, including targeting of the PHB pathway enzymes to the mesophyll plastid and their access to substrate. Results The small subunit of Rubisco from pea effectively targeted the PHB biosynthesis enzymes to both M and BS chloroplasts of sugarcane and switchgrass. PHB enzyme activity was retained following targeting to M plastids and was equivalent to that found in the BS plastids. Leaf total fatty acid content was not affected by PHB production. However, when fatty acid synthesis was chemically inhibited, polymer accumulated in M cells. Conclusions In this study, we provide evidence that access to substrate and neither poor targeting nor insufficient activity of the PHB biosynthetic enzymes may be the limiting factor for polymer production in mesophyll chloroplasts of C4 plants. PMID:25209261

  11. The response of mesophyll conductance to nitrogen and water availability differs between wheat genotypes.

    PubMed

    Barbour, Margaret M; Kaiser, Brent N

    2016-10-01

    Increased mesophyll conductance (gm) has been suggested as a target for selection for high productivity and high water-use efficiency in crop plants, and genotypic variability in gm has been reported in several important crop species. However, effective selection requires an understanding of how gm varies with growth conditions, to ensure that the ranking of genotypes is consistent across environments. We assessed the genotypic variability in gm and other leaf gas exchange traits, as well as growth and biomass allocation for six wheat genotypes under different water and nitrogen availabilities. The wheat genotypes differed in their response of gm to growth conditions, resulting in genotypic differences in the mesophyll limitation to photosynthesis and a significant increase in the mesophyll limitation to photosynthesis under drought. In this experiment, leaf intrinsic water-use efficiency was more closely related to stomatal conductance than to mesophyll conductance, and stomatal limitation to photosynthesis increased more in some genotypes than in others in response to drought. Screening for gm should be carried out under a range of growth conditions. PMID:27593470

  12. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    NASA Astrophysics Data System (ADS)

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  13. Effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of summer maize.

    PubMed

    Ren, Baizhao; Cui, Haiyan; Camberato, James J; Dong, Shuting; Liu, Peng; Zhao, Bin; Zhang, Jiwang

    2016-08-01

    A field experiment was conducted to study the effects of shading on the photosynthetic characteristics and mesophyll cell ultrastructure of two summer maize hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The ambient sunlight treatment was used as control (CK) and shading treatments (40 % of ambient sunlight) were applied at different growth stages from silking (R1) to physiological maturity (R6) (S1), from the sixth leaf stage (V6) to R1 (S2), and from seeding to R6 (S3), respectively. The net photosynthetic rate (P n) was significantly decreased after shading. The greatest reduction of P n was found at S3 treatment, followed by S1 and S2 treatments. P n of S3 was decreased by 59 and 48 % for DH605, and 39 and 43 % for ZD958 at tasseling and milk-ripe stages, respectively, compared to that of CK. Additionally, leaf area index (LAI) and chlorophyll content decreased after shading. In terms of mesophyll cell ultrastructure, chloroplast configuration of mesophyll cells dispersed, and part of chloroplast swelled and became circular. Meanwhile, the major characteristics of chloroplasts showed poorly developed thylakoid structure at the early growth stage, blurry lamellar structure, loose grana, and a large gap between slices and warping granum. Then, plasmolysis occurred in mesophyll cells and the endomembrane system was destroyed, which resulted in the dissolution of cell membrane, karyotheca, mitochondria, and some membrane structures. The damaged mesophyll cell ultrastructure led to the decrease of photosynthetic capacity, and thus resulted in significant yield reduction by 45, 11, and 84 % in S1, S2, and S3 treatments, respectively, compared to that of CK. PMID:27437706

  14. Biodegradation of a synthetic lubricant by Micrococcus roseus.

    PubMed Central

    Wright, M A; Taylor, F; Randles, S J; Brown, D E; Higgins, I J

    1993-01-01

    A bacterium that was able to utilize Emkarate 1550 (E1550), a synthetic lubricant ester, as the sole source of carbon was isolated. The isolate was tentatively identified as Micrococcus roseus. The components of the E1550 ester, octanoate, decanoate, and 1,1,1-tris(hydroxymethyl)propane (TMP), were detected in the culture medium of cells growing on the ester. The TMP tertiary alcohol accumulated during growth and was not utilized by this isolate. The detection of the components of the ester in the supernatant of cultures indicated that one of the first steps in its degradation was cleavage of the ester bonds. Esterase activity was significantly enhanced in cells grown on E1550 compared with esterase activity measured in cells grown on acetate. PMID:8476283

  15. Impact of mesophyll diffusion on estimated global land CO2 fertilization

    DOE PAGESBeta

    Sun, Ying; Gu, Lianhong; Dickinson, Robert E.; Norby, Richard J.; Pallardy, Stephen G.; Hoffman, Forrest M.

    2014-10-13

    In C3 plants, CO2 concentrations drop considerably along mesophyll diffusion pathways from substomatal cavities to chloroplasts where CO2 assimilation occurs. Global carbon cycle models have not explicitly represented this internal drawdown and so overestimate CO2 available for carboxylation and underestimate photosynthetic responsiveness to atmospheric CO2. An explicit consideration of mesophyll diffusion increases the modeled cumulative CO2 fertilization effect (CFE) for global gross primary production (GPP) from 915 PgC to 1057 PgC for the period of 1901 to 2010. This increase represents a 16% correction, large enough to explain the persistent overestimation of growth rates of historical atmospheric CO2 by Earthmore » System Models. Without this correction, the CFE for global GPP is underestimated by 0.05 PgC yr-1ppm-1. This finding implies that the contemporary terrestrial biosphere is more CO2-limited than previously thought.« less

  16. Impact of Mesophyll Diffusion on Estimated Global Land CO2 Fertilization

    NASA Astrophysics Data System (ADS)

    Sun, Y.; Gu, L.; Dickinson, R. E.

    2014-12-01

    In C3 plants, CO2 concentrations drop considerably along mesophyll diffusion pathways from substomatal cavities to chloroplasts where CO2 assimilation occurs. Global carbon cycle models have not explicitly represented this internal drawdown and so overestimate CO2 available for carboxylation and underestimate photosynthetic responsiveness to atmospheric CO2. An explicit consideration of mesophyll diffusion increases the modeled cumulative CO2 fertilization effect (CFE) for global gross primary production (GPP) from 915 PgC to 1057 PgC for the period of 1901 to 2010. This increase represents a 16% correction large enough to explain the persistent overestimation of growth rates of historical atmospheric CO2 by Earth System Models. Without this correction, the CFE for global GPP is underestimated by 0.05 PgC yr-1ppm-1. This finding implies that the contemporary terrestrial biosphere is more CO2-limited than previously thought.

  17. [Signal function of cytokinin 6-benzylaminopurine in the reaction of Triticum aestivum L. mesophyll cells to hyperthermia].

    PubMed

    Musiienko, M M; Zhuk, V V; Batsmanova, L M

    2014-01-01

    The signaling effect of 6-benzylaminopurine (BAP) on leaf mesophyll cells of Triticum aestivum L. under hyperthermic conditions was studied. It was found that BAP regulated photosynthetic pigment, hydrogen peroxide content and activity of antioxidant enzymes, namely superoxide dismutase, ascorbate peroxidase and catalase under high-temperature conditions. The additive effect of BAP and high temperature on the activation of cell antioxidant systems was demonstrated. BAP regulated reducing processes in mesophyll leaf cells under high-temperature conditions. PMID:25816607

  18. Rhabdobacter roseus gen. nov., sp. nov., isolated from soil.

    PubMed

    Dahal, Ram Hari; Kim, Jaisoo

    2016-01-01

    An aerobic, Gram-stain-negative, oxidase- and catalase-positive, non-motile, non-spore-forming, rod-shaped, pink-pigmented bacterium, designated strain R49T, was isolated from soil. Flexirubin-type pigments were absent. Phylogenetic analysis based on its 16S rRNA gene sequence revealed that strain R49T formed a lineage within the family Cytophagaceae of the phylum Bacteroidetes that was distinct from the most closely related genera Dyadobacter (91.98-93.85 % sequence similarity), Persicitalea (88.69 %) and Runella (84.79-85.81 %). The major isoprenoid quinone was menaquinone-7 (MK-7) and the major polar lipid was phosphatidylethanolamine. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), iso-C15 : 0, C16 : 1ω5c, C16 : 0 and iso-C17 : 0 3-OH. The DNA G+C content of strain R49T was 53.9 mol%. On the basis of phenotypic, genotypic and phylogenetic analysis, strain R49T represents a novel species of a new genus in the family Cytophagaceae, for which the name Rhabdobacter roseus gen. nov., sp. nov. is proposed. The type strain of Rhabdobacter roseus is R49T ( = KEMB 9005-318T = KACC 18395T = JCM 30685T). PMID:26508300

  19. Comparative Studies of Fluorescence from Mesophyll and Guard Cell Chloroplasts in Saxifraga cernua1

    PubMed Central

    Mawson, Bruce T.; Franklin, Angus; Filion, W. Gary; Cummins, W. Raymond

    1984-01-01

    The chlorophyll fluorescence induction curves from mesophyll and guard cell chloroplasts of Saxifraga cernua, including both the fast (O to P, the transients involved in the rise in variable fluorescence) and slow (P to steady state fluorescence due to quenching) components, were characterized over a range of excitation intensities using microspectrophotometry (with epi-lumination) equipped with apertures designed to eliminate cross contamination of the fluorescence signal between the two chloroplast types. At low excitation intensities, the fast fluorescence kinetics from guard cell plastids showed an extended I to D phase and a more rapid appearance of P while minimal quenching from P to steady state fluorescence was observed compared to the transients from mesophyll chloroplasts suggesting a lower activity of photochemical (electron movement via carriers between donor and acceptor sites) and nonphotochemical (such as membrane conformational changes) events which regulate the fluorescence induction curve kinetics. As the excitation intensity was increased, the quenching rates of guard cells were faster at initiating conditions for photophosphorylation and the fast and slow fluorescence kinetics from guard cells resembled those of the mesophyll cells. Guard cell chloroplasts of S. cernua from intact epidermal peels showed a low temperature (77 K) fluorescence emission spectrum having three major peaks (at 685, 695, and 730 nanometers when excited at 440 nanometers) which were qualitatively similar to those in the spectrum obtained from mesophyll tissue. These data suggest that S. cernua guard cell chloroplast photosystems I and II contribute to light-dependent stomatal activity only at high light intensities. Images Fig. 1 Fig. 3 PMID:16663448

  20. Auxin Reduces the Synthesis of Major Vacuolar Proteins in Tobacco Mesophyl Protoplast

    PubMed Central

    Meyer, Yves; Chartier, Yvette; Alibert, Gilbert

    1987-01-01

    We have established that polypeptides whose synthesis is reduced by 2,4-dichlorophenoxyacetic acid during in vitro culture of tobacco mesophyll protoplasts are secreted into the vacuole where they constitute the bulk of labeled proteins. In addition, these proteins continue to be synthesized in protoplast-derived cultured cells and their synthesis is strictly correlated with the size of the cell, i.e. with vacuolar size. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665313

  1. Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues

    PubMed Central

    Svozil, Julia; Gruissem, Wilhelm; Baerenfaller, Katja

    2015-01-01

    Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions. PMID:26074939

  2. The arc mutants of Arabidopsis with fewer large chloroplasts have a lower mesophyll conductance.

    PubMed

    Weise, Sean E; Carr, David J; Bourke, Ashley M; Hanson, David T; Swarthout, Debbie; Sharkey, Thomas D

    2015-04-01

    Photosynthetic cells of most land plant lineages have numerous small chloroplasts even though most algae, and even the early diverging land plant group the hornworts, tend to have one or a few large chloroplasts. One constraint that small chloroplasts could improve is the resistance to CO2 diffusion from the atmosphere to the chloroplast stroma. We examined the mesophyll conductance (inverse of the diffusion resistance) of mutant Arabidopsis thaliana plants with one or only a few large chloroplasts per cell. The accumulation and replication of chloroplasts (arc) mutants of A. thaliana were studied by model fitting to gas exchange data and (13)CO2 discrimination during carbon fixation. The two methods generally agreed, but the value of the CO2 compensation point of Rubisco (Γ *) used in the model had a large impact on the estimated photosynthetic parameters, including mesophyll conductance. We found that having only a few large chloroplasts per cell resulted in a 25-50 % reduction in the mesophyll conductance at ambient CO2. PMID:25733184

  3. Marked changes in volume of mesophyll protoplasts of pea (Pisum sativum) on exposure to growth hormones.

    PubMed

    Kolla, Venkat Apparao; Suhita, Dontamala; Raghavendra, Agepati S

    2004-05-01

    The present study reports quick and significant changes induced by plant hormones in the volume of mesophyll protoplasts of pea (Pisum sativum). Four plant hormones: gibberellic acid (GA3), indole 3-acetic acid (IAA), abscisic acid (ABA)(+/-) and methyl jasmonate (MJ), caused marked changes in the volume of mesophyll protoplasts. GA3 and IAA increased the volume of the protoplasts (up to 90%) whereas the ABA and MJ decreased (by about 40%) the volume. Aquaporins or water channels appear to play an important role in swelling/shrinkage of the protoplasts as indicated by the suppression of volume changes by HgCl2 and reversal by mercaptoethanol. The possible role of secondary messengers in volume changes induced by GA3 was investigated by using selected pharmacological reagents. The GA3 induced swelling was restricted by GDP-beta-S (G-protein antagonist), U73122 (phospholipase C inhibitor), and TFP (calmodulin antagonist), but was not affected by 1-butanol (phospholipase D inhibitor), GTP-gamma-S (G-protein agonist), or verapamil (calcium channel blocker). The results suggest that the mesophyll protoplasts can be a simple and useful system for further studies on volume changes in plant tissues. PMID:15202712

  4. Changes of mesophyll and the rubisco activity in pea plants grown in clinostat

    NASA Astrophysics Data System (ADS)

    Adamchuk, N. I.

    In earlier research, it was found that microgravity causes alteration of mesophyll cell parameters and dislication at the ultrastructural level (Kordyum et al., 1989, Nedukha et al., 1991, Kordyum, 1997, Adamchuk et al., 2002). Also, destruction of the fine structure of chloroplasts was reported by Abilov et al. (1986), Aliev et al. (1987), Kordyum et al. (1989), and Adamchuk et al. (1999). In addition, Abilov et al. (1986), Aliev et al. (1987), Brown et al. (1993) have discovered the decrease in starch volume. The objective of this work was to compare quantitative ultrastructural parameters of mesophyll cells (including properties of their chloroplasts) and the level of Rubisco activity detected in clinorotated and control plants of pea (Pisum sativum L.). Plants were grown for 12 days in the nutritional medium of Hogland on a clinostat (with 2 rev. min-1 speed of rotation) at a temperature of 23-25°C and illumination 230 μ mol per m-2s-1. The comparison of transversal cross-sections of leaves has revealed a significant increase of mesophyll cell volume and intercellular space under experimental conditions. This expansion of mesophyll cells has correlated with an increase of the number of chloroplasts. Essential ultrastructural changes have affected the total volume of thylakoids. Also, the value of the photosynthetic membranes development in the clinorotated plants was higher 17.11 ± 1.94 μ m3 then in control -- 12.65 ± 1.83 μ m3 due to extension of destacking thylakoids. Increase of the volume density of plastoglobuli in the clinorotated plants on the 1.63-fold suggested the effect of either greater accumulation of lipid or acceleration of chloroplasts senescence. Under influence of clinorotation, the partial volume of starch inclusions significantly decreased in the spongy mesophyll chloroplasts -- 10.46 ± 1.80 % to compare with control -- 31.34 ± 2.37 %. However, the clinorotation of plants resulted in an increase of the Rubisco activity. Intensities

  5. Effects of Nitrogen on Mesophyll Cell Division and Epidermal Cell Elongation in Tall Fescue Leaf Blades 1

    PubMed Central

    MacAdam, Jennifer W.; Volenec, Jeffrey J.; Nelson, Curtis J.

    1989-01-01

    Leaf elongation rate (LER) in grasses is dependent on epidermal cell supply (number) and on rate and duration of epidermal cell elongation. Nitrogen (N) fertilization increases LER. Longitudinal sections from two genotypes of tall fescue (Festuca arundinacea Schreb.), which differ by 50% in LER, were used to quantify the effects of N on the components of epidermal cell elongation and on mesophyll cell division. Rate and duration of epidermal cell elongation were determined by using a relationship between cell length and displacement velocity derived from the continuity equation. Rate of epidermal cell elongation was exponential. Relative rates of epidermal cell elongation increased by 9% with high N, even though high N increased LER by 89%. Duration of cell elongation was approximately 20 h longer in the high- than in the low-LER genotype regardless of N treatment. The percentage of mesophyll cells in division was greater in the high- than in the low-LER genotype. This increased with high N in both genotypes, indicating that LER increased with cell supply. Division of mesophyll cells adjacent to abaxial epidermal cells continued after epidermal cell division stopped, until epidermal cells had elongated to a mean length of 40 micrometers in the high-LER and a mean length of 50 micrometers in the low-LER genotype. The cell cycle length for mesophyll cells was calculated to be 12 to 13 hours. Nitrogen increased mesophyll cell number more than epidermal cell number: in both genotypes, the final number of mesophyll cells adjacent to each abaxial epidermal cell was 10 with low N and 14 with high N. A spatial model is used to describe three cell development processes relevant to leaf growth. It illustrates the overlap of mesophyll cell division and epidermal cell elongation, and the transition from epidermal cell elongation to secondary cell wall deposition. PMID:16666581

  6. Screening molecules for control of citrus huanglongbing using an optimized regeneration system for 'Candidatus Liberibacter asiaticus'-infected periwinkle (Catharanthus roseus) cuttings.

    PubMed

    Zhang, Muqing; Duan, Yongping; Zhou, Lijuan; Turechek, William W; Stover, Ed; Powell, Charles A

    2010-03-01

    Citrus huanglongbing is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of 'Candidatus Liberibacter', of which 'Ca. L. asiaticus' is the most widely distributed. An optimized system using 'Ca. L. asiaticus'-infected periwinkle cuttings was developed to screen chemical compounds effective for controlling the bacterial population while simultaneously assessing their phytotoxicity. The optimal regeneration conditions were determined to be the use of vermiculite as a growth medium for the cuttings, and a fertilization routine using half-strength Murashige and Tucker medium supplemented with both naphthalene acetic acid (4 microg/ml) and indole-3-butyric acid (4 microg/ml). This system allowed a plant regeneration rate of 60.6% for 'Ca. L. asiaticus'-infected cuttings in contrast to the <1% regeneration rate with water alone. Two chemical agents, penicillin G sodium and 2,2-dibromo-3-nitrilopropionamide (DBNPA), were found to be effective at eliminating or suppressing the 'Ca. L. asiaticus' bacterium in this periwinkle regeneration system. When treated with penicillin G sodium at 50 microg/ml, all plants regenerated from 'Ca. L. asiaticus'-infected cuttings were 'Ca. L. asiaticus' negative as determined by both nested polymerase chain reaction (PCR) and quantitative real-time PCR. In addition, DBNPA was also able to significantly reduce the percentage of 'Ca. L. asiaticus'-positive plants and the titer of the 'Ca. L. asiaticus' bacterium at 200 microl/liter. PMID:20128697

  7. Simple and rapid biosynthesis of stable silver nanoparticles using dried leaves of Catharanthus roseus. Linn. G. Donn and its anti microbial activity.

    PubMed

    Kotakadi, Venkata Subbaiah; Rao, Y Subba; Gaddam, Susmila Aparna; Prasad, T N V K V; Reddy, A Varada; Gopal, D V R Sai

    2013-05-01

    Nanoparticles have been used to alter and improve the pharmacokinetic and pharmacodynamic properties of various types of drug molecules. The plant extracts are eco-friendly, economical and cost effective for synthesis of large scale of nanoparticles. In this paper we represent the synthesis of silver nanoparticles (AgNPs) from room dried leaves of Vinca rosea. The AgNPs were characterized by UV-vis spectroscopy. The AgNPs are crystalline in nature, were determined from scanning electron microscopy (SEM) coupled with energy dispersive X-ray (EDX), X-ray diffraction patterns (XRD), and also the size of the NPs was calculated by using Hariba Nanoparticle analyzer and the stability was calculated by using the Zetapotential. The nanoparticles obtained from leaf extracts were of size 27±2 and 30±2 respectively and Zetapotential of AgNPs was found to be -63.1 mV, so it indicates the dispersion and stability. The synthesized AgNPs have very good antimicrobial activity. PMID:23376746

  8. Accumulation of Monoterpenoid Indole Alkaloids in Periwinkle Seedlings ("Catharanthus roseus") as a Model for the Study of Plant-Environment Interactions

    ERIC Educational Resources Information Center

    Miranda-Ham, Maria de Lourdes; Islas-Flores, Ignacio; Vazquez-Flota, Felipe

    2007-01-01

    Alkaloids are part of the chemical arsenal designed to protect plants against an adverse environment. Therefore, their synthesis and accumulation are frequently induced in response to certain environmental conditions and are mediated by chemical signals, which are formed as the first responses to the external stimulus. A set of experiments using…

  9. Online CO2 and H2 O oxygen isotope fractionation allows estimation of mesophyll conductance in C4 plants, and reveals that mesophyll conductance decreases as leaves age in both C4 and C3 plants.

    PubMed

    Barbour, Margaret M; Evans, John R; Simonin, Kevin A; von Caemmerer, Susanne

    2016-05-01

    Mesophyll conductance significantly, and variably, limits photosynthesis but we currently have no reliable method of measurement for C4 plants. An online oxygen isotope technique was developed to allow quantification of mesophyll conductance in C4 plants and to provide an alternative estimate in C3 plants. The technique is compared to an established carbon isotope method in three C3 species. Mesophyll conductance of C4 species was similar to that in the C3 species measured, and declined in both C4 and C3 species as leaves aged from fully expanded to senescing. In cotton leaves, simultaneous measurement of carbon and oxygen isotope discrimination allowed the partitioning of total conductance to the chloroplasts into cell wall and plasma membrane versus chloroplast membrane components, if CO2 was assumed to be isotopically equilibrated with cytosolic water, and the partitioning remained stable with leaf age. The oxygen isotope technique allowed estimation of mesophyll conductance in C4 plants and, when combined with well-established carbon isotope techniques, may provide additional information on mesophyll conductance in C3 plants. PMID:26778088

  10. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes.

    PubMed

    Sutton, F; Paul, S S; Wang, X Q; Assmann, S M

    2000-09-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels. PMID:10982437

  11. Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes

    NASA Technical Reports Server (NTRS)

    Sutton, F.; Paul, S. S.; Wang, X. Q.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels.

  12. Distinct Abscisic Acid Signaling Pathways for Modulation of Guard Cell versus Mesophyll Cell Potassium Channels Revealed by Expression Studies in Xenopus laevis Oocytes1

    PubMed Central

    Sutton, Fedora; Paul, Sunil S.; Wang, Xi-Qing; Assmann, Sarah M.

    2000-01-01

    Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K+ current (IKin) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K+ current (IKout). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, IKout was generated that displayed similar properties to IKout observed from direct analysis of mesophyll cell protoplasts. IKout expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K+ channel expressed in guard cells, resulted in IKin that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced IKin that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K+ channels. PMID:10982437

  13. A Modeling Approach to Quantify the Effects of Stomatal Behavior and Mesophyll Conductance on Leaf Water Use Efficiency.

    PubMed

    Moualeu-Ngangue, Dany P; Chen, Tsu-Wei; Stützel, Hartmut

    2016-01-01

    Water use efficiency (WUE) is considered as a determinant of yield under stress and a component of crop drought resistance. Stomatal behavior regulates both transpiration rate and net assimilation and has been suggested to be crucial for improving crop WUE. In this work, a dynamic model was used to examine the impact of dynamic properties of stomata on WUE. The model includes sub-models of stomatal conductance dynamics, solute accumulation in the mesophyll, mesophyll water content, and water flow to the mesophyll. Using the instantaneous value of stomatal conductance, photosynthesis, and transpiration rate were simulated using a biochemical model and Penman-Monteith equation, respectively. The model was parameterized for a cucumber leaf and model outputs were evaluated using climatic data. Our simulations revealed that WUE was higher on a cloudy than a sunny day. Fast stomatal reaction to light decreased WUE during the period of increasing light (e.g., in the morning) by up to 10.2% and increased WUE during the period of decreasing light (afternoon) by up to 6.25%. Sensitivity of daily WUE to stomatal parameters and mesophyll conductance to CO2 was tested for sunny and cloudy days. Increasing mesophyll conductance to CO2 was more likely to increase WUE for all climatic conditions (up to 5.5% on the sunny day) than modifications of stomatal reaction speed to light and maximum stomatal conductance. PMID:27379150

  14. A Modeling Approach to Quantify the Effects of Stomatal Behavior and Mesophyll Conductance on Leaf Water Use Efficiency

    PubMed Central

    Moualeu-Ngangue, Dany P.; Chen, Tsu-Wei; Stützel, Hartmut

    2016-01-01

    Water use efficiency (WUE) is considered as a determinant of yield under stress and a component of crop drought resistance. Stomatal behavior regulates both transpiration rate and net assimilation and has been suggested to be crucial for improving crop WUE. In this work, a dynamic model was used to examine the impact of dynamic properties of stomata on WUE. The model includes sub-models of stomatal conductance dynamics, solute accumulation in the mesophyll, mesophyll water content, and water flow to the mesophyll. Using the instantaneous value of stomatal conductance, photosynthesis, and transpiration rate were simulated using a biochemical model and Penman-Monteith equation, respectively. The model was parameterized for a cucumber leaf and model outputs were evaluated using climatic data. Our simulations revealed that WUE was higher on a cloudy than a sunny day. Fast stomatal reaction to light decreased WUE during the period of increasing light (e.g., in the morning) by up to 10.2% and increased WUE during the period of decreasing light (afternoon) by up to 6.25%. Sensitivity of daily WUE to stomatal parameters and mesophyll conductance to CO2 was tested for sunny and cloudy days. Increasing mesophyll conductance to CO2 was more likely to increase WUE for all climatic conditions (up to 5.5% on the sunny day) than modifications of stomatal reaction speed to light and maximum stomatal conductance. PMID:27379150

  15. Roles of mesophyll conductance and plant functional diversities in tropical photosynthesis

    NASA Astrophysics Data System (ADS)

    Gu, L.

    2013-12-01

    Tropical photosynthesis dominates global terrestrial gross primary production (GPP) and will likely play a defining role in determining how global GPP will respond to climate change. Yet, our current understanding of biological, ecological, edaphic and environmental controls on tropical photosynthesis is poor. The overly simplistic schemes that current Earth System Models use to simulate tropical photosynthesis cannot capture the functional diversities associated with high species diversities in the tropics. New approaches that explicitly represent the functional diversities of tropical photosynthesis in Earth System Models are needed in order to realistically model responses of tropical photosynthesis to increased atmospheric CO2 concentrations and associated climate changes. To establish a basis for such approaches, we conducted intensive field measurements of leaf photosynthesis at three forest sites along a strong rainfall gradient in Panama in 2012-2013. The three sites are Parque Natural Metropolitano, Gamboa, and Parque Nacional San Lorenzo. The Parque Natural Metropolitano receives an annual precipitation of less than 1800mm and Parque Nacional San Lorenzo over 3300 mm with Gamboa in between. The three sites differ in species diversity with Parque Nacional San Lorenzo having the highest species diversity and Parque Nacional San Lorenzo the lowest. At the three contrasting sites, we measured A/Ci curves, leaf traits and leaf nutrient (N and P) contents of about 100 species. We determined mesophyll conductance with the LeafWeb approach. From these measurements, we developed practical but realistic parameterizations of functional diversities of tropical plant species at the three sites and implemented these parameterizations in the latest version of the Community Land Model. We found that mesophyll conductance is key to representing functional diversities of tropical forest species. Without it, responses of tropical photosynthesis to increased atmospheric CO2

  16. Abscisic Acid Induces Rapid Reductions in Mesophyll Conductance to Carbon Dioxide.

    PubMed

    Sorrentino, Giuseppe; Haworth, Matthew; Wahbi, Said; Mahmood, Tariq; Zuomin, Shi; Centritto, Mauro

    2016-01-01

    The rate of photosynthesis (A) of plants exposed to water deficit is a function of stomatal (gs) and mesophyll (gm) conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA) plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci). Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis. PMID:26862904

  17. Abscisic Acid Induces Rapid Reductions in Mesophyll Conductance to Carbon Dioxide

    PubMed Central

    Sorrentino, Giuseppe; Haworth, Matthew; Wahbi, Said; Mahmood, Tariq; Zuomin, Shi; Centritto, Mauro

    2016-01-01

    The rate of photosynthesis (A) of plants exposed to water deficit is a function of stomatal (gs) and mesophyll (gm) conductance determining the availability of CO2 at the site of carboxylation within the chloroplast. Mesophyll conductance often represents the greatest impediment to photosynthetic uptake of CO2, and a crucial determinant of the photosynthetic effects of drought. Abscisic acid (ABA) plays a fundamental role in signalling and co-ordination of plant responses to drought; however, the effect of ABA on gm is not well-defined. Rose, cherry, olive and poplar were exposed to exogenous ABA and their leaf gas exchange parameters recorded over a four hour period. Application with ABA induced reductions in values of A, gs and gm in all four species. Reduced gm occurred within one hour of ABA treatment in three of the four analysed species; indicating that the effect of ABA on gm occurs on a shorter timescale than previously considered. These declines in gm values associated with ABA were not the result of physical changes in leaf properties due to altered turgor affecting movement of CO2, or caused by a reduction in the sub-stomatal concentration of CO2 (Ci). Increased [ABA] likely induces biochemical changes in the properties of the interface between the sub-stomatal air-space and mesophyll layer through the actions of cooporins to regulate the transport of CO2. The results of this study provide further evidence that gm is highly responsive to fluctuations in the external environment, and stress signals such as ABA induce co-ordinated modifications of both gs and gm in the regulation of photosynthesis. PMID:26862904

  18. Purification and properties of urease from Sporobolomyces roseus.

    PubMed

    Jahns, T

    1995-10-01

    Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme from Sporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (+/- 4) kDa. The M(r) of the native enzyme was estimated by size exclusion chromatography to be 340 (+/- 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 degrees C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen. PMID:8572678

  19. Impact of mesophyll diffusion on estimated global land CO2 fertilization

    SciTech Connect

    Sun, Ying; Gu, Lianhong; Dickinson, Robert E.; Norby, Richard J.; Pallardy, Stephen G.; Hoffman, Forrest M.

    2014-10-13

    In C3 plants, CO2 concentrations drop considerably along mesophyll diffusion pathways from substomatal cavities to chloroplasts where CO2 assimilation occurs. Global carbon cycle models have not explicitly represented this internal drawdown and so overestimate CO2 available for carboxylation and underestimate photosynthetic responsiveness to atmospheric CO2. An explicit consideration of mesophyll diffusion increases the modeled cumulative CO2 fertilization effect (CFE) for global gross primary production (GPP) from 915 PgC to 1057 PgC for the period of 1901 to 2010. This increase represents a 16% correction, large enough to explain the persistent overestimation of growth rates of historical atmospheric CO2 by Earth System Models. Without this correction, the CFE for global GPP is underestimated by 0.05 PgC yr-1ppm-1. This finding implies that the contemporary terrestrial biosphere is more CO2-limited than previously thought.

  20. Co-transport of Potassium and Sugars across the Plasmalemma of Mesophyll Protoplasts 1

    PubMed Central

    Huber, Steven C.; Moreland, Donald E.

    1981-01-01

    Sugars (sucrose + hexoses) produced photosynthetically by isolated mesophyll protoplasts of wheat and tobacco were effluxed across the plasma membrane (3 to 10 micromoles hexose equivalents per milligram chlorophyll per hour). The efflux was sensitive to uncouplers and oligomycin which indicated a requirement for energy. A proton gradient was probably not coupled directly to the transport because changing the proton gradient across the plasma membrane by varying the pH of the medium or by adding sodium acetate had no significant effect on the rate of sugar release. A release of K+ was associated with sugar efflux from the protoplasts. The molar ratio of K+ to sugar varied between 1.5 and 2.5, depending on the species. Exogenous CKl, RbCl, and LiCl (50 millimolar each), but not NaCl or CsCl, significantly inhibited sugar efflux. Conditions that reduced sugar efflux (exogenous KCl, LiCl, mersalyl, or oligomycin) also reduced K+ release and caused a time-dependent reduction in photosynthetic sucrose formation and increased amino acid and starch formation. Results obtained support the postulate that a K+ symport is involved in the transport of sugar across the energized plasmalemma of photosynthetically active mesophyll cells. PMID:16661619

  1. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue.

    PubMed

    Endo, Motomu; Shimizu, Hanako; Araki, Takashi

    2016-08-01

    To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays. PMID:27388555

  2. Evidence for a specific glutamate/H/sup +/ cotransport in isolated mesophyll cells. [Asparagus sprengeri

    SciTech Connect

    McCutcheon, S.L.; Bown, A.W.

    1987-03-01

    Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO/sub 4/. Immediate alkalinization of the medium occurred on the addition of 1 millimolar concentrations of L-glutamate (Glu) and its analog L-methionine-D,L-sulfoximine (L-MSO). D-Glu and the L isomers of the protein amino acids did not elicit alkalinization. L-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar L-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H/sup +//10/sup 6/ cells minute. L-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of L-(U-/sup 14/C)glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by L-MSO. L-Glu had no influence on K/sup +/ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific L-Glu/proton uptake process is present in Asparagus mesophyll cells.

  3. Methods of mesophyll conductance estimation: its impact on key biochemical parameters and photosynthetic limitations in phosphorus-stressed soybean across CO2.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Photosynthetic potential in C3 plants is largely limited by CO2 diffusion through stomata (Ls) and mesophyll (Lm) and photo-biochemical (Lb) processes. Accurate estimation of mesophyll conductance (gm) using gas exchange (GE) and chlorophyll fluorescence (CF) parameters of the photosynthetic proces...

  4. Mucilaginibacter roseus sp. nov., isolated from a freshwater river.

    PubMed

    Chen, Wen-Ming; Chen, Yi-Ling; Sheu, Shih-Yi

    2016-03-01

    A bacterial strain, designated TTM-1T, was isolated from a water sample taken from the Caohu River in Taiwan and characterized in a taxonomic study using a polyphasic approach. Cells of strain TTM-1T were Gram-stain-negative, aerobic, non-motile, rod-shaped and covered by large capsules, and formed pink-coloured colonies. Growth occurred at 10-37 °C (optimum 30-37 °C), at pH 6-8 (optimum pH 6-7) and with 0-2 % NaCl (optimum 0.5 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TTM-1T belonged to the genus Mucilaginibacter and was most closely related to Mucilaginibacter defluvii A5T with a 16S rRNA gene sequence similarity of 97.3 %. The predominant fatty acids of strain TTM-1T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c; 37.1 %) and iso-C15 : 0 (30.7 %). The polar lipid profile consisted of phosphatidylethanolamine and several uncharacterized aminophospholipids and phospholipids. The major isoprenoid quinone was MK-7. The DNA G+C content of the genomic DNA was 45.1 mol%. The DNA-DNA relatedness of strain TTM-1T with respect to recognized species of the genus Mucilaginibacter was less than 70 %. On the basis of the phylogenetic inference and phenotypic data, strain TTM-1T represents a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter roseus sp. nov. is proposed. The type strain is TTM-1T ( = LMG 28454T = KCTC 42273T). PMID:26652650

  5. Asymmetrical effects of mesophyll conductance on fundamental photosynthetic parameters and their relationships estimated from leaf gas exchange measurements

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Most previous analyses of leaf gas exchange measurements assumed an infinite value of mesophyll conductance (gm) and thus equaled CO2 partial pressures in the substomatal cavity and chloroplast. Yet an increasing number of studies have recognized that gm is finite and there is a drawdown of CO2 part...

  6. Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency.

    PubMed Central

    Morales, F.; Cerovic, Z. G.; Moya, I.

    1994-01-01

    The mesophyll of sugar beet (Beta vulgaris L.) leaves emits red (chlorophyll a) fluorescence and blue-green fluorescence when excited with ultraviolet light. The intensity of blue-green fluorescence was increased in mesophylls affected by iron deficiency. This increase was large and progressive. It was concomitant with a decrease of photosynthetic pigments per unit of leaf area. Most of the increase in blue-green fluorescence can be explained by the decrease of the screening of ultraviolet light by chlorophylls and carotenoids. In addition, chlorophylls selectively reabsorb blue fluorescence, which leads to a change in the form of the fluorescence emission spectra. This effect induces an increase of the blue-to-green fluorescence ratio in control mesophylls that was concomitant with the decrease of chlorophyll per unit of leaf area. Iron deficiency induced a decrease of the blue-to-green fluorescence ratio that may be attributed to an accumulation of flavins fluorescing in the green. Time-resolved fluorescence measurements indicate that they are mostly riboflavin and/or flavin mononucleotide phosphate. Our data also indicate that the blue-green fluorescence emitted from the mesophyll contains fluorescence of nicotinamide nucleotides. PMID:12232310

  7. Cold Transiently Activates Calcium-Permeable Channels in Arabidopsis Mesophyll Cells1[W

    PubMed Central

    Carpaneto, Armando; Ivashikina, Natalya; Levchenko, Victor; Krol, Elzbieta; Jeworutzki, Elena; Zhu, Jian-Kang; Hedrich, Rainer

    2007-01-01

    Living organisms are capable of discriminating thermal stimuli from noxious cold to noxious heat. For more than 30 years, it has been known that plant cells respond to cold with a large and transient depolarization. Recently, using transgenic Arabidopsis (Arabidopsis thaliana) expressing the calcium-sensitive protein aequorin, an increase in cytosolic calcium following cold treatment was observed. Applying the patch-clamp technique to Arabidopsis mesophyll protoplasts, we could identify a transient plasma membrane conductance induced by rapid cooling. This cold-induced transient conductance was characterized as an outward rectifying 33 pS nonselective cation channel. The permeability ratio between calcium and cesium was 0.7, pointing to a permeation pore >3.34 Å (ø of cesium). Our experiments thus provide direct evidence for the predicted but not yet measured cold-activated calcium-permeable channel in plants. PMID:17114272

  8. Ultrastructural analyses of somatic embryo initiation, development and polarity establishment from mesophyll cells of Dactylis glomerata

    NASA Technical Reports Server (NTRS)

    Vasilenko, A.; McDaniel, J. K.; Conger, B. V.

    2000-01-01

    Somatic embryos initiate and develop directly from single mesophyll cells in in vitro-cultured leaf segments of orchardgrass (Dactylis glomerata L.). Embryogenic cells establish themselves in the predivision stage by formation of thicker cell walls and dense cytoplasm. Electron microscopy observations for embryos ranging from the pre-cell-division stage to 20-cell proembryos confirm previous light microscopy studies showing a single cell origin. They also confirm that the first division is predominantly periclinal and that this division plane is important in establishing embryo polarity and in determining the embryo axis. If the first division is anticlinal or if divisions are in random planes after the first division, divisions may not continue to produce an embryo. This result may produce an embryogenic cell mass, callus formation, or no structure at all. Grant numbers: NAGW-3141, NAG10-0221.

  9. Kinetics of determination in the differentiation of isolated mesophyll cells of Zinnia elegans to tracheary elements

    NASA Technical Reports Server (NTRS)

    Church, D. L.; Galston, A. W.

    1988-01-01

    Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing 0.5 micromolar alpha-naphthaleneacetic acid and 0.5 micromolar benzyladenine. The cells do not differentiate when cultured in medium in which the concentration of auxin and/or cytokinin has been reduced to 0.005 micromolar. Cells require an initial 24-hour exposure to inductive cytokinin and 56-hour exposure to inductive auxin for differentiation at 72 hours of culture. Freshly isolated Zinnia cells can be maintained in medium having low concentrations of both auxin and cytokinin for only 1 day without significant loss of potential to differentiate upon transfer to inductive medium. Initial culture for up to 2 days in medium having high auxin and low cytokinin, or low auxin and high cytokinin, allows full differentiation on the third day after transfer to inductive medium and potentiates the early differentiation of some cells.

  10. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    PubMed Central

    Guo, Jianjun; Morrell-Falvey, Jennifer L.; Labbé, Jessy L.; Muchero, Wellington; Kalluri, Udaya C.; Tuskan, Gerald A.; Chen, Jin-Gui

    2012-01-01

    Background Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways. PMID:23028673

  11. Highly Efficient Isolation of Populus Mesophyll Protoplasts and Its Application in Transient Expression Assays

    SciTech Connect

    Guo, Jianjun; Morrell-Falvey, Jennifer L; Labbe, Jessy L; Muchero, Wellington; Kalluri, Udaya C; Tuskan, Gerald A; Chen, Jay

    2012-01-01

    Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.

  12. Isolation and identification of thermophilic and mesophylic proteolytic bacteria from shrimp paste "Terasi"

    NASA Astrophysics Data System (ADS)

    Murwani, R.; Supriyadi, Subagio, Trianto, A.; Ambariyanto

    2015-12-01

    Terasi is a traditional product generally made of fermented shrimp. There were many studies regarding lactic acid bacteria of terasi but none regarding proteolitic bacteria. This study was conducted to isolate and identify the thermophilic and mesophylic proteolytic bacteria from terasi. In addition, the effect of different salt concentrations on the growth of the isolated proteolytic bacteria with the greatest proteolytic activity was also studied. Terasi samples were obtained from the Northern coast region of Java island i.e. Jepara, Demak and Batang. The study obtained 34 proteolytic isolates. Four isolates were identified as Sulfidobacillus, three isolates as Vibrio / Alkaligenes / Aeromonas, two isolates as Pseudomonas, 21 isolates as Bacillus, three isolates as Kurthia/ Caryophanon and one isolates as Amphibacillus. The growth of proteolytic bacteria was affected by salt concentration. The largest growth was found at 0 ppm salt concentrations and growth was declined as salt concentration increased. Maximum growth at each salt concentration tested was found at 8 hours incubation.

  13. Post-transcriptional control of cell type-specific gene expression in bundle sheath and mesophyll chloroplasts of Amaranthus hypochondriacus.

    PubMed

    Boinski, J J; Wang, J L; Xu, P; Hotchkiss, T; Berry, J O

    1993-06-01

    Plants that utilize the highly efficient C4 photosynthetic pathway possess two types of specialized leaf cells, the mesophyll and bundle sheath. In mature leaves of amaranth, a dicotyledonous C4 plant, ribulose 1,5-bisphosphate carboxylase (Rubisco) is localized specifically to the chloroplasts of bundle sheath cells, and is not present in the chloroplasts of mesophyll cells. The cell type-specific expression of the chloroplast-encoded Rubisco large subunit (rbcL) gene, and other representative chloroplastic genes, was investigated by using separated bundle sheath and mesophyll chloroplasts prepared from mature amaranth leaves. One-dimensional SDS-polyacrylamide gel electrophoresis revealed several differences in the polypeptide compositions of the two chloroplast types. Western analysis demonstrated that, as in the intact leaves, the Rubisco LSU polypeptide was present only in chloroplast preparations from bundle sheath cells. Pyruvate orthophosphate dikinase (PPdK), a nuclear-encoded chloroplastic enzyme, was found only in the mesophyll chloroplast preparations. rbcL mRNA was present only in the bundle sheath chloroplast preparations, whereas transcripts for the chloroplast-encoded psbA, psaA-B, and rpl2 genes were present in both chloroplast types. Although the rbcL message accumulated only in bundle sheath chloroplasts, run-on transcription analysis indicated that the rbcL gene was transcribed in both bundle sheath and mesophyll chloroplast preparations. Therefore, differential rbcL gene expression in the isolated C4 chloroplasts is regulated, at least in part, at the post-transcriptional level. Possibly this control is mediated by differential processing or stabilization of the rbcL transcript. PMID:8329680

  14. The Role of Plasma Membrane Aquaporins in Regulating the Bundle Sheath-Mesophyll Continuum and Leaf Hydraulics1[C][W][OPEN

    PubMed Central

    Sade, Nir; Shatil-Cohen, Arava; Attia, Ziv; Maurel, Christophe; Boursiac, Yann; Kelly, Gilor; Granot, David; Yaaran, Adi; Lerner, Stephen; Moshelion, Menachem

    2014-01-01

    Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (Kleaf), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate Kleaf. To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (Pf), mesophyll conductance of CO2, photosynthesis, Kleaf, transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf and decreased Kleaf but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced Pf, but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed. PMID:25266632

  15. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages.

    PubMed

    Belkheir, Asma K; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  16. Benzophenone Synthase and Chalcone Synthase Accumulate in the Mesophyll of Hypericum perforatum Leaves at Different Developmental Stages

    PubMed Central

    Belkheir, Asma K.; Gaid, Mariam; Liu, Benye; Hänsch, Robert; Beerhues, Ludger

    2016-01-01

    The active medicinal constituents in Hypericum perforatum, used to treat depression and skin irritation, include flavonoids and xanthones. The carbon skeletons of these compounds are formed by chalcone synthase (CHS) and benzophenone synthase (BPS), respectively. Polyclonal antisera were raised against the polyketide synthases from Hypericum androsaemum and their IgG fractions were isolated. Immunoblotting and immunotitration were used to test the IgGs for crossreactivity and monospecificity in H. perforatum leaf protein extract. Immunofluorescence localization revealed that both CHS and BPS are located in the mesophyll. The maximum fluorescence levels were observed in approx. 0.5 and 1 cm long leaves, respectively. The fluorescence intensity observed for CHS significantly exceeded that for BPS. Using histochemical staining, flavonoids were detected in the mesophyll, indicating that the sites of biosynthesis and accumulation coincide. Our results help understand the biosynthesis and underlying regulation of active H. perforatum constituents. PMID:27446151

  17. CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis.

    PubMed Central

    Li, Hm.; Culligan, K.; Dixon, R. A.; Chory, J.

    1995-01-01

    Light plays a key role in the development and physiology of plants. One of the most profound effects of light on plant development is the derepression of expression of an array of light-responsive genes, including the genes encoding the chlorophyll a/b binding proteins (CAB) of photosystem II. To understand the mechanism by which light signals nuclear gene expression, we developed a genetic selection to identify mutants with reduced CAB transcription. Here, we describe a new Arabidopsis locus, CUE1 (for CAB underexpressed). Mutations at this locus result in defects in expression of several light-regulated genes, specifically in mesophyll but not in bundle-associated or epidermis cells. Reduced accumulation of CAB and other photosynthesis-related mRNAs in the mesophyll was correlated with defects in chloroplast development in these cells, resulting in a reticulate pattern with veins greener than the interveinal regions of leaves. Moreover, chalcone synthase mRNA, although known to be regulated by both phytochrome and a blue light receptor, accumulated normally in the leaf epidermis. Dark basal levels of CAB expression were unaffected in etiolated cue1 seedlings; however, induction of CAB transcription by pulses of red and blue light was reduced, suggesting that CUE1 acts downstream from both phytochrome and blue light photoreceptors. CUE1 appears to play a role in the primary derepression of mesophyll-specific gene expression in response to light, because cue1 mutants are severely deficient at establishing photoautotrophic growth. Based on this characterization, we propose that CUE1 is a cell-specific positive regulator linking light and intrinsic developmental programs in Arabidopsis leaf mesophyll cells. PMID:12242356

  18. Mesophyll conductance plays a central role in leaf functioning of Oleaceae species exposed to contrasting sunlight irradiance.

    PubMed

    Fini, Alessio; Loreto, Francesco; Tattini, Massimiliano; Giordano, Cristiana; Ferrini, Francesco; Brunetti, Cecilia; Centritto, Mauro

    2016-05-01

    The ability to modify mesophyll conductance (gm ) in response to changes in irradiance may be a component of the acclimation of plants to shade-sun transitions, thus influencing species-specific distributions along light-gradients, and the ecological niches for the different species. To test this hypothesis we grew three woody species of the Oleaceae family, the evergreen Phillyrea latifolia (sun-requiring), the deciduous Fraxinus ornus (facultative sun-requiring) and the hemi-deciduous Ligustrum vulgare (shade tolerant) at 30 or 100% sunlight irradiance. We show that neither mesophyll conductance calculated with combined gas exchange and chlorophyll fluorescence techniques (gm ) nor CO2 assimilation significantly varied in F. ornus because of sunlight irradiance. This corroborates previous suggestions that species with high plasticity for light requirements, do not need to undertake extensive reorganization of leaf conductances to CO2 diffusion to adapt to different light environments. On the other hand, gm steeply declined in L. vulgare and increased in P. latifolia exposed to full-sun conditions. In these two species, leaf anatomical traits are in part responsible for light-driven changes in gm , as revealed by the correlation between gm and mesophyll conductance estimated by anatomical parameters (gmA ). Nonetheless, gm was greatly overestimated by gmA when leaf metabolism was impaired because of severe light stress. We show that gm is maximum at the light intensity at which plant species have evolved and we conclude that gm actually plays a key role in the sun and shade adaptation of Mediterranean species. The limits of gmA in predicting mesophyll conductance are also highlighted. PMID:26537749

  19. The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar.

    PubMed

    Lawson, Tracy; Oxborough, Kevin; Morison, James I L; Baker, Neil R

    2003-07-01

    High resolution chlorophyll a fluorescence imaging was used to compare the photosynthetic efficiency of PSII electron transport (estimated by Fq'/Fm') in guard cell chloroplasts and the underlying mesophyll in intact leaves of six different species: Commelina communis, Vicia faba, Amaranthus caudatus, Polypodium vulgare, Nicotiana tabacum, and Tradescantia albifora. While photosynthetic efficiency varied between the species, the efficiencies of guard cells and mesophyll cells were always closely matched. As measurement light intensity was increased, guard cells from the lower leaf surfaces of C. communis and V. faba showed larger reductions in photosynthetic efficiency than those from the upper surfaces. In these two species, guard cell photosynthetic efficiency responded similarly to that of the mesophyll when either light intensity or CO2 concentration during either measurement or growth was changed. In all six species, reducing the O2 concentration from 21% to 2% reduced guard cell photosynthetic efficiency, even for the C4 species A. caudatus, although the mesophyll of the C4 species did not show any O2 modulation of photosynthetic efficiency. This suggests that Rubisco activity is significant in the guard cells of these six species. When C. communis plants were water-stressed, the guard cell photosynthetic efficiency declined in parallel with that of the mesophyll. It was concluded that the photosynthetic efficiency in guard cells is determined by the same factors that determine it in the mesophyll. PMID:12773521

  20. Stomatal responses to flooding of the intercellular air spaces suggest a vapor-phase signal between the mesophyll and the guard cells.

    PubMed

    Sibbernsen, Erik; Mott, Keith A

    2010-07-01

    Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO(2). These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K(+) in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light. PMID:20472750

  1. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing.

    PubMed

    Azoulay-Shemer, Tamar; Bagheri, Andisheh; Wang, Cun; Palomares, Axxell; Stephan, Aaron B; Kunz, Hans-Henning; Schroeder, Julian I

    2016-06-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  2. Phytochrome B in the Mesophyll Delays Flowering by Suppressing FLOWERING LOCUS T Expression in Arabidopsis Vascular Bundles

    PubMed Central

    Endo, Motomu; Nakamura, Satoshi; Araki, Takashi; Mochizuki, Nobuyoshi; Nagatani, Akira

    2005-01-01

    Light is one of the most important environmental factors that determine the timing of a plant's transition from the vegetative to reproductive, or flowering, phase. Not only daylength but also the spectrum of light greatly affect flowering. The shade of nearby vegetation reduces the ratio of red to far-red light and can trigger shade avoidance responses, including stem elongation and the acceleration of flowering. Phytochrome B (phyB) acts as a photoreceptor for this response. Physiological studies have suggested that leaves can perceive and respond to shade. However, little is known about the mechanisms involved in the processing of light signals within leaves. In this study, we used an enhancer-trap system to establish Arabidopsis thaliana transgenic lines that express phyB–green fluorescent protein (GFP) fusion protein in tissue-specific manners. The analysis of these lines demonstrated that phyB-GFP in mesophyll cells affected flowering, whereas phyB-GFP in vascular bundles did not. Furthermore, mesophyll phyB-GFP suppressed the expression of a key flowering regulator, FLOWERING LOCUS T, in the vascular bundles of cotyledons. Hence, a novel intertissue signaling from mesophyll to vascular bundles is revealed as a critical step for the regulation of flowering by phyB. PMID:15965119

  3. The photosynthetic capacity in 35 ferns and fern allies: mesophyll CO2 diffusion as a key trait.

    PubMed

    Tosens, Tiina; Nishida, Keisuke; Gago, Jorge; Coopman, Rafael Eduardo; Cabrera, Hernán Marino; Carriquí, Marc; Laanisto, Lauri; Morales, Loreto; Nadal, Miquel; Rojas, Roke; Talts, Eero; Tomas, Magdalena; Hanba, Yuko; Niinemets, Ülo; Flexas, Jaume

    2016-03-01

    Ferns and fern allies have low photosynthetic rates compared with seed plants. Their photosynthesis is thought to be limited principally by physical CO2 diffusion from the atmosphere to chloroplasts. The aim of this study was to understand the reasons for low photosynthesis in species of ferns and fern allies (Lycopodiopsida and Polypodiopsida). We performed a comprehensive assessment of the foliar gas-exchange and mesophyll structural traits involved in photosynthetic function for 35 species of ferns and fern allies. Additionally, the leaf economics spectrum (the interrelationships between photosynthetic capacity and leaf/frond traits such as leaf dry mass per unit area or nitrogen content) was tested. Low mesophyll conductance to CO2 was the main cause for low photosynthesis in ferns and fern allies, which, in turn, was associated with thick cell walls and reduced chloroplast distribution towards intercellular mesophyll air spaces. Generally, the leaf economics spectrum in ferns follows a trend similar to that in seed plants. Nevertheless, ferns and allies had less nitrogen per unit DW than seed plants (i.e. the same slope but a different intercept) and lower photosynthesis rates per leaf mass area and per unit of nitrogen. PMID:26508678

  4. Starch Biosynthesis in Guard Cells But Not in Mesophyll Cells Is Involved in CO2-Induced Stomatal Closing1[OPEN

    PubMed Central

    Stephan, Aaron B.; Schroeder, Julian I.

    2016-01-01

    Starch metabolism is involved in stomatal movement regulation. However, it remains unknown whether starch-deficient mutants affect CO2-induced stomatal closing and whether starch biosynthesis in guard cells and/or mesophyll cells is rate limiting for high CO2-induced stomatal closing. Stomatal responses to [CO2] shifts and CO2 assimilation rates were compared in Arabidopsis (Arabidopsis thaliana) mutants that were either starch deficient in all plant tissues (ADP-Glc-pyrophosphorylase [ADGase]) or retain starch accumulation in guard cells but are starch deficient in mesophyll cells (plastidial phosphoglucose isomerase [pPGI]). ADGase mutants exhibited impaired CO2-induced stomatal closure, but pPGI mutants did not, showing that starch biosynthesis in guard cells but not mesophyll functions in CO2-induced stomatal closing. Nevertheless, starch-deficient ADGase mutant alleles exhibited partial CO2 responses, pointing toward a starch biosynthesis-independent component of the response that is likely mediated by anion channels. Furthermore, whole-leaf CO2 assimilation rates of both ADGase and pPGI mutants were lower upon shifts to high [CO2], but only ADGase mutants caused impairments in CO2-induced stomatal closing. These genetic analyses determine the roles of starch biosynthesis for high CO2-induced stomatal closing. PMID:27208296

  5. AUXIN RESPONSE FACTOR7 Restores the Expression of Auxin-Responsive Genes in Mutant Arabidopsis Leaf Mesophyll ProtoplastsW⃞

    PubMed Central

    Wang, Shucai; Tiwari, Shiv B.; Hagen, Gretchen; Guilfoyle, Tom J.

    2005-01-01

    AUXIN RESPONSE FACTOR7 (ARF7) is one of five ARF transcriptional activators in Arabidopsis thaliana that is proposed to regulate auxin-responsive expression of genes containing TGTCTC auxin response elements in their promoters. An Arabidopsis mutant (nonphototropic hypocotyl4-1 [nph4-1]) that is a null for ARF7 showed strongly reduced expression of integrated auxin-responsive reporter genes and natural genes that were monitored in Arabidopsis leaf mesophyll protoplasts. Expression of the reporter and natural genes was restored in an auxin-dependent manner when protoplasts were transfected with a 35S:ARF7 effector gene, encoding a full-length ARF7 protein. Transfection of effector genes encoding other ARF activators restored auxin-responsive gene expression to varying degrees, but less than that observed with the ARF7 effector gene. Arabidopsis lines that were null for ARF6, ARF8, or ARF19 were not defective in expression of the reporter and natural auxin response genes assayed in mesophyll protoplasts, suggesting that ARF7 plays a major role in regulating expression of a subset of auxin response genes in leaf mesophyll cells. Auxin-responsive gene expression was induced in wild-type protoplasts and restored in nph4-1 protoplasts only with auxin and not with other hormones, including brassinolide. In the presence of auxin, however, brassinolide modestly enhanced auxin-responsive gene expression. PMID:15923351

  6. How succulent leaves of Aizoaceae avoid mesophyll conductance limitations of photosynthesis and survive drought

    PubMed Central

    Ripley, Brad S.

    2013-01-01

    In several taxa, increasing leaf succulence has been associated with decreasing mesophyll conductance (g M) and an increasing dependence on Crassulacean acid metabolism (CAM). However, in succulent Aizoaceae, the photosynthetic tissues are adjacent to the leaf surfaces with an internal achlorophyllous hydrenchyma. It was hypothesized that this arrangement increases g M, obviating a strong dependence on CAM, while the hydrenchyma stores water and nutrients, both of which would only be sporadically available in highly episodic environments. These predictions were tested with species from the Aizoaceae with a 5-fold variation in leaf succulence. It was shown that g M values, derived from the response of photosynthesis to intercellular CO2 concentration (A:C i), were independent of succulence, and that foliar photosynthate δ13C values were typical of C3, but not CAM photosynthesis. Under water stress, the degree of leaf succulence was positively correlated with an increasing ability to buffer photosynthetic capacity over several hours and to maintain light reaction integrity over several days. This was associated with decreased rates of water loss, rather than tolerance of lower leaf water contents. Additionally, the hydrenchyma contained ~26% of the leaf nitrogen content, possibly providing a nutrient reservoir. Thus the intermittent use of C3 photosynthesis interspersed with periods of no positive carbon assimilation is an alternative strategy to CAM for succulent taxa (Crassulaceae and Aizoaceae) which occur sympatrically in the Cape Floristic Region of South Africa. PMID:24127513

  7. Effects of potassium supply on limitations of photosynthesis by mesophyll diffusion conductance in Carya cathayensis.

    PubMed

    Jin, Song Heng; Huang, Jian Qin; Li, Xue Qin; Zheng, Bing Song; Wu, Jia Sen; Wang, Zheng Jia; Liu, Gen Hua; Chen, Miao

    2011-10-01

    Potassium (K) influences the photosynthesis process in a number of ways; however, the mechanisms underlying the photosynthetic response to differences in K supply are not well understood. Concurrent measurements of gas exchange and chlorophyll fluorescence were made to investigate the effect of K nutrition on photosynthetic efficiency and mesophyll conductance (g(m)) in hickory seedlings (Carya cathayensis Sarg.) in a greenhouse. The results show that leaf K concentrations < 0.7-0.8% appeared to limit the leaf net CO2 assimilation rate (A), and that the relative limitation of photosynthesis due to g(m) and stomatal conductance (g(s)) decreased with increasing supplies of K. However, a sensitivity analysis indicated that A was most sensitive to the maximum carboxylation rate of Rubisco (V(c,max)) and the maximum rate of electron transport (J(max)). These results indicate that the photosynthetic rate is primarily limited by the biochemical processes of photosynthesis (V(c,max) and J(max)), rather than by g(m) and g(s) in K-deficient plants. Additionally, g(m) was closely correlated with g(s) and the leaf dry mass per unit area (M(A)) in hickory seedlings, which indicates that decreased g(m) and g(s) may be a consequence of leaf anatomical adaptation. PMID:21990026

  8. How succulent leaves of Aizoaceae avoid mesophyll conductance limitations of photosynthesis and survive drought.

    PubMed

    Ripley, Brad S; Abraham, Trevor; Klak, Cornelia; Cramer, Michael D

    2013-12-01

    In several taxa, increasing leaf succulence has been associated with decreasing mesophyll conductance (g M) and an increasing dependence on Crassulacean acid metabolism (CAM). However, in succulent Aizoaceae, the photosynthetic tissues are adjacent to the leaf surfaces with an internal achlorophyllous hydrenchyma. It was hypothesized that this arrangement increases g M, obviating a strong dependence on CAM, while the hydrenchyma stores water and nutrients, both of which would only be sporadically available in highly episodic environments. These predictions were tested with species from the Aizoaceae with a 5-fold variation in leaf succulence. It was shown that g M values, derived from the response of photosynthesis to intercellular CO2 concentration (A:C i), were independent of succulence, and that foliar photosynthate δ(13)C values were typical of C3, but not CAM photosynthesis. Under water stress, the degree of leaf succulence was positively correlated with an increasing ability to buffer photosynthetic capacity over several hours and to maintain light reaction integrity over several days. This was associated with decreased rates of water loss, rather than tolerance of lower leaf water contents. Additionally, the hydrenchyma contained ~26% of the leaf nitrogen content, possibly providing a nutrient reservoir. Thus the intermittent use of C3 photosynthesis interspersed with periods of no positive carbon assimilation is an alternative strategy to CAM for succulent taxa (Crassulaceae and Aizoaceae) which occur sympatrically in the Cape Floristic Region of South Africa. PMID:24127513

  9. Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

    PubMed

    Omoto, Eiji; Iwasaki, Yugo; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2016-05-01

    The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress. PMID:26555406

  10. Microtubules in Mesophyll Cells of Nonacclimated and Cold-Acclimated Spinach 1

    PubMed Central

    Bartolo, Michael E.; Carter, John V.

    1991-01-01

    Responses of cortical microtubules in spinach (Spinacia oleracea L. cv Bloomsdale) mesophyll cells to freezing, thawing, supercooling, and dehydration were assessed. Microtubules were visualized using a modified procedure for indirect immunofluorescence microscopy. Leaf sections of nonacclimated and cold-acclimated spinach were slowly frozen to various temperatures, fixed while frozen, and microtubules immunolabelled. Both nonacclimated and cold-acclimated cells exhibited nearly complete microtubule depolymerization after ice formation. After 1 hour thawing at 23°C, microtubules in both nonacclimated and cold-acclimated cells repolymerized. With time, however, microtubules in nonacclimated cells again depolymerized. Since microtubules in cells of leaf tissue frozen slowly are subjected to dehydration as well as subzero temperatures, these stresses were applied separately and their effects on microtubules noted. Supercooling induced microtubule depolymerization in both nonacclimated and cold-acclimated cells, but to a smaller extent than did freezing. Exposing leaf sections to solutions of sorbitol (a cell wall-penetrating osmoticum) or polyethylene glycol 10,000 (a nonpenetrating osmoticum) at room temperature caused microtubule depolymerization. The effects of low temperature and dehydration are roughly additive in producing the observed microtubule responses during freezing. Only small differences in microtubule stability were resolved between nonacclimated and cold-acclimated cells. ImagesFigure 2 PMID:16668366

  11. Comparative proteomic analysis of amaranth mesophyll and bundle sheath chloroplasts and their adaptation to salt stress.

    PubMed

    Joaquín-Ramos, Ahuitzolt; Huerta-Ocampo, José Á; Barrera-Pacheco, Alberto; De León-Rodríguez, Antonio; Baginsky, Sacha; Barba de la Rosa, Ana P

    2014-09-15

    The effect of salt stress was analyzed in chloroplasts of Amaranthus cruentus var. Amaranteca, a plant NAD-malic enzyme (NAD-ME) type. Morphology of chloroplasts from bundle sheath (BSC) and mesophyll (MC) was observed by transmission electron microscopy (TEM). BSC and MC from control plants showed similar morphology, however under stress, changes in BSC were observed. The presence of ribulose bisphosphate carboxylase/oxygenase (RuBisCO) was confirmed by immunohistochemical staining in both types of chloroplasts. Proteomic profiles of thylakoid protein complexes from BSC and MC, and their changes induced by salt stress were analyzed by blue-native polyacrylamide gel electrophoresis followed by SDS-PAGE (2-D BN/SDS-PAGE). Differentially accumulated protein spots were analyzed by LC-MS/MS. Although A. cruentus photosynthetic tissue showed the Kranz anatomy, the thylakoid proteins showed some differences at photosystem structure level. Our results suggest that A. cruentus var. Amaranteca could be better classified as a C3-C4 photosynthetic plant. PMID:25046763

  12. Epidermal Micromorphology and Mesophyll Structure of Populus euphratica Heteromorphic Leaves at Different Development Stages

    PubMed Central

    Liu, Yubing; Li, Xinrong; Chen, Guoxiong; Li, Mengmeng; Liu, Meiling; Liu, Dan

    2015-01-01

    Leaf epidermal micromorphology and mesophyll structure during the development of Populus euphratica heteromorphic leaves, including linear, lanceolate, ovate, dentate ovate, dentate rhombic, dentate broad-ovate and dentate fan-shaped leaves, were studied by using electron and light microscopy. During development of heteromorphic leaves, epidermal appendages (wax crystals and trichomes) and special cells (mucilage cells and crystal idioblasts) increased in all leaf types while chloroplast ultrastructure and stomatal characters show maximum photosynthetic activity in dentate ovate and rhombic leaves. Also, functional analysis by subordinate function values shows that the maximum adaptability to adverse stress was exhibited in the broad type of mature leaves. The 12 heteromorphic leaf types are classified into three major groups by hierarchical cluster analysis: young, developing and mature leaves. Mature leaves can effectively obtain the highest stress resistance by combining the protection of xerophytic anatomy from drought stress, regulation of water uptake in micro-environment by mucilage and crystal idioblasts, and assistant defense of transpiration reduction through leaf epidermal appendages, which improves photosynthetic activity under arid desert conditions. Our data confirms that the main leaf function is differentiated during the developing process of heteromorphic leaves. PMID:26356300

  13. Ribulose-1,5-bisphosphate Carboxylase/Oxygenase content, assimilatory charge, and mesophyll conductance in leaves

    PubMed

    Eichelmann; Laisk

    1999-01-01

    The content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) (Et; EC 4.1.1.39) measured in different-aged leaves of sunflower (Helianthus annuus) and other plants grown under different light intensities, varied from 2 to 75 &mgr;mol active sites m-2. Mesophyll conductance (&mgr;) was measured under 1.5% O2, as well as postillumination CO2 uptake (assimilatory charge, a gas-exchange measure of the ribulose-1,5-bisphosphate pool). The dependence of &mgr; on Et saturated at Et = 30 &mgr;mol active sites m-2 and &mgr; = 11 mm s-1 in high-light-grown leaves. In low-light-grown leaves the dependence tended toward saturation at similar Et but reached a &mgr; of only 6 to 8 mm s-1. &mgr; was proportional to the assimilatory charge, with the proportionality constant (specific carboxylation efficiency) between 0.04 and 0.075 &mgr;M-1 s-1. Our data show that the saturation of the relationship between Et and &mgr; is caused by three limiting components: (a) the physical diffusion resistance (a minor limitation), (b) less than full activation of Rubisco (related to Rubisco activase and the slower diffusibility of Rubisco at high protein concentrations in the stroma), and (c) chloroplast metabolites, especially 3-phosphoglyceric acid and free inorganic phosphate, which control the reaction kinetics of ribulose-1,5-bisphosphate carboxylation by competitive binding to active sites. PMID:9880359

  14. Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

    PubMed Central

    Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

    2005-01-01

    Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

  15. Effects of Waterlogging on Leaf Mesophyll Cell Ultrastructure and Photosynthetic Characteristics of Summer Maize.

    PubMed

    Ren, Baizhao; Zhang, Jiwang; Dong, Shuting; Liu, Peng; Zhao, Bin

    2016-01-01

    A field experiment was performed to study the effects of waterlogging on the leaf mesophyll cell ultrastructure, chlorophyll content, gas exchange parameters, chlorophyll fluorescence, and malondialdehyde (MDA) content of summer maize (Zea mays L.) hybrids Denghai605 (DH605) and Zhengdan958 (ZD958). The waterlogging treatments were implemented for different durations (3 and 6 days) at the third leaf stage (V3), the sixth leaf stage (V6), and the 10th day after the tasseling stage (10VT). Leaf area index (LAI), chlorophyll content, photosynthetic rate (Pn), and actual photochemical efficiency (ΦPSII) were reduced after waterlogging, indicating that waterlogging significantly decreased photosynthetic capacity. The chloroplast shapes changed from long and oval to elliptical or circular after waterlogging. In addition, the internal structures of chloroplasts were degenerated after waterlogging. After waterlogging for 6 d at V3, the number of grana and grana lamellae of the third expanded leaf in DH605 were decreased by 26.83% and 55.95%, respectively, compared to the control (CK). Those in ZD958 were reduced by 30.08% and 31.94%, respectively. Waterlogging increased MDA content in both hybrids, suggesting an impact of waterlogging on membrane integrity and thus membrane deterioration. Waterlogging also damaged the biological membrane structure and mitochondria. Our results indicated that the physiological reactions to waterlogging were closely related to lower LAI, chlorophyll content, and Pn and to the destruction of chloroplast ultrastructure. These negative effects resulted in the decrease of grain yield in response to waterlogging. Summer maize was the most susceptible to damage when waterlogging occurred at V3, followed by V6 and 10VT, with damage increasing in the wake of waterlogging duration increasing. PMID:27583803

  16. Mesophyll Chloroplast Investment in C3, C4 and C2 Species of the Genus Flaveria.

    PubMed

    Stata, Matt; Sage, Tammy L; Hoffmann, Natalie; Covshoff, Sarah; Ka-Shu Wong, Gane; Sage, Rowan F

    2016-05-01

    The mesophyll (M) cells of C4 plants contain fewer chloroplasts than observed in related C3 plants; however, it is uncertain where along the evolutionary transition from C3 to C4 that the reduction in M chloroplast number occurs. Using 18 species in the genus Flaveria, which contains C3, C4 and a range of C3-C4 intermediate species, we examined changes in chloroplast number and size per M cell, and positioning of chloroplasts relative to the M cell periphery. Chloroplast number and coverage of the M cell periphery declined in proportion to increasing strength of C4 metabolism in Flaveria, while chloroplast size increased with increasing C4 cycle strength. These changes increase cytosolic exposure to the cell periphery which could enhance diffusion of inorganic carbon to phosphenolpyruvate carboxylase (PEPC), a cytosolic enzyme. Analysis of the transcriptome from juvenile leaves of nine Flaveria species showed that the transcript abundance of four genes involved in plastid biogenesis-FtsZ1, FtsZ2, DRP5B and PARC6-was negatively correlated with variation in C4 cycle strength and positively correlated with M chloroplast number per planar cell area. Chloroplast size was negatively correlated with abundance of FtsZ1, FtsZ2 and PARC6 transcripts. These results indicate that natural selection targeted the proteins of the contractile ring assembly to effect the reduction in chloroplast numbers in the M cells of C4 Flaveria species. If so, efforts to engineer the C4 pathway into C3 plants might evaluate whether inducing transcriptome changes similar to those observed in Flaveria could reduce M chloroplast numbers, and thus introduce a trait that appears essential for efficient C4 function. PMID:26985020

  17. Temperature response of carbon isotope discrimination and mesophyll conductance in tobacco.

    PubMed

    Evans, John R; von Caemmerer, Susanne

    2013-04-01

    The partial pressure of CO2 at the sites of carboxylation within chloroplasts depends on the conductance to CO2 diffusion from intercellular airspace to the sites of carboxylation, termed mesophyll conductance (gm ). We investigated the temperature response of gm in tobacco (Nicotiana tabacum) by combining gas exchange in high light, ambient CO2 in either 2 or 21% O2 with carbon isotope measurements using tuneable diode laser spectroscopy. The gm increased linearly with temperature in 2 or 21% O2 . In 21% O2 , isotope discrimination associated with gm decreased from 5.0 ± 0.2 to 1.8 ± 0.2‰ as temperature increased from 15 to 40 °C, but the photorespiratory contribution to the isotopic signal is significant. While the fractionation factor for photorespiration (f = 16.2 ± 0.7‰) was independent of temperature between 20 and 35 °C, discrimination associated with photorespiration increased from 1.1 ± 0.01 to 2.7 ± 0.02‰ from 15 to 40 °C. Other mitochondrial respiration contributed around 0.2 ± 0.03‰. The drawdown in CO2 partial pressure from ambient air to intercellular airspaces was nearly independent of leaf temperature. By contrast, the increase in gm with increasing leaf temperature resulted in the drawdown in CO2 partial pressure between intercellular airspaces and the sites of carboxylation decreasing substantially at high temperature. PMID:22882584

  18. Genomic Analysis of Melioribacter roseus, Facultatively Anaerobic Organotrophic Bacterium Representing a Novel Deep Lineage within Bacteriodetes/Chlorobi Group

    PubMed Central

    Kadnikov, Vitaly V.; Mardanov, Andrey V.; Podosokorskaya, Olga A.; Gavrilov, Sergey N.; Kublanov, Ilya V.; Beletsky, Alexey V.; Bonch-Osmolovskaya, Elizaveta A.; Ravin, Nikolai V.

    2013-01-01

    Melioribacter roseus is a moderately thermophilic facultatively anaerobic organotrophic bacterium representing a novel deep branch within Bacteriodetes/Chlorobi group. To better understand the metabolic capabilities and possible ecological functions of M. roseus and get insights into the evolutionary history of this bacterial lineage, we sequenced the genome of the type strain P3M-2T. A total of 2838 open reading frames was predicted from its 3.30 Mb genome. The whole proteome analysis supported phylum-level classification of M. roseus since most of the predicted proteins had closest matches in Bacteriodetes, Proteobacteria, Chlorobi, Firmicutes and deeply-branching bacterium Caldithrix abyssi, rather than in one particular phylum. Consistent with the ability of the bacterium to grow on complex carbohydrates, the genome analysis revealed more than one hundred glycoside hydrolases, glycoside transferases, polysaccharide lyases and carbohydrate esterases. The reconstructed central metabolism revealed pathways enabling the fermentation of complex organic substrates, as well as their complete oxidation through aerobic and anaerobic respiration. Genes encoding the photosynthetic and nitrogen-fixation machinery of green sulfur bacteria, as well as key enzymes of autotrophic carbon fixation pathways, were not identified. The M. roseus genome supports its affiliation to a novel phylum Ignavibateriae, representing the first step on the evolutionary pathway from heterotrophic ancestors of Bacteriodetes/Chlorobi group towards anaerobic photoautotrophic Chlorobi. PMID:23301019

  19. Phosphate translocator of mesophyll and bundle sheath chloroplasts of a C sub 4 plant, Panicum miliaceum L

    SciTech Connect

    Ohnishi, Junichi; Fluegge, U.I.; Heldt, H.W. )

    1989-12-01

    The phosphate translocator was identified in the envelope membranes of both mesophyll and bundle sheath chloroplasts of Panicum miliaceum L. by labeling with (1,2-{sup 3}H)1,2-(2,2{prime}-disulfo-4,4{prime}-diisothiocyano)diphenylethane (({sup 3}H)H{sub 2}DIDS) and by using SDS-PAGE. Assay of {sup 32}Pi uptake by the chloroplasts showed that the phosphate translocators of both types of chloroplasts have a higher affinity for phosphoenolpyruvate than the C{sub 3} counterpart and can be regarded as C{sub 4} types.

  20. Comparative Proteomic Insights into the Lactate Responses of Halophilic Salinicoccus roseus W12

    PubMed Central

    Wang, Hongyan; Wang, Limin; Yang, Han; Cai, Yumeng; Sun, Lifan; Xue, Yanfen; Yu, Bo; Ma, Yanhe

    2015-01-01

    Extremophiles use adaptive mechanisms to survive in extreme environments, which is of great importance for several biotechnological applications. A halophilic strain, Salinicoccus roseus W12, was isolated from salt lake in Inner Mongolia, China in this study. The ability of the strain to survive under high sodium conditions (including 20% sodium lactate or 25% sodium chloride, [w/v]) made it an ideal host to screen for key factors related to sodium lactate resistance. The proteomic responses to lactate were studied using W12 cells cultivated with or without lactate stress. A total of 1,656 protein spots in sodium lactate-treated culture and 1,843 spots in NaCl-treated culture were detected by 2-dimensional gel electrophoresis, and 32 of 120 significantly altered protein spots (fold change > 2, p < 0.05) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Among 21 successfully identified spots, 19 proteins were upregulated and 2 were downregulated. The identified proteins are mainly involved in metabolism, cellular processes and signaling, and information storage and processing. Transcription studies confirmed that most of the encoding genes were upregulated after the cells were exposed to lactate in 10 min. Cross-protecting and energy metabolism-related proteins played an important role in lactate tolerance for S. roseus W12. PMID:26358621

  1. Comparative Proteomic Insights into the Lactate Responses of Halophilic Salinicoccus roseus W12.

    PubMed

    Wang, Hongyan; Wang, Limin; Yang, Han; Cai, Yumeng; Sun, Lifan; Xue, Yanfen; Yu, Bo; Ma, Yanhe

    2015-01-01

    Extremophiles use adaptive mechanisms to survive in extreme environments, which is of great importance for several biotechnological applications. A halophilic strain, Salinicoccus roseus W12, was isolated from salt lake in Inner Mongolia, China in this study. The ability of the strain to survive under high sodium conditions (including 20% sodium lactate or 25% sodium chloride, [w/v]) made it an ideal host to screen for key factors related to sodium lactate resistance. The proteomic responses to lactate were studied using W12 cells cultivated with or without lactate stress. A total of 1,656 protein spots in sodium lactate-treated culture and 1,843 spots in NaCl-treated culture were detected by 2-dimensional gel electrophoresis, and 32 of 120 significantly altered protein spots (fold change > 2, p < 0.05) were identified by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. Among 21 successfully identified spots, 19 proteins were upregulated and 2 were downregulated. The identified proteins are mainly involved in metabolism, cellular processes and signaling, and information storage and processing. Transcription studies confirmed that most of the encoding genes were upregulated after the cells were exposed to lactate in 10 min. Cross-protecting and energy metabolism-related proteins played an important role in lactate tolerance for S. roseus W12. PMID:26358621

  2. Ectopic Expression of Rubisco Subunits in Maize Mesophyll Cells Does Not Overcome Barriers to Cell Type-Specific Accumulation1[C][W][OA

    PubMed Central

    Wostrikoff, Katia; Clark, Aimee; Sato, Shirley; Clemente, Tom; Stern, David

    2012-01-01

    In maize (Zea mays), Rubisco accumulates in bundle sheath but not mesophyll chloroplasts, but the mechanisms that underlie cell type-specific expression are poorly understood. To explore the coordinated expression of the chloroplast rbcL gene, which encodes the Rubisco large subunit (LS), and the two nuclear RBCS genes, which encode the small subunit (SS), RNA interference was used to reduce RBCS expression. This resulted in Rubisco deficiency and was correlated with translational repression of rbcL. Thus, as in C3 plants, LS synthesis depends on the presence of its assembly partner SS. To test the hypothesis that the previously documented transcriptional repression of RBCS in mesophyll cells is responsible for repressing LS synthesis in mesophyll chloroplasts, a ubiquitin promoter-driven RBCS gene was expressed in both bundle sheath and mesophyll cells. This did not lead to Rubisco accumulation in the mesophyll, suggesting that LS synthesis is impeded even in the presence of ectopic SS expression. To attempt to bypass this putative mechanism, a ubiquitin promoter-driven nuclear version of the rbcL gene was created, encoding an epitope-tagged LS that was expressed in the presence or absence of the Ubi-RBCS construct. Both transgenes were robustly expressed, and the tagged LS was readily incorporated into Rubisco complexes. However, neither immunolocalization nor biochemical approaches revealed significant accumulation of Rubisco in mesophyll cells, suggesting a continuing cell type-specific impairment of its assembly or stability. We conclude that additional cell type-specific factors limit Rubisco expression to bundle sheath chloroplasts. PMID:22744982

  3. Changes in Spectral Properties, Chlorophyll Content and Internal Mesophyll Structure of Senescing Populus balsamifera and Populus tremuloides Leaves

    PubMed Central

    Castro, Karen L.; Sanchez-Azofeifa, G. Arturo

    2008-01-01

    In this paper we compare leaf traits and spectral reflectance for sunlit and shafded leaves of Populus tremuloides and Populus balsamifera during autumn senescence using information derived from an Analytical Spectral Devise (ASD) Full Range spectrometer. The modified simple ratio (mSR705) and modified normalized difference index (mND705) were effective in describing changes in chlorophyll content over this period. Highly significant (P<0.01) correlation coefficients were found between the chlorophyll indices (mSR705, mND705)) and chlorophyll a, b, total chlorophyll and chlorophyll a/b. Changes in mesophyll structure were better described by the plant senescence reflectance index (PSRI) than by near-infrared wavebands. Overall, P. balsamifera exhibited lower total chlorophyll and earlier senescence than P. tremuloides. Leaves of P. balsamifera were also thicker, had a higher proportion of intercellular space in the spongy mesophyll, and higher reflectance at 800 nm. Further research, using larger sample sizes over a broader range of sites will extend our understanding of the spectral and temporal dynamics of senescence in P. tremuloides and P. balsamifera and will be particularly useful if species differences are detectable at the crown level using remotely sensed imagery.

  4. Photosynthetic Response of an Alpine Plant, Rhododendron delavayi Franch, to Water Stress and Recovery: The Role of Mesophyll Conductance

    PubMed Central

    Cai, Yanfei; Wang, Jihua; Li, Shifeng; Zhang, Lu; Peng, Lvchun; Xie, Weijia; Liu, Feihu

    2015-01-01

    Rhododendron delavayi Franch is an evergreen shrub or small tree with large scarlet flowers that makes it highly attractive as an ornamental species. The species is native to southwest China and southeast Asia, especially the Himalayan region, showing good adaptability, and tolerance to drought. To understand the water stress coping mechanisms of R. delavayi, we analyzed the plant's photosynthetic performance during water stress and recovery. In particular, we looked at the regulation of stomatal (gs) and mesophyll conductance (gm), and maximum rate of carboxylation (Vcmax). After 4 days of water stress treatment, the net CO2 assimilation rate (AN) declined slightly while gs and gm were not affected and stomatal limitation (SL) was therefore negligible. At this stage mesophyll conductance limitation (MCL) and biochemical limitation (BL) constituted the main limitation factors. After 8 days of water stress treatment, AN, gs, and gm had decreased notably. At this stage SL increased markedly and MCL even more so, while BL remained relatively constant. After re-watering, the recovery of AN, gs, and gm was rapid, although remaining below the levels of the control plants, while Vcmax fully regained control levels after 3 days of re-watering. MCL remained the main limitation factor irrespective of the degree of photosynthetic recovery. In conclusion, in our experiment MCL was the main photosynthetic limitation factor of R. delavayi under water stress and during the recovery phase, with the regulation of gm probably being the result of interactions between the environment and leaf anatomical features. PMID:26697043

  5. The Endogenous Sulfated Pentapeptide Phytosulfokine-α Stimulates Tracheary Element Differentiation of Isolated Mesophyll Cells of Zinnia1

    PubMed Central

    Matsubayashi, Yoshikatsu; Takagi, Leiko; Omura, Naomi; Morita, Akiko; Sakagami, Youji

    1999-01-01

    Dispersed zinnia (Zinnia elegans) mesophyll cells cannot differentiate into tracheary elements (TEs) at low cell density conditions even if auxin and cytokinin are present in the medium, indicating the involvement of intercellular interactions during the initiation and/or subsequent progresses in TE differentiation. When zinnia cells were incubated at a low density (2.5 × 104 cells mL−1) in TE-inductive medium in the presence of various concentrations of phytosulfokine (PSK)-α, which was originally identified as an intercellular signal peptide involved in cell proliferation, TE differentiation was strongly stimulated in a dose-dependent fashion; more than 35% of the living cells differentiated into TEs by 5 d of culture in the presence of 10 nm PSK-α. Enzyme-linked immunosorbent assay and mass spectroscopy confirmed that cultured zinnia cells produce nanomolar levels of PSKs under inductive conditions. These results suggest that PSK-α is a factor responsible for TE differentiation of zinnia mesophyll cells. PMID:10444087

  6. DIFFERENCES IN WHOLE CELL AND SINGLE CHANNEL ION CURRENTS ACROSS THE PLASMA MEMBRANE OF MESOPHYLL CELLS FROM TWO CLOSELY RELATED THLASPI SPECIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The patch clamp technique was used to study the physiology of ion transport in mesophyll cells from Thlaspi caerulescens, a heavy metal (Zn/Cd) hyperaccumulator species that can tolerate and accumulate very high levels of heavy metals in their leaf cells, and Thlaspi arvense, a related non-accumulat...

  7. A new mechanism for the regulation of stomatal aperture size in intact leaves: Accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba

    SciTech Connect

    Lu, Ping; Outlaw, W.H. Jr.; Smith, B.G.; Freed, G.A.

    1997-05-01

    At various times after pulse-labeling broad bean (Vicia faba L.) leaflets with {sup 14}CO{sub 2}, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents, whereas those from rinsed peels contained only symplastic contents. Sucrose (Suc)-specific radioactivity peaked (111 GBq mol{sup -1}) in palisade cells at 20 min. In contrast, the {sup 14}C content and Suc-specific radioactivity were very low in guard cells for 20 min, implying little CO, incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum Suc-specific radioactivity (204 GBq mol{sup -1}) and a high Suc influx rate (0.05 pmol stoma{sup -1} min{sup -1}). These and other comparisons implied the presence of (a) multiple Suc pools in mesophyll cells, M a localized mesophyll-apoplast region that exchanges with phloem and stomata, and mesophyll-derived Suc in guard-cell walls sufficient to diminish stomatal opening by approximately 3 pm. Factors expected to enhance Suc accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and N high apoplastic Suc concentration, which is elevated when mesophyll Suc efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal aperture size by this previously unrecognized mechanism. 50 refs., 9 figs.

  8. Compartmentation of malic acid in mesophyll cells of Kalanchoee daigremontiana: indications of a intracellular cytosolic vesicle transport mechanism

    SciTech Connect

    Balsamo, R.A.; Uribe, E.G.

    1987-04-01

    Leaf tissue was harvested over a 24hr period at one to three hour intervals. The malic acid levels in the tissue were assayed spectrophotometrically and the percent cell volume occupied by cytosolic vesicles in replicate samples was determined. The total volume of the cytosolic vesicles fluctuated throughout the photoperiod concommitantly with malic acid concentrations present in the tissue. An intact leaf tissue section (10.2cm/sup 2/) was radiolabeled with /sup 14/CO/sub 2/ seven hours into the dark period for thirty minutes. Two dimensional thin layer chromatography and electrophoresis of the tissue determined that 96% of the label was incorporated into malic acid. A freeze substitution procedure was initiated followed by microautoradiography (Fisher 1971) which allowed for the tracing of intracellular malic acid migration and compartmentation within the mesophyll cells. The results and interpretation of this experiment will be presented.

  9. Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study

    PubMed Central

    Łabuz, Justyna; Samardakiewicz, Sławomir; Hermanowicz, Paweł; Wyroba, Elżbieta; Pilarska, Maria; Gabryś, Halina

    2016-01-01

    Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca2+ after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca2+, and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca2+ was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca2+ homeostasis in mesophyll cells. PMID:26957564

  10. Anatomical basis of variation in mesophyll resistance in eastern Australian sclerophylls: news of a long and winding path

    PubMed Central

    Tosens, Tiina

    2012-01-01

    In sclerophylls, photosynthesis is particularly strongly limited by mesophyll diffusion resistance from substomatal cavities to chloroplasts (r m), but the controls on diffusion limits by integral leaf variables such as leaf thickness, density, and dry mass per unit area and by the individual steps along the diffusion pathway are imperfectly understood. To gain insight into the determinants of r m in leaves with varying structure, the full CO2 physical diffusion pathway was analysed in 32 Australian species sampled from sites contrasting in soil nutrients and rainfall, and having leaf structures from mesophytic to strongly sclerophyllous. r m was estimated based on combined measurements of gas exchange and chlorophyll fluorescence. In addition, r m was modelled on the basis of detailed anatomical measurements to separate the importance of different serial resistances affecting CO2 diffusion into chloroplasts. The strongest sources of variation in r m were S c/S, the exposed surface area of chloroplasts per unit leaf area, and mesophyll cell wall thickness, t cw. The strong correlation of r m with t cw could not be explained by cell wall thickness alone, and most likely arose from a further effect of cell wall porosity. The CO2 drawdown from intercellular spaces to chloroplasts was positively correlated with t cw, suggesting enhanced diffusional limitations in leaves with thicker cell walls. Leaf thickness and density were poorly correlated with S c/S, indicating that widely varying combinations of leaf anatomical traits occur at given values of leaf integrated traits, and suggesting that detailed anatomical studies are needed to predict r m for any given species. PMID:22888123

  11. Threshold response of mesophyll CO2 conductance to leaf hydraulics in highly transpiring hybrid poplar clones exposed to soil drying

    PubMed Central

    Pepin, Steeve

    2014-01-01

    Mesophyll conductance (g m) has been shown to impose significant limitations to net CO2 assimilation (A) in various species during water stress. Net CO2 assimilation is also limited by stomatal conductance to water (g sw), both having been shown to co-vary with leaf hydraulic conductance (K leaf). Lately, several studies have suggested a close functional link between K leaf, g sw, and g m. However, such relationships could only be circumstantial since a recent study has shown that the response of g m to drought could merely be an artefactual consequence of a reduced intercellular CO2 mole fraction (C i). Experiments were conducted on 8-week-old hybrid poplar cuttings to determine the relationship between K leaf, g sw, and g m in clones of contrasting drought tolerance. It was hypothesized that changes in g sw and K leaf in response to drought would not impact on g m over most of its range. The results show that K leaf decreased in concert with g sw as drought proceeded, whereas g m measured at a normalized C i remained relatively constant up to a g sw threshold of ~0.15mol m–2 s–1. This delayed g m response prevented a substantial decline in A at the early stage of the drought, thereby enhancing water use efficiency. Reducing the stomatal limitation of droughted plants by diminishing the ambient CO2 concentration of the air did not modify g m or K leaf. The relationship between gas exchange and leaf hydraulics was similar in both drought-tolerant and drought-sensitive clones despite their contrasting vulnerability to stem cavitation and stomatal response to soil drying. The results support the hypothesis of a partial hydraulic isolation of the mesophyll from the main transpiration pathway. PMID:24368507

  12. The paraveinal mesophyll of soybean leaves in relation to assimilate transfer and compartmentation : II. Structural, metabolic and compartmental changes during reproductive growth.

    PubMed

    Franceschi, V R; Giaquinta, R T

    1983-04-01

    Nitrogen and carbohydrate assimilates were temporally and spatially compartmented among various cell types in soybean (Glycine max L., Merr.) leaves during seed filling. The paraveinal mesophyll (PVM), a unique cell layer found in soybean, was demonstrated to function in the synthesis, compartmentation and remobilization of nitrogen reserves prior to and during the seed-filling stages. At anthesis, the PVM vacuoles contain substantial protein which completely disappears by two weeks into the seed filling. Distinct changes in the PVM cytoplasm, tonoplast and organelles were correlated with the presence or absence of the vacuolar material. Microautoradiography following the accumulation of several radiolabeled sugars and amino acids demonstrated the glycoprotein nature of the vacuolar material. Incorporation of methionine, leucine, glucose, and glucosamine resulted in heavy labelling of the PVM vacuole, in contrast to galactose, proline, and mannose which resulted in a much reduced labelling pattern. In addition, starch is unequally compartmented and degraded among the various leaf cells during seed filling. At the end of the photoperiod at the flowering stage, the highest starch accumulation was in the second palisade layer followed by the spongy mesophyll and the first (uppermost) palisade layer. Starch in the first palisade layer was completely degraded during the dark whereas the starch in the second palisade and spongy mesophyll was not remobilized to any appreciable extent. By mid-podfilling (approximately five weeks postanthesis) starch was absent in the first palisade layer at the end of the photoperiod while the second palisade and spongy mesophyll layers contained substantial starch. Starch was remobilized from these latter cells during the remainder of seed filling when current photosynthetic production is low. Structural changes associated with cell senescence first appear in the upper palisade layer and then progress (excluding the PVM) to the second

  13. A novel method of measuring leaf epidermis and mesophyll stiffness shows the ubiquitous nature of the sandwich structure of leaf laminas in broad-leaved angiosperm species.

    PubMed

    Onoda, Yusuke; Schieving, Feike; Anten, Niels P R

    2015-05-01

    Plant leaves commonly exhibit a thin, flat structure that facilitates a high light interception per unit mass, but may increase risks of mechanical failure when subjected to gravity, wind and herbivory as well as other stresses. Leaf laminas are composed of thin epidermis layers and thicker intervening mesophyll layers, which resemble a composite material, i.e. sandwich structure, used in engineering constructions (e.g. airplane wings) where high bending stiffness with minimum weight is important. Yet, to what extent leaf laminas are mechanically designed and behave as a sandwich structure remains unclear. To resolve this issue, we developed and applied a novel method to estimate stiffness of epidermis- and mesophyll layers without separating the layers. Across a phylogenetically diverse range of 36 angiosperm species, the estimated Young's moduli (a measure of stiffness) of mesophyll layers were much lower than those of the epidermis layers, indicating that leaf laminas behaved similarly to efficient sandwich structures. The stiffness of epidermis layers was higher in evergreen species than in deciduous species, and strongly associated with cuticle thickness. The ubiquitous nature of sandwich structures in leaves across studied species suggests that the sandwich structure has evolutionary advantages as it enables leaves to be simultaneously thin and flat, efficiently capturing light and maintaining mechanical stability under various stresses. PMID:25675956

  14. A novel method of measuring leaf epidermis and mesophyll stiffness shows the ubiquitous nature of the sandwich structure of leaf laminas in broad-leaved angiosperm species

    PubMed Central

    Onoda, Yusuke; Schieving, Feike; Anten, Niels P. R.

    2015-01-01

    Plant leaves commonly exhibit a thin, flat structure that facilitates a high light interception per unit mass, but may increase risks of mechanical failure when subjected to gravity, wind and herbivory as well as other stresses. Leaf laminas are composed of thin epidermis layers and thicker intervening mesophyll layers, which resemble a composite material, i.e. sandwich structure, used in engineering constructions (e.g. airplane wings) where high bending stiffness with minimum weight is important. Yet, to what extent leaf laminas are mechanically designed and behave as a sandwich structure remains unclear. To resolve this issue, we developed and applied a novel method to estimate stiffness of epidermis- and mesophyll layers without separating the layers. Across a phylogenetically diverse range of 36 angiosperm species, the estimated Young’s moduli (a measure of stiffness) of mesophyll layers were much lower than those of the epidermis layers, indicating that leaf laminas behaved similarly to efficient sandwich structures. The stiffness of epidermis layers was higher in evergreen species than in deciduous species, and strongly associated with cuticle thickness. The ubiquitous nature of sandwich structures in leaves across studied species suggests that the sandwich structure has evolutionary advantages as it enables leaves to be simultaneously thin and flat, efficiently capturing light and maintaining mechanical stability under various stresses. PMID:25675956

  15. Vacuolar Localization of Proteases and Degradation of Chloroplasts in Mesophyll Protoplasts from Senescing Primary Wheat Leaves 1

    PubMed Central

    Wittenbach, Vernon A.; Lin, Willy; Hebert, Richard R.

    1982-01-01

    Mesophyll protoplasts isolated from primary leaves of wheat seedlings were used to follow the localization of proteases and the breakdown of chloroplasts during dark-induced senescence. Protoplasts were readily obtained from leaf tissue, even after 80% of the chlorophyll and protein had been lost. Intact chloroplasts and vacuoles could be isolated from the protoplasts at all stages of senescence. All the proteolytic activity associated with the degradation of ribulose bisphosphate carboxylase in the protoplasts could be accounted for by that localized within the vacuole. Moreover, this localization was retained late into senescence. Protoplasts isolated during leaf senescence first showed a decline in photosynthesis, then a decline in ribulose bisphosphate carboxylase activity, followed by a decline in chloroplast number. There was a close correlation between the decline in chloroplast number and the loss of chlorophyll and soluble protein per protoplast, suggesting a sequential degradation of chloroplasts during senescence. Ultrastructural studies indicated a movement of chloroplasts in toward the center of the protoplasts during senescence. Thus, within senescing protoplasts, chloroplasts appeared either to move into invaginations of the vacuole or to be taken up into the vacuole. Images PMID:16662193

  16. 4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements

    NASA Technical Reports Server (NTRS)

    Church, D. L.; Galston, A. W.

    1988-01-01

    When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.

  17. Asymmetrical effects of mesophyll conductance on fundamental photosynthetic parameters and their relationships estimated from leaf gas exchange measurements.

    PubMed

    Sun, Ying; Gu, Lianhong; Dickinson, Robert E; Pallardy, Stephen G; Baker, John; Cao, Yonghui; DaMatta, Fábio Murilo; Dong, Xuejun; Ellsworth, David; Van Goethem, Davina; Jensen, Anna M; Law, Beverly E; Loos, Rodolfo; Martins, Samuel C Vitor; Norby, Richard J; Warren, Jeffrey; Weston, David; Winter, Klaus

    2014-04-01

    Worldwide measurements of nearly 130 C3 species covering all major plant functional types are analysed in conjunction with model simulations to determine the effects of mesophyll conductance (g(m)) on photosynthetic parameters and their relationships estimated from A/Ci curves. We find that an assumption of infinite g(m) results in up to 75% underestimation for maximum carboxylation rate V(cmax), 60% for maximum electron transport rate J(max), and 40% for triose phosphate utilization rate T(u) . V(cmax) is most sensitive, J(max) is less sensitive, and T(u) has the least sensitivity to the variation of g(m). Because of this asymmetrical effect of g(m), the ratios of J(max) to V(cmax), T(u) to V(cmax) and T(u) to J(max) are all overestimated. An infinite g(m) assumption also limits the freedom of variation of estimated parameters and artificially constrains parameter relationships to stronger shapes. These findings suggest the importance of quantifying g(m) for understanding in situ photosynthetic machinery functioning. We show that a nonzero resistance to CO2 movement in chloroplasts has small effects on estimated parameters. A non-linear function with gm as input is developed to convert the parameters estimated under an assumption of infinite gm to proper values. This function will facilitate gm representation in global carbon cycle models. PMID:24117476

  18. Progress of lignification mediated by intercellular transportation of monolignols during tracheary element differentiation of isolated Zinnia mesophyll cells.

    PubMed

    Hosokawa, M; Suzuki, S; Umezawa, T; Sato, Y

    2001-09-01

    Tracheary element (TE) differentiation is a typical example of programmed cell death (PCD) in higher plants, and maturation of TEs is completed by degradation of all cell contents. However, lignification of TEs progresses even after PCD. We investigated how and whence monolignols are supplied to TEs which have undergone PCD during differentiation of isolated Zinnia mesophyll cells into TEs. Higher densities of cell culture induced greater lignification of TEs. Whereas the continuous exchanging of culture medium suppressed lignification of TEs, further addition of coniferyl alcohol into the exchanging medium reduced the suppression of lignification. Analysis of the culture medium by HPLC and GC-MS showed that coniferyl alcohol, coniferaldehyde, and sinapyl alcohol accumulated in TE inductive culture. The concentration of coniferyl alcohol peaked at the beginning of secondary wall thickening, decreased rapidly during secondary wall thickening, then increased again. These results indicated that lignification on TEs progresses by supply of monolignols from not only TEs themselves but also surrounding xylem parenchyma-like cells through medium in vitro. PMID:11577190

  19. Proton/l-Glutamate Symport and the Regulation of Intracellular pH in Isolated Mesophyll Cells 1

    PubMed Central

    Snedden, Wayne A.; Chung, Induk; Pauls, Randy H.; Bown, Alan W.

    1992-01-01

    Addition of l-[U-14C]glutamate to a suspension of mechanically isolated asparagus (Asparagus sprengeri Regel) mesophyll cells results in (a) alkalinization of the medium, (b) uptake of l-[U-14C]glutamate, and (c) efflux of [14C]4-aminobutyrate, a product of glutamate decarboxylation. All three phenomena were eliminated by treatment with 1 millimolar aminooxyacetate. In vitro glutamate decarboxylase (GAD) assays showed that (a) 2 millimolar aminooxyacetate eliminated enzyme activity, (b) activity was pyridoxal phosphate-dependent, and (c) activity exhibited a sharp pH optimum at 6.0 that decreased to 20% of optimal activity at pH 5.0 and 7.0. Addition of 1.5 millimolar sodium butyrate or sodium acetate to cell suspensions caused immediate alkalinization of the medium followed by a resumption of acidification of the medium at a rate approximately double the initial rate. The data indicate that (a) continued H+/l-glutamate contransport is dependent upon GAD activity, (b) the pH-dependent properties of GAD are consistent with a role in a metabolic pH-stat, and (c) the regulation of intracellular pH during H+/l-Glu symport may involve both H+ consumption during 4-aminobutyrate production and ATP-driven H+ efflux. PMID:16668938

  20. Influence of Drought on Mesophyll Resistance to CO2 Diffusion and its Impact on Water-Use Efficiency in Trees

    NASA Astrophysics Data System (ADS)

    Guo, J.; Beverly, D.; Cook, C.; Ewers, B. E.; Williams, D. G.

    2015-12-01

    The resistance to CO2 diffusion inside leaves (mesophyll resistance; rm) during photosynthesis is often comparable in magnitude to stomatal diffusion resistance, and varies among species and across environmental conditions. Consequently, photosynthesis is strongly limited by rm at low internal CO2 partial pressures, such that its variation may determine patterns of leaf water-use efficiency (WUE). Reduction in stomatal conductance with drought typically increases WUE, but also decreases photosynthesis. In theory, the decrease in photosynthesis could be countered by reduction in rm while maintaining high WUE. It is still uncertain how drought-related changes in rm affect short- and long-term WUE strategies of different tree species. We conducted field observations of instantaneous WUE and 13C discrimination in two dominant conifer species (Pinus contorta and Picea engelmannii) in SE Wyoming over the seasonal dry-down period in the summer of 2015. rm was examined by on-line 13C discrimination using isotope laser spectroscopy. Controlled environment studies on three conifer species (P. contorta, P. engelmannii, and Abies lasiocarpa) and one angiosperm (Populus tremuloides) are in progress. We hypothesize that the plasticity of rm in response to drought accounts for significant adjustments in photosynthetic capacity and WUE. Needle leaf conifers are known to have relatively high rm, and we expect them to show greater improvements in photosynthesis and WUE when rm is decreased compared to angiosperm tree species.

  1. Asymmetrical effects of mesophyll conductance on fundamental photosynthetic parameters and their relationships estimated from leaf gas exchange measurements

    SciTech Connect

    Sun, Ying; Gu, Lianhong

    2013-01-01

    Worldwide measurements of nearly 130 C3 species covering all major plant functional types are analyzed in conjunction with model simulations to determine the effects of mesophyll conductance (gm) on photosynthetic parameters and their relationships estimated from A/Ci curves. We find that an assumption of infinite gm results in up to 75% underestimation for maximum carboxylation rate Vcmax, 60% for maximum electron transport rate Jmax, and 40% for triose phosphate utilization rate Tu. Vcmax is most sensitive, Jmax is less sensitive, and Tu has the least sensitivity to the variation of gm. Due to this asymmetrical effect of gm, the ratios of Jmax to Vcmax, Tu to Vcmax, and Tu to Jmax are all overestimated. An infinite gm assumption also limits the freedom of variation of estimated parameters and artificially constrains parameter relationships to stronger shapes. These findings suggest the importance of quantifying gm for understanding in-situ photosynthetic machinery functioning. We show that a nonzero resistance to CO2 movement in chloroplasts has small effects on estimated parameters. A nonlinear function with gm as input is developed to convert the parameters estimated under an assumption of infinite gm to proper values. This function will facilitate gm representation in global carbon cycle models.

  2. Mesophyll conductance to CO2 and Rubisco as targets for improving intrinsic water use efficiency in C3 plants.

    PubMed

    Flexas, J; Díaz-Espejo, A; Conesa, M A; Coopman, R E; Douthe, C; Gago, J; Gallé, A; Galmés, J; Medrano, H; Ribas-Carbo, M; Tomàs, M; Niinemets, Ü

    2016-05-01

    Water limitation is a major global constraint for plant productivity that is likely to be exacerbated by climate change. Hence, improving plant water use efficiency (WUE) has become a major goal for the near future. At the leaf level, WUE is the ratio between photosynthesis and transpiration. Maintaining high photosynthesis under water stress, while improving WUE requires either increasing mesophyll conductance (gm ) and/or improving the biochemical capacity for CO2 assimilation-in which Rubisco properties play a key role, especially in C3 plants at current atmospheric CO2 . The goals of the present analysis are: (1) to summarize the evidence that improving gm and/or Rubisco can result in increased WUE; (2) to review the degree of success of early attempts to genetically manipulate gm or Rubisco; (3) to analyse how gm , gsw and the Rubisco's maximum velocity (Vcmax ) co-vary across different plant species in well-watered and drought-stressed conditions; (4) to examine how these variations cause differences in WUE and what is the overall extent of variation in individual determinants of WUE; and finally, (5) to use simulation analysis to provide a theoretical framework for the possible control of WUE by gm and Rubisco catalytic constants vis-à-vis gsw under water limitations. PMID:26297108

  3. Differential positioning of C(4) mesophyll and bundle sheath chloroplasts: recovery of chloroplast positioning requires the actomyosin system.

    PubMed

    Kobayashi, Hiroaki; Yamada, Masahiro; Taniguchi, Mitsutaka; Kawasaki, Michio; Sugiyama, Tatsuo; Miyake, Hiroshi

    2009-01-01

    In C(4) plants, bundle sheath (BS) chloroplasts are arranged in the centripetal position or in the centrifugal position, although mesophyll (M) chloroplasts are evenly distributed along cell membranes. To examine the molecular mechanism for the intracellular disposition of these chloroplasts, we observed the distribution of actin filaments in BS and M cells of the C(4) plants finger millet (Eleusine coracana) and maize (Zea mays) using immunofluorescence. Fine actin filaments encircled chloroplasts in both cell types, and an actin network was observed adjacent to plasma membranes. The intracellular disposition of both chloroplasts in finger millet was disrupted by centrifugal force but recovered within 2 h in the dark. Actin filaments remained associated with chloroplasts during recovery. We also examined the effects of inhibitors on the rearrangement of chloroplasts. Inhibitors of actin polymerization, myosin-based activities and cytosolic protein synthesis blocked migration of chloroplasts. In contrast, a microtubule-depolymerizing drug had no effect. These results show that C(4) plants possess a mechanism for keeping chloroplasts in the home position which is dependent on the actomyosin system and cytosolic protein synthesis but not tubulin or light. PMID:19022806

  4. Relationships of Leaf Net Photosynthesis, Stomatal Conductance, and Mesophyll Conductance to Primary Metabolism: A Multispecies Meta-Analysis Approach.

    PubMed

    Gago, Jorge; Daloso, Danilo de Menezes; Figueroa, Carlos María; Flexas, Jaume; Fernie, Alisdair Robert; Nikoloski, Zoran

    2016-05-01

    Plant metabolism drives plant development and plant-environment responses, and data readouts from this cellular level could provide insights in the underlying molecular processes. Existing studies have already related key in vivo leaf gas-exchange parameters with structural traits and nutrient components across multiple species. However, insights in the relationships of leaf gas-exchange with leaf primary metabolism are still limited. We investigated these relationships through a multispecies meta-analysis approach based on data sets from 17 published studies describing net photosynthesis (A) and stomatal (gs) and mesophyll (gm) conductances, alongside the 53 data profiles from primary metabolism of 14 species grown in different experiments. Modeling results highlighted the conserved patterns between the different species. Consideration of species-specific effects increased the explanatory power of the models for some metabolites, including Glc-6-P, Fru-6-P, malate, fumarate, Xyl, and ribose. Significant relationships of A with sugars and phosphorylated intermediates were observed. While gs was related to sugars, organic acids, myo-inositol, and shikimate, gm showed a more complex pattern in comparison to the two other traits. Some metabolites, such as malate and Man, appeared in the models for both conductances, suggesting a metabolic coregulation between gs and gm The resulting statistical models provide the first hints for coregulation patterns involving primary metabolism plus leaf water and carbon balances that are conserved across plant species, as well as species-specific trends that can be used to determine new biotechnological targets for crop improvement. PMID:26977088

  5. Variability of mesophyll conductance and its relationship with water use efficiency in cotton leaves under drought pretreatment.

    PubMed

    Han, Ji-Mei; Meng, Hao-Feng; Wang, Sai-Yu; Jiang, Chuang-Dao; Liu, Feng; Zhang, Wang-Feng; Zhang, Ya-Li

    2016-05-01

    Drought slows net photosynthetic rate (AN) but increases water use efficiency (WUE). Farmers give an artificial drought pretreatment to some crops in the early growth stage and find that yield increases accompanying with the improvement of WUE. We conducted well-watered, non-drought, mild drought and moderate drought pretreatments of potted cotton cultivars. The aims of the present study were to analyse the importance of mesophyll conductance (gm) as a factor that may simultaneously improve AN and WUE under drought pretreatment conditions, and to analyse the role of anatomical structure and biochemical mechanism in the variability of gm. Our results showed that significant variability of gm estimated by gas exchange and chlorophyll fluorescence was observed between non-drought pretreatment and drought pretreatment associated with change in AN and WUE. There was great difference in anatomical structure and expression of aquaporins (GhAQP1) among all the treatments. In addition, expression of carbonic anhydrase (CA) may not be important in the regulation of gm under drought pretreatment conditions. We concluded that the variability of gm offers a potential target for improving leaf AN and WUE simultaneously by the regulation of anatomical structure and GhAQP1. PMID:27101723

  6. An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells[OPEN

    PubMed Central

    Burgess, Steven J.; Reyna-Llorens, Ivan; Knerova, Jana; Stanley, Susan

    2016-01-01

    C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5′ untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5′ UTRs and six 3′ UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5′ UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5′ UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis. PMID:26772995

  7. Transgenic Rice Expressing Ictb and FBP/Sbpase Derived from Cyanobacteria Exhibits Enhanced Photosynthesis and Mesophyll Conductance to CO2

    PubMed Central

    Gong, Han Yu; Li, Yang; Fang, Gen; Hu, Dao Heng; Jin, Wen Bin; Wang, Zhao Hai; Li, Yang Sheng

    2015-01-01

    To find a way to promote the rate of carbon flux and further improve the photosynthetic rate in rice, two CO2-transporting and fixing relevant genes, Ictb and FBP/Sbpase, which were derived from cyanobacteria with the 35SCaMV promotor in the respective constructs, were transformed into rice. Three homologous transgenic groups with Ictb, FBP/Sbpase and the two genes combined were constructed in parallel, and the functional effects of these two genes were investigated by physiological, biochemical and leaf anatomy analyses. The results indicated that the mesophyll conductance and net photosynthetic rate were higher at approximately 10.5–36.8% and 13.5–34.6%, respectively, in the three groups but without any changes in leaf anatomy structure compared with wild type. Other physiological and biochemical parameters increased with the same trend in the three groups, which showed that the effect of FBP/SBPase on improving photosynthetic capacity was better than that of ICTB and that there was an additive effect in ICTB+FBP/SBPase. ICTB localized in the cytoplasm, whereas FBP/SBPase was successfully transported to the chloroplast. The two genes might show a synergistic interaction to promote carbon flow and the assimilation rate as a whole. The multigene transformation engineering and its potential utility for improving the photosynthetic capacity and yield in rice were discussed. PMID:26488581

  8. Spatio-temporal decoupling of stomatal and mesophyll conductance induced by vein cutting in leaves of Helianthus annuus

    PubMed Central

    Hanson, David T.; Green, Laura E.; Pockman, William T.

    2013-01-01

    Reduction of hydraulic conductance to the canopy has been shown to result in stomatal responses to limit transpiration. To test for similar responses to perturbations of the hydraulic network in leaves, we simultaneously measured leaf gas exchange with spatially explicit chlorophyll-a fluorescence and leaf temperature to examine the effects of cutting a primary leaf vein in Helianthus annuus. We repeated the leaf treatment at each of three different vapor pressure deficits and monitored the short-term dynamics of gas exchange following the treatment. Immediately after treatment, photosynthesis and stomatal conductance (gs) showed a transient “wrong way” response in which photosynthesis declined despite increased gs. Comparisons of fluorescence and temperature across the leaf showed that both photosynthesis and gs were transiently patchy across the measured leaf area, but that the patchiness of the two processes did not correspond in space or time. This suggests that photosynthesis and gs respond to vein cutting-induced cavitation via different mechanisms. Because the stomatal response varied by vapor pressure difference condition but photosynthesis did not, it is likely that gs, but not photosynthesis, responded to a hydraulic signal. In contrast, we hypothesize that photosynthesis declined due to a wound-induced electrical signal that has recently been shown to transiently decrease mesophyll conductance to CO2. The interaction of epidermal hydraulics and the electrical signal across the leaf likely created a patchy pattern of chlorophyll fluorescence and leaf temperature that cannot be explained through the action of a single signal. PMID:24065972

  9. Deregulation of Maize C4 Photosynthetic Development in a Mesophyll Cell-Defective Mutant1[C][W][OA

    PubMed Central

    Covshoff, Sarah; Majeran, Wojciech; Liu, Peng; Kolkman, Judith M.; van Wijk, Klaas J.; Brutnell, Thomas P.

    2008-01-01

    During maize (Zea mays) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood, but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize high chlorophyll fluorescence (Hcf136) homolog that in Arabidopsis (Arabidopsis thaliana) functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zmhcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type-specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis. PMID:18258693

  10. Transgenic Rice Expressing Ictb and FBP/Sbpase Derived from Cyanobacteria Exhibits Enhanced Photosynthesis and Mesophyll Conductance to CO2.

    PubMed

    Gong, Han Yu; Li, Yang; Fang, Gen; Hu, Dao Heng; Jin, Wen Bin; Wang, Zhao Hai; Li, Yang Sheng

    2015-01-01

    To find a way to promote the rate of carbon flux and further improve the photosynthetic rate in rice, two CO2-transporting and fixing relevant genes, Ictb and FBP/Sbpase, which were derived from cyanobacteria with the 35SCaMV promotor in the respective constructs, were transformed into rice. Three homologous transgenic groups with Ictb, FBP/Sbpase and the two genes combined were constructed in parallel, and the functional effects of these two genes were investigated by physiological, biochemical and leaf anatomy analyses. The results indicated that the mesophyll conductance and net photosynthetic rate were higher at approximately 10.5-36.8% and 13.5-34.6%, respectively, in the three groups but without any changes in leaf anatomy structure compared with wild type. Other physiological and biochemical parameters increased with the same trend in the three groups, which showed that the effect of FBP/SBPase on improving photosynthetic capacity was better than that of ICTB and that there was an additive effect in ICTB+FBP/SBPase. ICTB localized in the cytoplasm, whereas FBP/SBPase was successfully transported to the chloroplast. The two genes might show a synergistic interaction to promote carbon flow and the assimilation rate as a whole. The multigene transformation engineering and its potential utility for improving the photosynthetic capacity and yield in rice were discussed. PMID:26488581

  11. Artifactual responses of mesophyll conductance to CO2 and irradiance estimated with the variable J and online isotope discrimination methods

    SciTech Connect

    Gu, Lianhong; Sun, Ying

    2013-01-01

    Studies with the variable J method have reported that mesophyll conductance (gm) rapidly decreases with increasing intercellular CO2 partial pressures (Ci) or decreasing irradiance. Similar responses have been suggested with the online isotope discrimination method, although with less consistency. Here we show that even when the true gm is constant, the variable J method can produce an artifactual dependence of gm on Ci or irradiance similar to those reported in previous studies for any of the following factors: day respiration and chloroplastic CO2 photocompensation point are estimated with Laisk method; Ci or electron transport rate is positively biased; net photosynthetic rate is negatively biased; insufficient NADPH is assumed while insufficient ATP limits RuBP regeneration. The isotopic method produces similar artifacts if fractionation of carboxylation or Ci are positively biased or 13 negatively biased. A nonzero chloroplastic resistance to CO2 movement results in a qualitatively different dependence of gm on Ci or irradiance and this dependence is only sensitive at low Ci. We thus cannot rule out the possibility that previously reported dependence of gm on Ci or irradiance is a methodological artifact. Recommendations are made to take advantage of sensitivities of the variable J and isotopic methods for estimating gm.

  12. Geminicoccus roseus gen. nov., sp. nov., an aerobic phototrophic Alphaproteobacterium isolated from a marine aquaculture biofilter.

    PubMed

    Foesel, Bärbel U; Gössner, Anita S; Drake, Harold L; Schramm, Andreas

    2007-12-01

    A Gram-negative, strictly aerobic, diplococcoid bacterium (strain D2-3(T)) was isolated from the biofilter of a recirculating marine aquaculture system. Phylogenetic analysis of the 16S rRNA gene sequence of D2-3(T) indicated that the new organism occupied a novel lineage within the alpha-1 subclass of Proteobacteria and was related to the genera Rhodothalassium, Azospirillum, Craurococcus, Acidiphilium, and Tistrella. The highest sequence similarity (90.8%) of the 16S rRNA gene sequence of D2-3(T) was to that of Candidatus "Alysiosphaera europaea". D2-3(T) was mesophilic, heterotrophic, required sea salt, and had a pH optimum of 8.0. Growth in the presence of light resulted in the formation of pink colonies, a 25% increased cell yield, and a slightly increased growth rate. D2-3(T) contained carotenoids and low amounts of bacteriochlorophyll a. Membranes of D2-3(T) contained b-type cytochromes. The G+C content of the DNA was 60.3+/-0.1mol%. Phylogenetic, morphological, physiological, and biochemical analyses demonstrated that D2-3(T) represented a new aerobic phototrophic genus, for which the name Geminicoccus roseus gen. nov., sp. nov. is proposed for the type species (D2-3(T)=DSM 18922(T)=ATCC BAA-1445(T)). PMID:17643894

  13. Pedobacter roseus sp. nov., isolated from a hypertrophic pond, and emended description of the genus Pedobacter.

    PubMed

    Hwang, Chung Yeon; Choi, Dong Han; Cho, Byung Cheol

    2006-08-01

    A Gram-negative, pink-coloured, rod-shaped, non-flagellated bacterium, designated CL-GP80(T), was isolated from a hypertrophic pond located within the campus of Seoul National University, Korea. Analysis of its 16S rRNA gene sequence revealed that strain CL-GP80(T) belongs to the family Sphingobacteriaceae and is closely related to Pedobacter heparinus ATCC 13125(T) (95.8 % sequence similarity) and to other members of the genus Pedobacter (90.8-95.3 % similarity). Temperature and pH ranges for growth were 5-33 degrees C and pH 6-8, respectively. The DNA G+C content was 41.3 mol%. The major fatty acids were iso-C(15 : 0) (37.0 %), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (24.5 %), and iso-C(17 : 0) 3-OH (11.3 %). Phenotypic, chemotaxonomic and phylogenetic analyses indicated that strain CL-GP80(T) could be assigned to the genus Pedobacter, but distinguished from recognized species of the genus. Strain CL-GP80(T) (=KCCM 42272(T)=JCM 13399(T)) is therefore proposed as the type strain of a novel species, for which the name Pedobacter roseus sp. nov. is proposed. PMID:16902016

  14. Greater efficiency of water use in poplar clones having a delayed response of mesophyll conductance to drought.

    PubMed

    Théroux Rancourt, Guillaume; Éthier, Gilbert; Pepin, Steeve

    2015-02-01

    Improvement of water use efficiency is a key objective to improve the sustainability of cultivated plants, especially fast growing species with high water consumption like poplar. It is well known that water use efficiency (WUE) varies considerably among poplar genotypes, and it was recently suggested that the use of the mesophyll-to-stomatal conductance ratio (gm/gs) would be an appropriate trait to improve WUE. The responses of 7-week-old cuttings of four hybrid poplar clones and one native Balsam poplar (Populus balsamifera L.) to a water stress-recovery cycle were examined to evaluate the relation between the gm/gs ratio and transpiration efficiency (TE), a leaf-level component of WUE. A contrasting gs response to water stress was observed among the five clones, from stomatal closure early on during soil drying up to limited closure in Balsam poplar. However in the hybrids, the decline in gm was consistently delayed by a few days compared with gs. Moreover, in the most water use-efficient hybrids, the recovery following rehydration occurred faster for gm than for gs. Thus, the delay in the response of gm to drought and its faster recovery upon rewatering increased the gm/gs of the hybrids and this ratio scaled positively with TE. Our results support the use of the gm/gs ratio to select genotypes with improved WUE, and the notion that breeding strategies focusing mainly on stomatal responses to soil drying should also look for a strong curvilinearity between net carbon assimilation rate and gs, the indication of a significant increase in gm/gs in the earlier stages of stomatal closure. PMID:25721370

  15. Temperature response of mesophyll conductance. Implications for the determination of Rubisco enzyme kinetics and for limitations to photosynthesis in vivo.

    PubMed

    Bernacchi, Carl J; Portis, Archie R; Nakano, Hiromi; von Caemmerer, Susanne; Long, Stephen P

    2002-12-01

    CO(2) transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (g(m)), is finite. Therefore, it will limit photosynthesis when CO(2) is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of g(m). The process dominating g(m) is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO(2) in water have all been suggested. The response of g(m) to temperature (10 degrees C-40 degrees C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q(10)) of approximately 2.2 for g(m), suggesting control by a protein-facilitated process because the Q(10) for diffusion of CO(2) in water is about 1.25. Further, g(m) values are maximal at 35 degrees C to 37.5 degrees C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of g(m) to calculate CO(2) at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10 degrees C to 40 degrees C. Using these parameters, we determined the limitation imposed on photosynthesis by g(m). Despite an exponential rise with temperature, g(m) does not keep pace with increased capacity for CO(2) uptake at the site of Rubisco. The fraction of the total limitations to CO(2) uptake within the leaf attributable to g(m) rose from 0.10 at 10 degrees C to 0.22 at 40 degrees C. This shows that transfer of CO(2) from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature. PMID:12481082

  16. Temperature Response of Mesophyll Conductance. Implications for the Determination of Rubisco Enzyme Kinetics and for Limitations to Photosynthesis in Vivo

    PubMed Central

    Bernacchi, Carl J.; Portis, Archie R.; Nakano, Hiromi; von Caemmerer, Susanne; Long, Stephen P.

    2002-01-01

    CO2 transfer conductance from the intercellular airspaces of the leaf into the chloroplast, defined as mesophyll conductance (gm), is finite. Therefore, it will limit photosynthesis when CO2 is not saturating, as in C3 leaves in the present atmosphere. Little is known about the processes that determine the magnitude of gm. The process dominating gm is uncertain, though carbonic anhydrase, aquaporins, and the diffusivity of CO2 in water have all been suggested. The response of gm to temperature (10°C–40°C) in mature leaves of tobacco (Nicotiana tabacum L. cv W38) was determined using measurements of leaf carbon dioxide and water vapor exchange, coupled with modulated chlorophyll fluorescence. These measurements revealed a temperature coefficient (Q10) of approximately 2.2 for gm, suggesting control by a protein-facilitated process because the Q10 for diffusion of CO2 in water is about 1.25. Further, gm values are maximal at 35°C to 37.5°C, again suggesting a protein-facilitated process, but with a lower energy of deactivation than Rubisco. Using the temperature response of gm to calculate CO2 at Rubisco, the kinetic parameters of Rubisco were calculated in vivo from 10°C to 40°C. Using these parameters, we determined the limitation imposed on photosynthesis by gm. Despite an exponential rise with temperature, gm does not keep pace with increased capacity for CO2 uptake at the site of Rubisco. The fraction of the total limitations to CO2 uptake within the leaf attributable to gm rose from 0.10 at 10°C to 0.22 at 40°C. This shows that transfer of CO2 from the intercellular air space to Rubisco is a very substantial limitation on photosynthesis, especially at high temperature. PMID:12481082

  17. Meiothermus roseus sp. nov., a thermophilic bacterium isolated from a geothermal area.

    PubMed

    Ming, Hong; Duan, Yan-Yan; Guo, Qian-Qian; Yin, Yi-Rui; Zhou, En-Min; Liu, Lan; Li, Shuai; Nie, Guo-Xing; Li, Wen-Jun

    2015-10-01

    Two closely related thermophilic bacterial strains, designated YIM 71031(T) and YIM 71039, were isolated from a hot spring in Tengchong county, Yunnan province, south-western China. The novel isolates were observed to be Gram-negative, aerobic, rod-shaped and yellow-pigmented bacteria. The strains were found to be able to grow at 37-65 °C, pH 6.0-9.0 and with a NaCl tolerance up to 1.0 % (w/v). Phylogenetic analysis based on 16S rRNA gene sequences placed these two isolates in the genus Meiothermus. They were found to be closely related to Meiothermus timidus DSM 17022(T) (98.6 % similarity), and formed a cluster with this species. The predominant menaquinone was identified as MK-8 and the major fatty acids (>10 %) as anteiso-C15:0, iso-C15:0, anteiso-C17:0, iso-C16:0 and C16:0. The genomic DNA G+C contents of strains YIM 71031(T) and YIM 71039 were determined to be 64.0 and 65.4 mol%, respectively. DNA-DNA hybridizations showed low values between strains YIM 71031(T) and YIM 71039 and their closely related neighbour M. timidus DSM 17022(T). Morphological phylogenetic and chemotaxonomic results suggest that strains YIM 71031(T) and YIM 71039 are representatives of a new species within the genus Meiothermus, for which the name Meiothermus roseus sp. nov. is proposed. The type strain is YIM 71031(T) (=KCTC 42495(T) =NBRC 110900(T)). PMID:26219565

  18. Roseivivax roseus sp. nov., an alphaproteobacterium isolated from a solar saltern soil sample.

    PubMed

    Zhang, Yu-Qin; Lee, Jae-Chan; Park, Dong-Jin; Lu, Xin-Xin; Mou, Xiao-Zhen; Kim, Chang-Jin

    2014-05-01

    A pink, Gram-stain-negative, motile, halotolerant bacterium with subpolar flagellum, designated strain BH87090T, was isolated from a saline soil sample collected from the south-west coastal area of South Korea (125° 58' 58.08″ E 34° 45' 37.32″ N). The isolate formed opaque pink to red colonies on marine agar plates at 30 °C. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylcholine and one unidentified phospholipid. The sole respiratory quinone was ubiquinone-10 (Q-10). The major cellular fatty acids were C18:1ω7c, C19:0 cyclo ω8c, C16:0 and 11-methyl C18:1ω7c. The genomic DNA G+C content was 61.8 mol%. These chemotaxonomic characteristics were all consistent with specific properties of the genus Roseivivax. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate affiliated to the cluster with members of the genus Roseivivax in the Roseobacter clade, which suggested that the strain belonged to the genus Roseivivax. However, the low 16S rRNA gene similarities (93.5-95.3%) of strain BH87090T with all the members of the genus Roseivivax indicated that it represented a novel species of the genus Roseivivax. On the basis of phenotypic and genotypic data, strain BH87090T should be classified as a novel species of the genus Roseivivax. The name Roseivivax roseus sp. nov. is proposed, with strain BH87090T (=DSM 23042T=KCTC 22650T) as the type strain. PMID:24554641

  19. Seasonal time-course of gradients of photosynthetic capacity and mesophyll conductance to CO2 across a beech (Fagus sylvatica L.) canopy.

    PubMed

    Montpied, Pierre; Granier, André; Dreyer, Erwin

    2009-01-01

    Leaf photosynthesis is known to acclimate to the actual irradiance received by the different layers of a canopy. This acclimation is usually described in terms of changes in leaf structure, and in photosynthetic capacity. Photosynthetic capacity is likely to be affected by mesophyll conductance to CO(2) which has seldom been assessed in tree species, and whose plasticity in response to local irradiance is still poorly known. Structural [N and chlorophyll content, leaf mass to area ratio (LMA)] and functional leaf traits [maximum carboxylation rate (V(cmax)), maximum light-driven electron flux (J(max)), and mesophyll conductance (g(i))] were assessed by measuring leaf response curves of net CO(2) assimilation versus intercellular CO(2) partial pressure, along a vertical profile across a beech canopy, and by fitting a version of the Farquhar model including g(i). The measurements were repeated five times during a growth season to catch potential seasonal variation. Irradiance gradients resulted in large decreasing gradients of LMA, g(i), V(cmax), and J(max). Relative allocation of leaf N to the different photosynthetic processes was only slightly affected by local irradiance. Seasonal changes after leaf expansion and before induction of leaf senescence were only minor. Structural equation modelling confirmed that LMA was the main driving force for changes in photosynthetic traits, with only a minor contribution of leaf Nitrogen content. In conclusion, mesophyll conductance to CO(2) displays a large plasticity that scales with photosynthetic capacity across a tree canopy, and that it is only moderately (if at all) affected by seasonal changes in the absence of significant soil water depletion. PMID:19457983

  20. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    PubMed Central

    Smith-Moritz, Andreia M.; Hao, Zhao; Fernández-Niño, Susana G.; Fangel, Jonatan U.; Verhertbruggen, Yves; Holman, Hoi-Ying N.; Willats, William G. T.; Ronald, Pamela C.; Scheller, Henrik V.; Heazlewood, Joshua L.; Vega-Sánchez, Miguel E.

    2015-01-01

    The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion. PMID:26347754

  1. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    SciTech Connect

    Smith-Moritz, Andreia M.; Hao, Zhao; Fernández-Nino, Susana G.; Fangel, Jonatan U.; Verhertbruggen, Yves; Holman, Hoi-Ying N.; Willats, William G. T.; Ronald, Pamela C.; Scheller, Henrik V.; Heazlewood, Joshua L.; Vega-Sanchez, Miguel E.

    2015-08-18

    The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Finally, taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

  2. Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls

    DOE PAGESBeta

    Smith-Moritz, Andreia M.; Hao, Zhao; Fernández-Nino, Susana G.; Fangel, Jonatan U.; Verhertbruggen, Yves; Holman, Hoi-Ying N.; Willats, William G. T.; Ronald, Pamela C.; Scheller, Henrik V.; Heazlewood, Joshua L.; et al

    2015-08-18

    The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to testmore » the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Finally, taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.« less

  3. Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts, vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana.

    PubMed

    Balsamo, R A; Uribe, E G

    1988-02-01

    Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H(+)-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces. PMID:24226399

  4. Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

    PubMed Central

    De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

    2015-01-01

    Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth

  5. Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells *S⃞

    PubMed Central

    Majeran, Wojciech; Zybailov, Boris; Ytterberg, A. Jimmy; Dunsmore, Jason; Sun, Qi; van Wijk, Klaas J.

    2008-01-01

    Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C4 photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b6f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/Pi translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available

  6. Height-related decreases in mesophyll conductance, leaf photosynthesis and compensating adjustments associated with leaf nitrogen concentrations in Pinus densiflora.

    PubMed

    Han, Qingmin

    2011-09-01

    Hydraulic limitations associated with increasing tree height result in reduced foliar stomatal conductance (g(s)) and light-saturated photosynthesis (A(max)). However, it is unclear whether the decline in A(max) is attributable to height-related modifications in foliar nitrogen concentration (N), to mesophyll conductance (g(m)) or to biochemical capacity for photosynthesis (maximum rate of carboxylation, V(cmax)). Simultaneous measurements of gas exchange and chlorophyll fluorescence were made to determine g(m) and V(cmax) in four height classes of Pinus densiflora Sieb. & Zucc. trees. As the average height of growing trees increased from 3.1 to 13.7 m, g(m) decreased from 0.250 to 0.107 mol m(-2) s(-1), and the CO(2) concentration from the intercellular space (C(i)) to the site of carboxylation (C(c)) decreased by an average of 74 µmol mol(-1). Furthermore, V(cmax) estimated from C(c) increased from 68.4 to 112.0 µmol m(-2) s(-1) with the increase in height, but did not change when it was calculated based on C(i). In contrast, A(max) decreased from 14.17 to 10.73 µmol m(-2) s(-1). Leaf dry mass per unit area (LMA) increased significantly with tree height as well as N on both a dry mass and an area basis. All of these parameters were significantly correlated with tree height. In addition, g(m) was closely correlated with LMA and g(s), indicating that increased diffusive resistance for CO(2) may be the inevitable consequence of morphological adaptation. Foliar N per unit area was positively correlated with V(cmax) based on C(c) but negatively with A(max), suggesting that enhancement of photosynthetic capacity is achieved by allocating more N to foliage in order to minimize the declines in A(max). Increases in the N cost associated with carbon gain because of the limited water available to taller trees lead to a trade-off between water use efficiency and photosynthetic nitrogen use efficiency. In conclusion, the height-related decrease in photosynthetic

  7. Inorganic carbon uptake during photosynthesis. II. Uptake by isolated Asparagus mesophyll cells during isotopic disequilibrium. [Asparagus sprengeri

    SciTech Connect

    Espie, G.S.; Owttrim, G.W.; Colman, B.

    1986-04-01

    The species of inorganic carbon (CO/sub 2/ or HCO/sub 3//sup -/) taken up as a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO/sub 2/ or HCO/sub 3//sup -/ transport, during steady state photosynthesis, is achieved by monitoring the changes (by /sup 14/C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO/sub 2/ or HCO/sub 3//sup -/ transport) and experimental time-courses of /sup 14/C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO/sub 2/, rather than HCO/sub 3//sup -/, is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO/sub 2/ is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO/sub 3//sup -/ transport, as the incorporation of /sup 14/C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO/sub 2/ uptake alone. The contribution of HCO/sub 3//sup -/ to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO/sub 3//sup -/ concentration. The evidence for direct HCO/sub 3//sup -/ transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of /sup 14/C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO/sub 2/, which is partially alleviated by a high extracellular concentration of HCO/sub 3//sup -/.

  8. Nectar replenishment and pollen receipt interact in their effects on seed production of Penstemon roseus.

    PubMed

    Ornelas, Juan Francisco; Lara, Carlos

    2009-07-01

    Resource supply and pollen delivery are often thought to equally limit seed production in animal-pollinated plants. At equilibrium, plants should show no response to experimental pollen supplementation because resources limit seed set above the current level of pollen attraction, while experimental reduction in pollen deposition below the equilibrium level would reduce seed set. The predicted equilibrium may be disrupted, however, if plants expend additional energy to replenish removed nectar. We investigated the combined effects of nectar removal and pollen delivery on female reproductive success of Penstemon roseus (Plantaginaceae), a hummingbird-pollinated plant that replenishes removed nectar. We first documented that the frequency of experimental nectar removal was correlated with total nectar secretion; and increased frequency of nectar removal resulted in increased female reproductive costs to the plant. Trade-offs between investing resources in nectar and investing resources in seeds were then investigated in two contrasting natural populations by removing nectar from flowers at increasing frequencies while simultaneously hand-pollinating flowers with increasing amounts of pollen. Seed set was lowest at low levels of pollen deposition, highest at medium-sized pollen loads, and intermediate when pollen loads were highest. At both sites, the frequency of nectar removal and pollen deposition had an interactive effect on seed production, in that intermediate levels of nectar removal result in the absolute highest seed set, but only at intermediate pollen loads. At high pollen loads, seed set was higher following little to no nectar removal, and at low pollen loads, all rates of nectar removal affected fecundity equally. Seed mass responded to nectar removal and pollination differently than did seed set. High levels of nectar removal and pollen delivery both lowered seed mass, with little interaction between main effects. Our findings are among the first to

  9. Importance of leaf anatomy in determining mesophyll diffusion conductance to CO2 across species: quantitative limitations and scaling up by models

    PubMed Central

    Tomás, Magdalena; Flexas, Jaume; Copolovici, Lucian; Galmés, Jeroni; Hallik, Lea; Medrano, Hipólito; Ribas-Carbó, Miquel; Tosens, Tiina; Vislap, Vivian; Niinemets, Ülo

    2013-01-01

    Foliage photosynthetic and structural traits were studied in 15 species with a wide range of foliage anatomies to gain insight into the importance of key anatomical traits in the limitation of diffusion of CO2 from substomatal cavities to chloroplasts. The relative importance of different anatomical traits in constraining CO2 diffusion was evaluated using a quantitative model. Mesophyll conductance (g m) was most strongly correlated with chloroplast exposed surface to leaf area ratio (S c/S) and cell wall thickness (T cw), but, depending on foliage structure, the overall importance of g m in constraining photosynthesis and the importance of different anatomical traits in the restriction of CO2 diffusion varied. In species with mesophytic leaves, membrane permeabilities and cytosol and stromal conductance dominated the variation in g m. However, in species with sclerophytic leaves, g m was mostly limited by T cw. These results demonstrate the major role of anatomy in constraining mesophyll diffusion conductance and, consequently, in determining the variability in photosynthetic capacity among species. PMID:23564954

  10. Analyzing the Light Energy Distribution in the Photosynthetic Apparatus of C4 Plants Using Highly Purified Mesophyll and Bundle-Sheath Thylakoids.

    PubMed Central

    Pfundel, E.; Nagel, E.; Meister, A.

    1996-01-01

    The chlorophyll fluorescence characteristics of mesophyll and bundle-sheath thylakoids from plant species with the C4 dicarboxylic acid pathway of photosynthesis were investigated using flow cytometry. Ten species with the NADP-malic enzyme (NADP-ME) biochemical type of C4 photosynthesis were tested: Digitaria sanguinalis (L.) Scop., Euphorbia maculata L., Portulaca grandiflora Hooker, Saccharum officinarum L., Setaria viridis (L.) Beauv., Zea mays L., and four species of the genus Flaveria. This study also included three species with NAD-ME biochemistry (Atriplex rosea L., Atriplex spongiosa F. Muell., and Portulaca oleracea L.). Two C4 species of unknown biochemical type were investigated: Cyperus papyrus L. and Atriplex tatarica L. Pure mesophyll and bundle-sheath thylakoids were prepared by flow cytometry and characterized by low-temperature fluorescence spectroscopy. In pure bundle-sheath thylakoids from many species with C4 photosynthesis of the NADP-ME type, significant amounts of photosystem II (PSII) emission can be detected by fluorescence spectroscopy. Simulation of fluorescence excitation spectra of these thylakoids showed that PSII light absorption contributes significantly to the apparent excitation spectrum of photosystem I. Model calculations indicated that the excitation energy of PSII is efficiently transferred to photosystem I in bundle-sheath thylakoids of many NADP-ME species. PMID:12226432

  11. Rapid responses of mesophyll conductance to changes of CO2 concentration, temperature and irradiance are affected by N supplements in rice.

    PubMed

    Xiong, Dongliang; Liu, Xi; Liu, Limin; Douthe, Cyril; Li, Yong; Peng, Shaobing; Huang, Jianliang

    2015-12-01

    Photosynthesis in C3 plants is significantly limited by mesophyll conductance (gm ), which can vary with leaf anatomical traits and nitrogen (N) supplements. Several studies have investigated the response of gm to N supplements; however, none examined the implications of N supplements on the response of gm to rapid environmental changes. Here we investigated the effect of N supplement on gm and the response of gm to change of CO2 , temperature and irradiance in rice. High N supplement (HN) increased mesophyll cell wall surface area and chloroplast surface area exposed to intercellular airspace per leaf area, and reduced cell wall thickness. These changes resulted in increased gm . The gm of leaves with HN was more sensitive to changes in CO2 concentration, temperature and irradiance. The difference in leaf structural features between low N supplement and HN indicates that a rapid change in gm is related to the regulation of diffusion through biological membranes rather than leaf structural features. These results will contribute to an understanding of the determinants of gm response to rapid changes in environmental factors. PMID:25923314

  12. Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels.

    PubMed

    Wang, Shu-Wei; Li, Ying; Zhang, Xiao-Lu; Yang, Hai-Qiang; Han, Xue-Fei; Liu, Zhao-Hui; Shang, Zhong-Lin; Asano, Tomoya; Yoshioka, Yasushi; Zhang, Chun-Guang; Chen, Yu-Ling

    2014-09-01

    Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells. PMID:24506786

  13. Biotransformations with plant tissue cultures.

    PubMed

    Carew, D P; Bainbridge, T

    1976-01-01

    Suspension cultures of Catharanthus roseus, Apocynum cannabinum and Conium maculatum were examined for their capacity to transform aniline, anisole, acetanilide, benzoic acid and coumarin. None of the cultures transformed acetanilide but each produced acetanilide when fed aniline. All three cultures converted benzoic acid to its para-hydroxy derivative. Coumarin was selectively hydroxylated at the 7-position by Catharanthus and Conium and anisole was O-demethylated only by older Catharanthus tissue. PMID:1084950

  14. Methods of mesophyll conductance estimation: its impact on key biochemical parameters and photosynthetic limitations in phosphorus-stressed soybean across CO2.

    PubMed

    Singh, Shardendu K; Reddy, Vangimalla R

    2016-06-01

    Despite the development of various methods, the rapid estimation of mesophyll conductance (gm ) for a large number of samples is still a daunting challenge. Although the accurate estimation of gm is critical to partition photosynthetic limitations by stomatal (Ls ) and mesophyll (Lm ) conductance and by photo-biochemical (Lb ) processes, the impact of various gm estimation methods on this is ambiguous. As phosphorus (P) starvation and elevated CO2 (eCO2 ) strongly affect photosynthetic processes, their combined effect on the proportional changes in these limitations are not well understood. To investigate this, while also evaluating distinct recent methods of gm estimation sharing few common theories and assumptions, soybean was grown under a range of P nutrition at ambient and eCO2 . Methods significantly affected gm and carboxylation efficiency (VCmax ) but not other photosynthetic parameters. In all the methods, all photosynthetic parameters responded similarly to treatments. However, the percentage difference between VCmax assuming finite and infinite gm was highly inconsistent among methods. The primary mechanism responsible for P limitation to soybean photosynthesis was not CO2 diffusion limitations but Lb comprised of reduced chlorophyll, photochemistry and biochemical processes. The eCO2 decreased Lb but increased Lm without affecting Ls across leaf P concentration. Although each method explored advances of our understanding about gm variability, they all require assumptions of varying degrees, which lead to the discrepancy in the gm values. Among the methods, the oxygen sensitivity-based gm estimation appeared to be suitable for the quick assessment of a large number of samples or genotypes. Digital tools are provided for the easy estimation of gm for some methods. PMID:26806194

  15. Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source.

    PubMed

    Kytridis, Velissarios-Phaedon; Manetas, Yiannis

    2006-01-01

    The hypothesis that anthocyanins in red leaves may be potential in vivo antioxidants whose efficiency is linked to their proximity with the oxy-radical source was tested. Advantage was taken of intra-individual and intra-species variations in the anthocyanic trait and green and red leaves on the same individuals or leaves of green and red phenotypes were compared for the extent of PSII damage by reactive oxygen species generated by methyl viologen treatment in the light. Two species possessing anthocyanins in the mesophyll (Cistus creticus and Photinia x fraseri) and two in the epidermis (Rosa sp. and Ricinus communis) were used, while red actinic light (which is not absorbed by anthocyanins) allowed discrimination between an indirect sunscreen and a direct antioxidant function. Red leaves whose anthocyanins were located in the mesophyll were more resistant to methyl viologen treatment than their green counterparts. In one of these species (Cistus creticus), where anthocyanins are induced in some individuals within the natural population after bright cool days in winter, both green and future-red morphs displayed the same sensitivity to methyl viologen before anthocyanin induction. Immediately after reddening, however, resistance to methyl viologen was considerably increased in the red morphs. By contrast, red leaves whose anthocyanins were restricted to epidermal cells were more sensitive to the herbicide. Total leaf phenolic levels in green/red pairs were similar. The results indicate that vacuolar anthocyanins may be an effective in vivo target for oxy-radicals, provided that the oxy-radical source and the anthocyanic detoxifying sink are in close vicinity. PMID:16714309

  16. Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.

    PubMed

    Qu, Yang; Easson, Michael L A E; Froese, Jordan; Simionescu, Razvan; Hudlicky, Tomas; De Luca, Vincenzo

    2015-05-12

    Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine. PMID:25918424

  17. Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast

    PubMed Central

    Qu, Yang; Easson, Michael L. A. E.; Froese, Jordan; Simionescu, Razvan; Hudlicky, Tomas; De Luca, Vincenzo

    2015-01-01

    Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine. PMID:25918424

  18. Metabolic costs of avian flight in relation to flight velocity: a study in Rose Coloured Starlings (Sturnus roseus, Linnaeus).

    PubMed

    Engel, Sophia; Biebach, Herbert; Visser, G Henk

    2006-06-01

    The metabolic costs of flight at a natural range of speeds were investigated in Rose Coloured Starlings (Sturnus roseus, Linnaeus) using doubly labelled water. Eight birds flew repeatedly and unrestrained for bouts of 6 h at speeds from 9 to 14 m s(-1) in a low-turbulence wind tunnel, corresponding to travel distances between 200 and 300 km, respectively. This represents the widest speed range where we could obtain voluntarily sustained flights. From a subset of these flights, data on the wing beat frequency (WBF) and intermittent flight behaviour were obtained. Over the range of speeds that were tested, flight costs did not change with velocity and were on an average 8.17+/-0.64 W or 114 W kg(-1). Body mass was the only parameter with a significant (positive) effect on flight costs, which can be described as EE(f)=0.741 M(0.554). WBF changed slightly with speed, but correlated better with body mass. Birds showed both types of intermittent flight, undulating and bounding, but their frequencies did not systematically change with flight speed. PMID:16425018

  19. Effect of composites based nickel foam anode in microbial fuel cell using Acetobacter aceti and Gluconobacter roseus as a biocatalysts.

    PubMed

    Karthikeyan, Rengasamy; Krishnaraj, Navanietha; Selvam, Ammaiyappan; Wong, Jonathan Woon-Chung; Lee, Patrick K H; Leung, Michael K H; Berchmans, Sheela

    2016-10-01

    This study explores the use of materials such as chitosan (chit), polyaniline (PANI) and titanium carbide (TC) as anode materials for microbial fuel cells. Nickel foam (NF) was used as the base anode substrate. Four different types of anodes (NF, NF/PANI, NF/PANI/TC, NF/PANI/TC/Chit) are thus prepared and used in batch type microbial fuel cells operated with a mixed consortium of Acetobacter aceti and Gluconobacter roseus as the biocatalysts and bad wine as a feedstock. A maximum power density of 18.8Wm(-3) (≈2.3 times higher than NF) was obtained in the case of the anode modified with a composite of PANI/TC/Chit. The MFCs running under a constant external resistance of (50Ω) yielded 14.7% coulombic efficiency with a maximum chemical oxygen demand (COD) removal of 87-93%. The overall results suggest that the catalytic materials embedded in the chitosan matrix show the best performance and have potentials for further development. PMID:26970695

  20. Characterization of Melioribacter roseus gen. nov., sp. nov., a novel facultatively anaerobic thermophilic cellulolytic bacterium from the class Ignavibacteria, and a proposal of a novel bacterial phylum Ignavibacteriae.

    PubMed

    Podosokorskaya, Olga A; Kadnikov, Vitaly V; Gavrilov, Sergey N; Mardanov, Andrey V; Merkel, Alexander Y; Karnachuk, Olga V; Ravin, Nikolay V; Bonch-Osmolovskaya, Elizaveta A; Kublanov, Ilya V

    2013-06-01

    A novel moderately thermophilic, facultatively anaerobic chemoorganotrophic bacterium strain P3M-2(T) was isolated from a microbial mat developing on the wooden surface of a chute under the flow of hot water (46°C) coming out of a 2775-m-deep oil exploration well (Tomsk region, Russia). Strain P3M-2(T) is a moderate thermophile and facultative anaerobe growing on mono-, di- or polysaccharides by aerobic respiration, fermentation or by reducing diverse electron acceptors [nitrite, Fe(III), As(V)]. Its closest cultivated relative (90.8% rRNA gene sequence identity) is Ignavibacterium album, the only chemoorganotrophic member of the phylum Chlorobi. New genus and species Melioribacter roseus are proposed for isolate P3M-2(T) . Together with I. album, the new organism represents the class Ignavibacteria assigned to the phylum Chlorobi. The revealed group includes a variety of uncultured environmental clones, the 16S rRNA gene sequences of some of which have been previously attributed to the candidate division ZB1. Phylogenetic analysis of M. roseus and I. album based on their 23S rRNA and RecA sequences confirmed that these two organisms could represent an even deeper, phylum-level lineage. Hence, we propose a new phylum Ignavibacteriae within the Bacteroidetes-Chlorobi group with a sole class Ignavibacteria, two families Ignavibacteriaceae and Melioribacteraceae and two species I. album and M. roseus. This proposal correlates with chemotaxonomic data and phenotypic differences of both organisms from other cultured representatives of Chlorobi. The most essential differences, supported by the analyses of complete genomes of both organisms, are motility, facultatively anaerobic and obligately organotrophic mode of life, the absence of chlorosomes and the apparent inability to grow phototrophically. PMID:23297868

  1. Determining Photosynthetic Parameters from Leaf CO2 Exchange and Chlorophyll Fluorescence (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Specificity Factor, Dark Respiration in the Light, Excitation Distribution between Photosystems, Alternative Electron Transport Rate, and Mesophyll Diffusion Resistance.

    PubMed

    Laisk, A.; Loreto, F.

    1996-03-01

    Using simultaneous measurements of leaf gas exchange and chlorophyll fluorescence, we determined the excitation partitioning to photosystem II (PSII), the CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase, the dark respiration in the light, and the alternative electron transport rate to acceptors other than bisphosphoglycerate, and the transport resistance for CO2 in the mesophyll cells for individual leaves of herbaceous and tree species. The specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase for CO2 was determined from the slope of the O2 dependence of the CO2 compensation point between 1.5 and 21% O2. Its value, on the basis of dissolved CO2 and O2 concentrations at 25.5[deg]C, varied between 86 and 89. Dark respiration in the light, estimated from the difference between the CO2 compensation point and the CO2 photocompensation point, was about 20 to 50% of the respiration rate in the dark. The excitation distribution to PSII was estimated from the extrapolation of the dependence of the PSII quantum yield on F/Fm to F = 0, where F is steady-state and Fm is pulse-satuarated fluorescence, and varied between 0.45 and 0.6. The alternative electron transport rate was found as the difference between the electron transport rates calculated from fluorescence and from gas exchange, and at low CO2 concentrations and 10 to 21% O2, it was 25 to 30% of the maximum electron transport. The calculated mesophyll diffusion resistance accounted for about 20 to 30% of the total mesophyll resistance, which also includes carboxylation resistance. Whole-leaf photosynthesis is limited by gas phase, mesophyll diffusion, and carboxylation resistances in nearly the same proportion in both herbaceous species and trees. PMID:12226229

  2. Ultradian variation of isoprene emission, photosynthesis, mesophyll conductance, and optimum temperature sensitivity for isoprene emission in water-stressed Eucalyptus citriodora saplings.

    PubMed

    Brilli, Federico; Tsonev, Tsonko; Mahmood, Tariq; Velikova, Violeta; Loreto, Francesco; Centritto, Mauro

    2013-01-01

    Water availability is a major limiting factor on plant growth and productivity. Considering that Eucalyptus spp. are among the few plant species able to produce both isoprene and monoterpenes, experiments were designed to investigate the response of isoprene emission and isoprenoid concentrations in Eucalyptus citriodora saplings exposed to decreasing fraction of transpirable soil water (FTSW). In particular, this study aimed to assess: (a) the kinetic of water stress-induced variations in photosynthesis, isoprene emission, and leaf isoprenoid concentrations during progressive soil water shortage as a function of FTSW; (b) the ultradian control of isoprene emission and photosynthesis under limited soil water availability; and (c) the optimum temperature sensitivity of isoprene emission and photosynthesis under severe water stress. The optimum temperature for isoprene emission did not change under progressive soil water deficit. However, water stress induced a reallocation of carbon through the MEP/DOXP pathway resulting in a qualitative change of the stored isoprenoids. The ultradian trend of isoprene emission was also unaffected under water stress, and a similar ultradian trend of stomatal and mesophyll conductances was also observed, highlighting a tight coordination between diffusion limitations to photosynthesis during water stress. The kinetics of photosynthetic parameters and isoprene emission in response to decreasing FTSW in E. citriodora are strikingly similar to those measured in other plant functional types. These findings may be useful to refine the algorithms employed in process-based models aiming to precisely up-scale carbon assimilation and isoprenoid emissions at regional and global scales. PMID:23293347

  3. Regulation of photosynthesis and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins

    PubMed Central

    Perez-Martin, Alfonso; Michelazzo, Chiara; Torres-Ruiz, Jose M.; Flexas, Jaume; Fernández, José E.; Sebastiani, Luca; Diaz-Espejo, Antonio

    2014-01-01

    The hypothesis that aquaporins and carbonic anhydrase (CA) are involved in the regulation of stomatal (g s) and mesophyll (g m) conductance to CO2 was tested in a short-term water-stress and recovery experiment in 5-year-old olive plants (Olea europaea) growing outdoors. The evolution of leaf gas exchange, chlorophyll fluorescence, and plant water status, and a quantitative analysis of photosynthesis limitations, were followed during water stress and recovery. These variables were correlated with gene expression of the aquaporins OePIP1.1 and OePIP2.1, and stromal CA. At mild stress and at the beginning of the recovery period, stomatal limitations prevailed, while the decline in g m accounted for up to 60% of photosynthesis limitations under severe water stress. However, g m was restored to control values shortly after rewatering, facilitating the recovery of the photosynthetic rate. CA was downregulated during water stress and upregulated after recovery. The use of structural equation modelling allowed us to conclude that both OePIP1.1 and OePIP2.1 expression could explain most of the variations observed for g s and g m. CA expression also had a small but significant effect on g m in olive under water-stress conditions. PMID:24799563

  4. Cadmium sorption, influx, and efflux at the mesophyll layer of leaves from ecotypes of the Zn/Cd hyperaccumulator Thlaspi caerulescens.

    PubMed

    Ebbs, Stephen D; Zambrano, M Clemencia; Spiller, Shawna M; Newville, Matthew

    2009-01-01

    Differential sorption and transport characteristics of the leaf mesophyll layer of the Prayon and Ganges ecotypes of the hyperaccumulator Thlaspi caerulescens were examined. (109)Cd influx and efflux experiments were conducted with leaf sections, and X-ray absorption near edge structure (XANES) data were collected from leaves as a general comparison of in vivo cadmium (Cd) coordination. There were modest differences in cell wall sorption of Cd between ecotypes. There were obvious differences in time- and concentration-dependent Cd influx, including a greater V(MAX) for Prayon but a lower K(M) for Ganges for concentration-dependent Cd uptake and a notably greater Cd uptake by Ganges leaf sections at 1000 microm Cd. Leaf sections of Prayon had a greater Cd efflux than Ganges. The XANES spectra from the two ecotypes suggested differences in Cd coordination. The fundamental differences observed between the two ecotypes may reflect differential activity and/or expression of plasma membrane and tonoplast transporters. More detailed study of these transporters and the in vivo coordination of Cd are needed to determine the contribution of these processes to metal homeostasis and tolerance. PMID:19054336

  5. Ultrastructural localization of photosynthetic and photorespiratory enzymes in epidermal, mesophyll, bundle sheath, and vascular bundle cells of the C4 dicot Amaranthus viridis.

    PubMed

    Ueno, O

    2001-05-01

    In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells. PMID:11432917

  6. Light acclimation of photosynthesis in two closely related firs (Abies pinsapo Boiss. and Abies alba Mill.): the role of leaf anatomy and mesophyll conductance to CO2.

    PubMed

    Peguero-Pina, José Javier; Sancho-Knapik, Domingo; Flexas, Jaume; Galmés, Jeroni; Niinemets, Ülo; Gil-Pelegrín, Eustaquio

    2016-03-01

    Leaves growing in the forest understory usually present a decreased mesophyll conductance (gm) and photosynthetic capacity. The role of leaf anatomy in determining the variability in gm among species is known, but there is a lack of information on how the acclimation of gm to shade conditions is driven by changes in leaf anatomy. Within this context, we demonstrated that Abies pinsapo Boiss. experienced profound modifications in needle anatomy to drastic changes in light availability that ultimately led to differential photosynthetic performance between trees grown in the open field and in the forest understory. In contrast to A. pinsapo, its congeneric Abies alba Mill. did not show differences either in needle anatomy or in photosynthetic parameters between trees grown in the open field and in the forest understory. The increased gm values found in trees of A. pinsapo grown in the open field can be explained by occurrence of stomata at both needle sides (amphistomatous needles), increased chloroplast surface area exposed to intercellular airspace, decreased cell wall thickness and, especially, decreased chloroplast thickness. To the best of our knowledge, the role of such drastic changes in ultrastructural needle anatomy in explaining the response of gm to the light environment has not been demonstrated in field conditions. PMID:26543153

  7. Sustained enhancement of photosynthesis in coffee trees grown under free-air CO2 enrichment conditions: disentangling the contributions of stomatal, mesophyll, and biochemical limitations.

    PubMed

    DaMatta, Fábio M; Godoy, Alice G; Menezes-Silva, Paulo E; Martins, Samuel C V; Sanglard, Lílian M V P; Morais, Leandro E; Torre-Neto, André; Ghini, Raquel

    2016-01-01

    Coffee (Coffea spp.), a globally traded commodity, is a slow-growing tropical tree species that displays an improved photosynthetic performance when grown under elevated atmospheric CO2 concentrations ([CO2]). To investigate the mechanisms underlying this response, two commercial coffee cultivars (Catuaí and Obatã) were grown using the first free-air CO2 enrichment (FACE) facility in Latin America. Measurements were conducted in two contrasting growth seasons, which were characterized by the high (February) and low (August) sink demand. Elevated [CO2] led to increases in net photosynthetic rates (A) in parallel with decreased photorespiration rates, with no photochemical limitations to A. The stimulation of A by elevated CO2 supply was more prominent in August (56% on average) than in February (40% on average). Overall, the stomatal and mesophyll conductances, as well as the leaf nitrogen and phosphorus concentrations, were unresponsive to the treatments. Photosynthesis was strongly limited by diffusional constraints, particularly at the stomata level, and this pattern was little, if at all, affected by elevated [CO2]. Relative to February, starch pools (but not soluble sugars) increased remarkably (>500%) in August, with no detectable alteration in the maximum carboxylation capacity estimated on a chloroplast [CO2] basis. Upregulation of A by elevated [CO2] took place with no signs of photosynthetic downregulation, even during the period of low sink demand, when acclimation would be expected to be greatest. PMID:26503540

  8. Enhanced Photosynthesis and Growth in atquac1 Knockout Mutants Are Due to Altered Organic Acid Accumulation and an Increase in Both Stomatal and Mesophyll Conductance.

    PubMed

    Medeiros, David B; Martins, Samuel C V; Cavalcanti, João Henrique F; Daloso, Danilo M; Martinoia, Enrico; Nunes-Nesi, Adriano; DaMatta, Fábio M; Fernie, Alisdair R; Araújo, Wagner L

    2016-01-01

    Stomata control the exchange of CO2 and water vapor in land plants. Thus, whereas a constant supply of CO2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions. PMID:26542441

  9. Sustained enhancement of photosynthesis in coffee trees grown under free-air CO2 enrichment conditions: disentangling the contributions of stomatal, mesophyll, and biochemical limitations

    PubMed Central

    DaMatta, Fábio M.; Godoy, Alice G.; Menezes-Silva, Paulo E.; Martins, Samuel C.V.; Sanglard, Lílian M.V.P.; Morais, Leandro E.; Torre-Neto, André; Ghini, Raquel

    2016-01-01

    Coffee (Coffea spp.), a globally traded commodity, is a slow-growing tropical tree species that displays an improved photosynthetic performance when grown under elevated atmospheric CO2 concentrations ([CO2]). To investigate the mechanisms underlying this response, two commercial coffee cultivars (Catuaí and Obatã) were grown using the first free-air CO2 enrichment (FACE) facility in Latin America. Measurements were conducted in two contrasting growth seasons, which were characterized by the high (February) and low (August) sink demand. Elevated [CO2] led to increases in net photosynthetic rates (A) in parallel with decreased photorespiration rates, with no photochemical limitations to A. The stimulation of A by elevated CO2 supply was more prominent in August (56% on average) than in February (40% on average). Overall, the stomatal and mesophyll conductances, as well as the leaf nitrogen and phosphorus concentrations, were unresponsive to the treatments. Photosynthesis was strongly limited by diffusional constraints, particularly at the stomata level, and this pattern was little, if at all, affected by elevated [CO2]. Relative to February, starch pools (but not soluble sugars) increased remarkably (>500%) in August, with no detectable alteration in the maximum carboxylation capacity estimated on a chloroplast [CO2] basis. Upregulation of A by elevated [CO2] took place with no signs of photosynthetic downregulation, even during the period of low sink demand, when acclimation would be expected to be greatest. PMID:26503540

  10. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine. [Hordeum vulgare L

    SciTech Connect

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E. )

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using {sup 14}C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.

  11. Regulation of photosynthesis and stomatal and mesophyll conductance under water stress and recovery in olive trees: correlation with gene expression of carbonic anhydrase and aquaporins.

    PubMed

    Perez-Martin, Alfonso; Michelazzo, Chiara; Torres-Ruiz, Jose M; Flexas, Jaume; Fernández, José E; Sebastiani, Luca; Diaz-Espejo, Antonio

    2014-07-01

    The hypothesis that aquaporins and carbonic anhydrase (CA) are involved in the regulation of stomatal (g s) and mesophyll (g m) conductance to CO2 was tested in a short-term water-stress and recovery experiment in 5-year-old olive plants (Olea europaea) growing outdoors. The evolution of leaf gas exchange, chlorophyll fluorescence, and plant water status, and a quantitative analysis of photosynthesis limitations, were followed during water stress and recovery. These variables were correlated with gene expression of the aquaporins OePIP1.1 and OePIP2.1, and stromal CA. At mild stress and at the beginning of the recovery period, stomatal limitations prevailed, while the decline in g m accounted for up to 60% of photosynthesis limitations under severe water stress. However, g m was restored to control values shortly after rewatering, facilitating the recovery of the photosynthetic rate. CA was downregulated during water stress and upregulated after recovery. The use of structural equation modelling allowed us to conclude that both OePIP1.1 and OePIP2.1 expression could explain most of the variations observed for g s and g m. CA expression also had a small but significant effect on g m in olive under water-stress conditions. PMID:24799563

  12. The Metabolite Pathway between Bundle Sheath and Mesophyll: Quantification of Plasmodesmata in Leaves of C3 and C4 Monocots[OPEN

    PubMed Central

    Quick, William Paul

    2016-01-01

    C4 photosynthesis is characterized by a CO2-concentrating mechanism between mesophyll (M) and bundle sheath (BS) cells of leaves. This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). Quantification of these symplastic fluxes for modeling studies requires accurate quantification of PD, which has proven difficult using transmission electron microscopy. Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. 3D immunolocalization of callose at pitfields using confocal microscopy showed that pitfield area per M-BS interface area was 5 times greater in C4 species. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm−2; maize, 7.5 PD µm−2; rice 1.0 PD µm−2; wheat, 2.6 PD µm−2). Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 acid flux per PD across the M-BS interface. These quantitative data are essential for modeling studies and gene discovery strategies needed to introduce aspects of C4 photosynthesis to C3 crops. PMID:27288224

  13. The relationship between turgor pressure and titratable acidity in mesophyll cells of intact leaves of a Crassulacean-acid-metabolism plant, Kalanchoe daigremontiana Hamet et Perr.

    PubMed

    Rygol, J; Winter, K; Zimmermann, U

    1987-12-01

    Day/night changes in turgor pressure (P) and titratable acidity content were investigated in the (Crassulacean-acid-metabolism (CAM) plant Kalanchoe daigremontiana. Measurements of P were made on individual mesophyll cells of intact attached leaves using the pressure-probe technique. Under conditions of high relative humidity, when transpiration rates were minimal, changes in P correlated well with changes in the level of titratable acidity. During the standard 12 h light/12 h dark cycle, maximum turgor pressure (0.15 MPa) occurred at the end of the dark period when the level of titratable acidity was highest (about 300 μeq H(+)·g(-1) fresh weight). A close relationship between P and titratable acidity was also seen in leaves exposed to perturbations of the standard light/dark cycle. (The dark period was either prolonged, or else only CO2-free air was supplied in this period). In plants deprived of irrigation for five weeks, diurnal changes in titratable acidity of the leaves were reduced (ΔH=160 μeq H(+)·g(-1) fresh weight) and P increased from essentially zero at the end of the light period to 0.02 MPa at the end of the dark period. Following more severe water stress (experiments were made on leaves which had been detached for five weeks), P was zero throughout day and night, yet small diurnal changes in titratable acidity were still measured. These findings are discussed in relation to a hypothesis by Lüttge et al. 1975 (Plant Physiol. 56,613-616) for the role of P in the regulation of acidification/de-acidification cycles of plants exhibiting CAM. PMID:24226067

  14. Interactive effects of soil water deficit and air vapour pressure deficit on mesophyll conductance to CO2 in Vitis vinifera and Olea europaea.

    PubMed

    Perez-Martin, A; Flexas, J; Ribas-Carbó, M; Bota, J; Tomás, M; Infante, J M; Diaz-Espejo, A

    2009-01-01

    The present work aims to study the interactive effect of drought stress and high vapour pressure deficit (VPD) on leaf gas exchange, and especially on mesophyll conductance to CO(2) (g(m)), in two woody species of great agronomical importance in the Mediterranean basin: Vitis vinifera L. cv. Tempranillo and Olea europaea L. cv. Manzanilla. Plants were grown in specially designed outdoor chambers with ambient and below ambient VPD, under both well-irrigated and drought conditions. g(m) was estimated by the variable J method from simultaneous measurements of gas exchange and fluorescence. In both species, the response to soil water deficit was larger in g(s) than in g(m), and more important than the response to VPD. Olea europaea was apparently more sensitive to VPD, so that plants growing in more humid chambers showed higher g(s) and g(m). In V. vinifera, in contrast, soil water deficit dominated the response of g(s) and g(m). Consequently, changes in g(m)/g(s) were more related to VPD in O. europaea and to soil water deficit in V. vinifera. Most of the limitations of photosynthesis were diffusional and especially due to stomatal closure. No biochemical limitation was detected. The results showed that structural parameters played an important role in determining g(m) during the acclimation process. Although the relationship between leaf mass per unit area (M(A)) with g(m) was scattered, it imposed a limitation to the maximum g(m) achievable, with higher values of M(A) in O. europaea at lower g(m) values. M(A) decreased under water stress in O. europaea but it increased in V. vinifera. This resulted in a negative relationship between M(A) and the CO(2) draw-down between substomatal cavities and chloroplasts in O. europaea, while being positive in V. vinifera. PMID:19457982

  15. Auxin-induced regulation of protein synthesis in tobacco mesophyll protoplasts cultivated in vitro: I. Characteristics of auxin-sensitive proteins.

    PubMed

    Meyer, Y; Aspart, L; Chartier, Y

    1984-08-01

    The presence of auxin (2,4-D), in the culture medium of tobacco (Nicotiana tabacum var Maryland) mesophyll protoplasts is necessary both for cell wall regeneration and for passage of the cells from phase G(0) to phase G(1) of the cell cycle. Among about 250 proteins synthesized by protoplasts and characterized by their migration in a two-dimensional electrophoresis gel, 2,4-dichlorophenoxyacetic acid affects the synthesis of 11.Nine proteins are synthesized at a reduced level in the presence of the hormone, of which three are rapidly labeled and short-lived, while the others, which are long-lived, become detectable only after 2 hours of radioactive labeling, suggesting that they undergo slow posttranslational maturation. These nine proteins are proline-rich but the proline radicals are not strongly hydroxylated. The synthesis of these proteins is no longer inhibited by auxin if dichlorobenzonitril, a weed-killer which inhibits cell wall reformation of tobacco protoplasts, is added to the culture medium.Two proteins are only synthesized if protoplasts are cultivated in an auxin-containing medium. These polypeptides are rapidly labeled, and are long-lived. The inhibition of cell wall reformation by dichlorobenzonitril does not modify their synthesis.These results suggest that proteins whose synthesis is reduced by auxin are related to cell wall reformation and that they do not play a role in the induction of the cell cycle. In contrast, proteins whose synthesis is stimulated in the presence of auxin are good candidates for a role in the induction of the cell cycle. PMID:16663728

  16. Assessing sex-related chick provisioning in greater flamingo Phoenicopterus roseus parents using capture-recapture models.

    PubMed

    Rendón, Miguel A; Garrido, Araceli; Rendón-Martos, Manuel; Ramírez, José M; Amat, Juan A

    2014-03-01

    In sexually dimorphic species, the parental effort of the smaller sex may be reduced due to competitive exclusion in the feeding areas by the larger sex or physiological constraints. However, to determine gender effects on provisioning patterns, other intrinsic and extrinsic factors affecting parental effort should be accounted for. Greater flamingos (Phoenicopterus roseus) exhibit sexual size dimorphism. In Fuente de Piedra colony, the lake dries out almost completely during the breeding season and both parents commute between breeding and foraging sites >130 km away during the chick-rearing period. Applying multistate capture-recapture models to daily observations of marked parents, we determined the effects of sex, and their interactions with other intrinsic and extrinsic factors, on the probability of chick desertion and sojourn in the colony and feeding areas. Moreover, using stable isotopes in the secretions that parents produce to feed their chicks, we evaluated sex-specific use of wetlands. The probability of chick attendance (complementary to chick desertion) was >0.98. Chick desertion was independent of parental sex, but decreased with parental age. Females stayed in the feeding areas for shorter periods [mean: 7.5 (95% CI: 6.0-9.4) days] than males [9.2 (7.3-11.8) days]. Isotopic signatures of secretions did not show sex differences in δ(13)C, but males' secretions were enriched in δ(15)N, suggesting they fed on prey of higher trophic levels than females. Both parents spent approximately 1 day in the colony, but females prolonged their mean stay when the lake dried out. Females also allocated more time to foraging in the flooded areas remaining in the colony, likely because they were energetically more stressed than males. The results indicate that sex-specific provisioning behaviour in greater flamingo is related to differential effects of both intrinsic and extrinsic factors. Males seem forage less efficiently than females, whereas females' body

  17. High quality draft genome sequence and analysis of Pontibacter roseus type strain SRC-1T (DSM 17521T) isolated from muddy waters of a drainage system in Chandigarh, India

    SciTech Connect

    Mukherjee, Supratim; Lapidus, Alla; Shapiro, Nicole; Cheng, Jan -Fang; Han, James; Reddy, T. B. K.; Huntemann, Marcel; Ivanova, Natalia; Mikhailova, Natalia; Chen, Amy; Palaniappan, Krishna; Spring, Stefan; Goker, Markus; Markowitz, Victor; Woyke, Tanja; Tindall, Brian J.; Klenk, Hans -Peter; Kyrpides, Nikos C.; Pati, Amrita

    2015-02-09

    Pontibacter roseus is a member of genus Pontibacter family Cytophagaceae, class Cytophagia. While the type species of the genus Pontibacter actiniarum was isolated in 2005 from a marine environment, subsequent species of the same genus have been found in different types of habitats ranging from seawater, sediment, desert soil, rhizosphere, contaminated sites, solar saltern and muddy water. Here we describe the features of Pontibacter roseus strain SRC-1T along with its complete genome sequence and annotation from a culture of DSM 17521T. In conclusion, the 4,581,480 bp long draft genome consists of 12 scaffolds with 4,003 protein-coding and 50 RNA genes and is a part of Genomic Encyclopedia of Type Strains: KMG-I project.

  18. High quality draft genome sequence and analysis of Pontibacter roseus type strain SRC-1T (DSM 17521T) isolated from muddy waters of a drainage system in Chandigarh, India

    SciTech Connect

    Mukherjee, Supratim; Lapidus, Alla; Shapiro, Nicole; Cheng, Jan-Fang; Han, James; Reddy, T. B.K.; Huntemann, Marcel; Ivanova, Natalia; Mikhailova, Natalia; Chen, Amy; Palaniappan, Krishna; Spring, Stefan; Gö ker, Markus; Woyke, Tanja; Tindall, Brian J.; Klenk, Hans-Peter; Pati, Amrita

    2014-11-26

    Pontibacter roseus Suresh et al 2006 is a member of genus Pontibacter family Cytophagaceae, class Cytophagia. While the type species of the genus Pontibacter actiniarum was isolated in 2005 from a marine environment, subsequent species of the same genus have been found in different types of habitats ranging from seawater, sediment, desert soil, rhizosphere, contaminated sites, solar saltern and muddy water. Here we describe the features of Pontibacter roseus strain SRC-1T along with its complete genome sequence and annotation from a culture of DSM 17521T. The 4,581,480 bp long draft genome consists of 12 scaffolds with 4,003 protein-coding and 50 RNA genes and is a part of Genomic encyclopedia of Type Strains, Phase I: the one thousand microbial genomes (KMG-I) project.

  19. High quality draft genome sequence and analysis of Pontibacter roseus type strain SRC-1T (DSM 17521T) isolated from muddy waters of a drainage system in Chandigarh, India

    DOE PAGESBeta

    Mukherjee, Supratim; Lapidus, Alla; Shapiro, Nicole; Cheng, Jan -Fang; Han, James; Reddy, T. B. K.; Huntemann, Marcel; Ivanova, Natalia; Mikhailova, Natalia; Chen, Amy; et al

    2015-02-09

    Pontibacter roseus is a member of genus Pontibacter family Cytophagaceae, class Cytophagia. While the type species of the genus Pontibacter actiniarum was isolated in 2005 from a marine environment, subsequent species of the same genus have been found in different types of habitats ranging from seawater, sediment, desert soil, rhizosphere, contaminated sites, solar saltern and muddy water. Here we describe the features of Pontibacter roseus strain SRC-1T along with its complete genome sequence and annotation from a culture of DSM 17521T. In conclusion, the 4,581,480 bp long draft genome consists of 12 scaffolds with 4,003 protein-coding and 50 RNA genesmore » and is a part of Genomic Encyclopedia of Type Strains: KMG-I project.« less

  20. High quality draft genome sequence and analysis of Pontibacter roseus type strain SRC-1T (DSM 17521T) isolated from muddy waters of a drainage system in Chandigarh, India

    PubMed Central

    2015-01-01

    Pontibacter roseus is a member of genus Pontibacter family Cytophagaceae, class Cytophagia. While the type species of the genus Pontibacter actiniarum was isolated in 2005 from a marine environment, subsequent species of the same genus have been found in different types of habitats ranging from seawater, sediment, desert soil, rhizosphere, contaminated sites, solar saltern and muddy water. Here we describe the features of Pontibacter roseus strain SRC-1T along with its complete genome sequence and annotation from a culture of DSM 17521T. The 4,581,480 bp long draft genome consists of 12 scaffolds with 4,003 protein-coding and 50 RNA genes and is a part of Genomic Encyclopedia of Type Strains: KMG-I project. PMID:26203325

  1. Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat : Effect of electron transfer inhibitors and uncouplers.

    PubMed

    Goller, M; Hampp, R; Ziegler, H

    1982-12-01

    Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448-455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the

  2. Valine-Resistance, a Potential Marker in Plant Cell Genetics. II. Optimization of Uv Mutagenesis and Selection of Valine-Resistant Colonies Derived from Tobacco Mesophyll Protoplasts.

    PubMed

    Grandbastien, M A; Bourgin, J P; Caboche, M

    1985-02-01

    The induction and selection of valine-resistant mutants from haploid tobacco (Nicotiana tabacum L.) mesophyll protoplast-derived cells have been studied. Using cells from an original mutant plant obtained previously, we performed reconstruction experiments in order to determine the best conditions for the recovery of resistant cells among a population of sensitive cells. Optimal selective conditions were shown to depend on various factors including cell density, time of addition of valine and seasonal variations affecting the mother plants.-Using cell densities of approximately 10( 4) cells/ml, we defined efficient selective conditions: more than 25% of the putative mutant clones selected from UV-mutagenized protoplasts were reproducibly confirmed to be valine resistant. Further characterization of some regenerated mutant plants indicated that valine-resistance was associated with an uptake deficiency, as in the case of the original mutant plant of the Val(r)-2 line used for reconstruction experiments. Spontaneous mutation rates for valine-resistance were below accurately detectable levels, i.e., less than 10(-6) per cell per generation. Induced mutation frequency varied nonlinearily with UV dose from 10(-5) to 5 x 10(-4) resistant clones per surviving colony. Two independent loci (vr2 and vr3) were previously shown to be involved in valine-resistance due to amino acid uptake deficiency. Haploid tobacco plants were produced through anther culture from an F(1) double-heterozygous plant obtained from a cross between the original mutant plant and a wild-type plant. Study of the level of resistance to valine of protoplast-derived cells allowed the classification of these haploid plants in four types: sensitive, resistant and two intermediary resistant types believed to result from the presence of a mutant allele at only one of the two loci involved. The frequencies of UV-induced mutations in cells derived from haploid plants of one of the intermediary types were

  3. Comparative Proteomics of Chloroplasts Envelopes from Bundle Sheath and Mesophyll Chloroplasts Reveals Novel Membrane Proteins with a Possible Role in C4-Related Metabolite Fluxes and Development

    PubMed Central

    Manandhar-Shrestha, K.; Tamot, B.; Pratt, E. P. S.; Saitie, S.; Bräutigam, A.; Weber, A. P. M.; Hoffmann-Benning, Susanne

    2013-01-01

    As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second “green revolution” will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2–15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene

  4. Plant, cell, and molecular mechanisms of abscisic-acid regulation of stomatal apertures. A new mechanism for the regulation of stomatal-aperture size in intact leaves: Accumulation of mesophyll-derived sucrose in the guard-cell wall of Vicia faba L.

    SciTech Connect

    Lu, P.; Outlaw, W.H. Jr.; Smith, B.G.; Freed, G.A.

    1996-12-31

    At various times after pulse labeling Vicia faba L. leaflets with {sup 14}CO{sub 2}, whole-leaf pieces and rinsed epidermal peels were harvested and subsequently processed for histochemical analysis. Cells dissected from whole leaf retained apoplastic contents whereas those from rinsed peels contained only cytoplastic contents. Sucrose specific radioactivity peaked in palisade cells, 111 GBq{center_dot}mol{sup {minus}1}, at 20 min. In contrast, the {sup 14}C content and sucrose specific radioactivity were very low in guard cells for 20 min, implying little CO{sub 2} incorporation; both then peaked at 40 min. The guard-cell apoplast had a high maximum sucrose specific radioactivity and a high sucrose influx rate. These and other comparisons implied the presence of (a) multiple sucrose pools in mesophyll cells, (b) a localized mesophyll-apoplast region that exchanges with phloem and stomata, and (c) mesophyll-derived sucrose in guard-cell walls sufficient to diminish stomatal opening by {approximately} 4 {micro}m. Factors expected to enhance sucrose accumulation in guard-cell walls are (a) high transpiration rate, which closes stomata, and (b) high apoplastic sucrose concentration, which is elevated when mesophyll-sucrose efflux exceeds translocation. Therefore, multiple physiological factors are integrated in the attenuation of stomatal-aperture size by this previously unrecognized mechanism.

  5. Seed transmission of Candidatus Liberibacter asiaticus in periwinkle and dodder resulted in low bacterial titer and very mild disease in periwinkle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Canadidatus Liberibacter asiaticus (Las) is the most widely-distributed of three species of Liberibacter that are associated with citrus Huanglongbing (HLB), a lethal disease of citrus worldwide. In addition to citrus, periwinkle (Catharanthus roseus) and dodder (Cuscuta pentagona) are two experime...

  6. The Operation of Two Decarboxylases, Transamination, and Partitioning of C4 Metabolic Processes between Mesophyll and Bundle Sheath Cells Allows Light Capture To Be Balanced for the Maize C4 Pathway1[W

    PubMed Central

    Bellasio, Chandra; Griffiths, Howard

    2014-01-01

    The C4 photosynthesis carbon-concentrating mechanism in maize (Zea mays) has two CO2 delivery pathways to the bundle sheath (BS; via malate or aspartate), and rates of phosphoglyceric acid reduction, starch synthesis, and phosphoenolpyruvate regeneration also vary between BS and mesophyll (M) cells. The theoretical partitioning of ATP supply between M and BS cells was derived for these metabolic activities from simulated profiles of light penetration across a leaf, with a potential 3-fold difference in the fraction of ATP produced in the BS relative to M (from 0.29 to 0.96). A steady-state metabolic model was tested using varying light quality to differentially stimulate M or BS photosystems. CO2 uptake, ATP production rate (JATP; derived with a low oxygen/chlorophyll fluorescence method), and carbon isotope discrimination were measured on plants under a low light intensity, which is considered to affect C4 operating efficiency. The light quality treatments did not change the empirical ATP cost of gross CO2 assimilation (JATP/GA). Using the metabolic model, measured JATP/GA was compared with the predicted ATP demand as metabolic functions were varied between M and BS. Transamination and the two decarboxylase systems (NADP-malic enzyme and phosphoenolpyruvate carboxykinase) were critical for matching ATP and reduced NADP demand in BS and M when light capture was varied under contrasting light qualities. PMID:24254314

  7. Analysis toward innovative herbal antibacterial and antifungal drugs.

    PubMed

    Dhankhar, Sandeep; Dhankhar, Seema; Kumar, Manish; Ruhil, Sonam; Balhara, Meenakshi; Chhillar, Anil K

    2012-12-01

    The antimicrobial activities of four medicinal plants Argemona mexicana, Achyranthes aspera, Catharanthus roseus, and Syzygium cumini were evaluated against Escherichia coli, Vibrio cholerae, Klebsiella pneumoniae, Proteus vulgaris, Bacillus subtilis, Salmonella typhi and three Aspergillus species. Extracts from Achyranthes aspera and Catharanthus roseus showed the highest antimicrobial potential (MIC 0.375-0.750 mg/ml) while extract from Argemona mexicana and Syzygium cumini, showed less activity. In disc diffusion assay, only eight out of twenty extracts showed antimicrobial activity at a concentration of 25.0 μg/ disc. The GCMS investigation reveals the existence of 2-bornanone; 1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester; hexadecanoic acid, methyl ester and hexatriacontane in water extract fraction of C. roseus. The present research article provides a review of some medicinal plants incorporating antimicrobial drugs, together with recent advances in emerging therapeutics in clinical development and related patents for exploitation of herbal medicine. PMID:23072646

  8. Inconsistency of mesophyll conductance estimate causes the inconsistency for the estimates of maximum rate of Rubisco carboxylation among the linear, rectangular and non-rectangular hyperbola biochemical models of leaf photosynthesis--a case study of CO₂ enrichment and leaf aging effects in soybean.

    PubMed

    Sun, Jindong; Feng, Zhaozhong; Leakey, Andrew D B; Zhu, Xinguang; Bernacchi, Carl J; Ort, Donald R

    2014-09-01

    The responses of CO2 assimilation to [CO2] (A/Ci) were investigated at two developmental stages (R5 and R6) and in several soybean cultivars grown under two levels of CO2, the ambient level of 370 μbar versus the elevated level of 550 μbar. The A/Ci data were analyzed and compared by either the combined iterations or the separated iterations of the Rubisco-limited photosynthesis (Ac) and/or the RuBP-limited photosynthesis (Aj) using various curve-fitting methods: the linear 2-segment model; the non-rectangular hyperbola model; the rectangular hyperbola model; the constant rate of electron transport (J) method and the variable J method. Inconsistency was found among the various methods for the estimation of the maximum rate of carboxylation (Vcmax), the mitochondrial respiration rate in the light (Rd) and mesophyll conductance (gm). The analysis showed that the inconsistency was due to inconsistent estimates of gm values that decreased with an instantaneous increase in [CO2], and varied with the transition Ci cut-off between Rubisco-limited photosynthesis and RuBP-regeneration-limited photosynthesis, and due to over-parameters for non-linear curve-fitting with gm included. We proposed an alternate solution to A/Ci curve-fitting for estimates of Vcmax, Rd, Jmax and gm with the various A/Ci curve-fitting methods. The study indicated that down-regulation of photosynthetic capacity by elevated [CO2] and leaf aging was due to partially the decrease in the maximum rates of carboxylation and partially the decrease in gm. Mesophyll conductance lowered photosynthetic capacity by 18% on average for the case of soybean plants. PMID:25113450

  9. Structural determinants of reductive terpene cyclization in iridoid biosynthesis.

    PubMed

    Kries, Hajo; Caputi, Lorenzo; Stevenson, Clare E M; Kamileen, Mohammed O; Sherden, Nathaniel H; Geu-Flores, Fernando; Lawson, David M; O'Connor, Sarah E

    2016-01-01

    The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (from Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homolog progesterone 5β-reductase are highlighted. PMID:26551396

  10. Structural determinants of reductive terpene cyclization in iridoid biosynthesis

    PubMed Central

    Stevenson, Clare E. M.; Kamileen, Mohammed O.; Sherden, Nathaniel H.; Geu-Flores, Fernando; Lawson, David M.; O’Connor, Sarah E.

    2015-01-01

    The carbon skeleton of ecologically and pharmacologically important iridoid monoterpenes is formed in a reductive cyclization reaction unrelated to canonical terpene cyclization. Here we report the crystal structure of the recently discovered iridoid cyclase (Catharanthus roseus) bound to a mechanism-inspired inhibitor that illuminates substrate binding and catalytic function of the enzyme. Key features that distinguish iridoid synthase from its close homologue, progesterone 5β-reductase, are highlighted. PMID:26551396

  11. ZCT1 and ZCT2 transcription factors repress the activity of a gene promoter from the methyl erythritol phosphate pathway in Madagascar periwinkle cells.

    PubMed

    Chebbi, Mouadh; Ginis, Olivia; Courdavault, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Clastre, Marc; Papon, Nicolas; Gaillard, Cécile; Atanassova, Rossitza; St-Pierre, Benoit; Giglioli-Guivarc'h, Nathalie; Courtois, Martine; Oudin, Audrey

    2014-10-15

    In Catharanthus roseus, accumulating data highlighted the existence of a coordinated transcriptional regulation of structural genes that takes place within the secoiridoid biosynthetic branch, including the methyl erythritol phosphate (MEP) pathway and the following steps leading to secologanin. To identify transcription factors acting in these pathways, we performed a yeast one-hybrid screening using as bait a promoter region of the hydroxymethylbutenyl 4-diphosphate synthase (HDS) gene involved in the responsiveness of C. roseus cells to hormonal signals inducing monoterpene indole alkaloid (MIA) production. We identified that ZCT2, one of the three members of the zinc finger Catharanthus protein (ZCT) family, can bind to a HDS promoter region involved in hormonal responsiveness. By trans-activation assays, we demonstrated that ZCT1 and ZCT2 but not ZCT3 repress the HDS promoter activity. Gene expression analyses in C. roseus cells exposed to methyljasmonate revealed a persistence of induction of ZCT2 gene expression suggesting the existence of feed-back regulatory events acting on HDS gene expression in correlation with the MIA production. PMID:25108262

  12. Review of the taxonomy of the genus Arthrobacter, emendation of the genus Arthrobacter sensu lato, proposal to reclassify selected species of the genus Arthrobacter in the novel genera Glutamicibacter gen. nov., Paeniglutamicibacter gen. nov., Pseudoglutamicibacter gen. nov., Paenarthrobacter gen. nov. and Pseudarthrobacter gen. nov., and emended description of Arthrobacter roseus.

    PubMed

    Busse, Hans-Jürgen

    2016-01-01

    In this paper, the taxonomy of the genus Arthrobacter is discussed, from its first description in 1947 to the present state. Emphasis is given to intrageneric phylogeny and chemotaxonomic characteristics, concentrating on quinone systems, peptidoglycan compositions and polar lipid profiles. Internal groups within the genus Arthrobacter indicated from homogeneous chemotaxonomic traits and corresponding to phylogenetic grouping and/or high 16S rRNA gene sequence similarities are highlighted. Furthermore, polar lipid profiles and quinone systems of selected species are shown, filling some gaps concerning these chemotaxonomic traits. Based on phylogenetic groupings, 16S rRNA gene sequence similarities and homogeneity in peptidoglycan types, quinone systems and polar lipid profiles, a description of the genus Arthrobacter sensu lato and an emended description of Arthrobacter roseus are provided. Furthermore, reclassifications of selected species of the genus Arthrobacter into novel genera are proposed, namely Glutamicibacter gen. nov. (nine species), Paeniglutamicibacter gen. nov. (six species), Pseudoglutamicibacter gen. nov. (two species), Paenarthrobacter gen. nov. (six species) and Pseudarthrobacter gen. nov. (ten species). PMID:26486726

  13. Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry.

    PubMed

    Zhou, Hui; Tai, Yuanpo; Sun, Cuirong; Pan, Yuanjiang

    2005-01-01

    A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts. PMID:16223089

  14. In Vivo and Real-time Monitoring of Secondary Metabolites of Living Organisms by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hu, Bin; Wang, Lei; Ye, Wen-Cai; Yao, Zhong-Ping

    2013-07-01

    Secondary metabolites are compounds that are important for the survival and propagation of animals and plants. Our current understanding on the roles and secretion mechanism of secondary metabolites is limited by the existing techniques that typically cannot provide transient and dynamic information about the metabolic processes. In this manuscript, by detecting venoms secreted by living scorpion and toad upon attack and variation of alkaloids in living Catharanthus roseus upon stimulation, which represent three different sampling methods for living organisms, we demonstrated that in vivo and real-time monitoring of secondary metabolites released from living animals and plants could be readily achieved by using field-induced direct ionization mass spectrometry.

  15. Analysis of several iridoid and indole precursors of terpenoid indole alkaloids with a single HPLC run.

    PubMed

    Dagnino, D; Schripsema, J; Verpoorte, R

    1996-06-01

    An isocratic HPLC system is described which allows the separation of the iridoid and indole precursors of terpenoid indole alkaloids, which are present in a single crude extract. The system consists of a column of LiChrospher 60 RP select B 5 microm, 250 x 4 mm (Merck) with an eluent of 1% formic acid-acetonitrile-trichloroacetic acid (100:10:0.25, v:v:w) at a flow of 1.2 ml/min. In the suspension cultures of Catharanthus roseus secologanin and tryptophan were detected. in the cultures of Tabernaemontana divaricata loganin, tryptophan, and tryptamine accumulated. PMID:17252445

  16. Inhibition of anthocyanin formation in seedlings and flowers by the enantiomers of α-aminooxy-β-phenylpropionic acid and their N-benzyloxycarbonyl derivatives.

    PubMed

    Amrhein, N; Holländer, H

    1979-01-01

    Both enantiomers of α-aminooxy-β-phenylpropionic acid (AOPP), potent inhibitors of L-phenylalanine ammonia-lyase, and their N-benzyloxycarbonyl (N-BOC) derivatives inhibit anthocyanin formation in developing flowers of Ipomoea tricolor Cav. and Catharanthus roseus Don. as well as in seedlings of Brassica oleracea var. caulo-rapa DC (kohlrabi) and B. oleracea var. capitata L. (red cabbage) with little interference with their normal development. Kohlrabi seedlings tolerate up to 0.3 mM L-AOPP and N-BOC-L-AOPP without a reduction of fresh weight or chlorophyll content, while anthocyanin is reduced to less than 20%. PMID:24407328

  17. Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification, Subcellular Localization, and Occurrence in the Plant Kingdom).

    PubMed Central

    Wissing, J. B.; Behrbohm, H.

    1993-01-01

    Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom. PMID:12231900

  18. A novel method for rapid and non-invasive detection of plants senescence using delayed fluorescence technique

    NASA Astrophysics Data System (ADS)

    Zhang, Lingrui; Xing, Da; Wang, Junsheng; Zeng, Lizhang; Li, Qiang

    2007-05-01

    Plants senescence is a phase of plants ontogeny marked by declining photosynthetic activity that is paralleled by a decline in chloroplast function. The photosystem II ( PSII ) in a plant is considered the primary site where light-induced delayed fluorescence (DF) is produced. With the leaves of Catharanthus roseus (Catharanthus roseus (L.) G.Don) as testing models, we have studied the effects of plants senescence induced by dark and/or exogenous hormones treatments on characteristics of DF by using a home-made portable DF detection system, which can enable various DF parameters, such as DF decay kinetic curve and DF intensity, to be rapidly produced for the plants in a short time. The results show that the changes in DF intensity of green plants can truly reflect the changes in photosynthetic capacity and chlorophyll content. Therefore, DF may be used an important means of evaluating in vivo plants senescence physiology. The changes in DF intensity may provide a new approach for the rapid and early detection of plants senescence caused by age or other senescence-related factors. DF technique could be potential useful for high throughput screening and less time-consuming and automated identifying the interesting mutants with genetic modifications that change plants senescence progress.

  19. Phytochemical genomics of the Madagascar periwinkle: Unravelling the last twists of the alkaloid engine.

    PubMed

    Dugé de Bernonville, Thomas; Clastre, Marc; Besseau, Sébastien; Oudin, Audrey; Burlat, Vincent; Glévarec, Gaëlle; Lanoue, Arnaud; Papon, Nicolas; Giglioli-Guivarc'h, Nathalie; St-Pierre, Benoit; Courdavault, Vincent

    2015-05-01

    The Madagascar periwinkle produces a large palette of Monoterpenoid Indole Alkaloids (MIAs), a class of complex alkaloids including some of the most valuable plant natural products with precious therapeutical values. Evolutionary pressure on one of the hotspots of biodiversity has obviously turned this endemic Malagasy plant into an innovative alkaloid engine. Catharanthus is a unique taxon producing vinblastine and vincristine, heterodimeric MIAs with complex stereochemistry, and also manufactures more than 100 different MIAs, some shared with the Apocynaceae, Loganiaceae and Rubiaceae members. For over 60 years, the quest for these powerful anticancer drugs has inspired biologists, chemists, and pharmacists to unravel the chemistry, biochemistry, therapeutic activity, cell and molecular biology of Catharanthus roseus. Recently, the "omics" technologies have fuelled rapid progress in deciphering the last secret of strictosidine biosynthesis, the central precursor opening biosynthetic routes to several thousand MIA compounds. Dedicated C. roseus transcriptome, proteome and metabolome databases, comprising organ-, tissue- and cell-specific libraries, and other phytogenomic resources, were developed for instance by PhytoMetaSyn, Medicinal Plant Genomic Resources and SmartCell consortium. Tissue specific library screening, orthology comparison in species with or without MIA-biochemical engines, clustering of gene expression profiles together with various functional validation strategies, largely contributed to enrich the toolbox for plant synthetic biology and metabolic engineering of MIA biosynthesis. PMID:25146650

  20. Apocynaceae species with antiproliferative and/or antiplasmodial properties: a review of ten genera.

    PubMed

    Chan, Eric Wei Chiang; Wong, Siu Kuin; Chan, Hung Tuck

    2016-07-01

    Apocynaceae is a large family of tropical trees, shrubs and vines with most species producing white latex. Major metabolites of species are triterpenoids, iridoids, alkaloids and cardenolides, which are known for a wide range of biological and pharmacological activities such as cardioprotective, hepatoprotective, neuroprotective, anti-inflammatory, anticancer and antimalarial properties. Prompted by their anticancer and antimalarial properties, the current knowledge on ten genera (Allamanda, Alstonia, Calotropis, Catharanthus, Cerbera, Dyera, Kopsia, Nerium, Plumeria and Vallaris) is updated. Major classes of metabolites are described using some species as examples. Species with antiproliferative (APF) and/or antiplasmodial (APM) properties have been identified. With the exception of the genus Dyera, nine genera of 22 species possess APF activity. Seven genera (Alstonia, Calotropis, Catharanthus, Dyera, Kopsia, Plumeria and Vallaris) of 13 species have APM properties. Among these species, Alstonia angustiloba, Alstonia macrophylla, Calotropis gigantea, Calotropis procera, Catharanthus roseus, Plumeria alba and Vallaris glabra displayed both APF and APM properties. The chemical constituents of these seven species are compiled for assessment and further research. PMID:27417173

  1. Genome-guided investigation of plant natural product biosynthesis.

    PubMed

    Kellner, Franziska; Kim, Jeongwoon; Clavijo, Bernardo J; Hamilton, John P; Childs, Kevin L; Vaillancourt, Brieanne; Cepela, Jason; Habermann, Marc; Steuernagel, Burkhard; Clissold, Leah; McLay, Kirsten; Buell, Carol Robin; O'Connor, Sarah E

    2015-05-01

    The medicinal plant Madagascar periwinkle, Catharanthus roseus (L.) G. Don, produces hundreds of biologically active monoterpene-derived indole alkaloid (MIA) metabolites and is the sole source of the potent, expensive anti-cancer compounds vinblastine and vincristine. Access to a genome sequence would enable insights into the biochemistry, control, and evolution of genes responsible for MIA biosynthesis. However, generation of a near-complete, scaffolded genome is prohibitive to small research communities due to the expense, time, and expertise required. In this study, we generated a genome assembly for C. roseus that provides a near-comprehensive representation of the genic space that revealed the genomic context of key points within the MIA biosynthetic pathway including physically clustered genes, tandem gene duplication, expression sub-functionalization, and putative neo-functionalization. The genome sequence also facilitated high resolution co-expression analyses that revealed three distinct clusters of co-expression within the components of the MIA pathway. Coordinated biosynthesis of precursors and intermediates throughout the pathway appear to be a feature of vinblastine/vincristine biosynthesis. The C. roseus genome also revealed localization of enzyme-rich genic regions and transporters near known biosynthetic enzymes, highlighting how even a draft genome sequence can empower the study of high-value specialized metabolites. PMID:25759247

  2. "Self" and "non-self" in the control of phytoalexin biosynthesis: plant phospholipases A2 with alkaloid-specific molecular fingerprints.

    PubMed

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-02-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the "self" plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate "self-made" from "foreign" alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  3. “Self” and “Non-Self” in the Control of Phytoalexin Biosynthesis: Plant Phospholipases A2 with Alkaloid-Specific Molecular Fingerprints

    PubMed Central

    Heinze, Michael; Brandt, Wolfgang; Marillonnet, Sylvestre; Roos, Werner

    2015-01-01

    The overproduction of specialized metabolites requires plants to manage the inherent burdens, including the risk of self-intoxication. We present a control mechanism that stops the expression of phytoalexin biosynthetic enzymes by blocking the antecedent signal transduction cascade. Cultured cells of Eschscholzia californica (Papaveraceae) and Catharanthus roseus (Apocynaceae) overproduce benzophenanthridine alkaloids and monoterpenoid indole alkaloids, respectively, in response to microbial elicitors. In both plants, an elicitor-responsive phospholipase A2 (PLA2) at the plasma membrane generates signal molecules that initiate the induction of biosynthetic enzymes. The final alkaloids produced in the respective plant inhibit the respective PLA, a negative feedback that prevents continuous overexpression. The selective inhibition by alkaloids from the class produced in the “self” plant could be transferred to leaves of Nicotiana benthamiana via recombinant expression of PLA2. The 3D homology model of each PLA2 displays a binding pocket that specifically accommodates alkaloids of the class produced by the same plant, but not of the other class; for example, C. roseus PLA2 only accommodates C. roseus alkaloids. The interaction energies of docked alkaloids correlate with their selective inhibition of PLA2 activity. The existence in two evolutionary distant plants of phospholipases A2 that discriminate “self-made” from “foreign” alkaloids reveals molecular fingerprints left in signal enzymes during the evolution of species-specific, cytotoxic phytoalexins. PMID:25670767

  4. Four botanical extracts are toxic to the hispine beetle, Brontispa longissima, in laboratory and semi-field trials.

    PubMed

    Lv, Chaojun; Zhong, Baozhu; Zhong Guohua; Weng, Qunfang; Chen, Shaohua; Hu, Meiying; Sun, Xiaodong; Qin, Weiquan

    2012-01-01

    The potential of botanical extracts such as Celosia argenea L. (Caryophyllales: Amaranthaceae), Ricinus communis L. (Malpighiales: Euphorbiaceae), Mikania micrantha Humboldt, Bonpland & Kunth (Astrales: Asteraceae), and Catharanthus roseus (L.) G. Don (Gentianales: Apocynaceae) for the control of Brontispa longissima Gestro was evaluated in a bioassay and semi-field trial. Dose-response bioassay showed no significant difference in oral-toxicity among the extracts of C. argenea, M. micrantha, and C. roseus to larvae and adult of B. longissima. All extracts tested decreased the hatchability of B. longissima eggs. In particular, the extract of M. micrantha showed higher activity than others at the concentration of 5 mg/mL. In an antifeedant bioassay, the extract of C. argenea showed higher activity against the 1(st) larvae than that of other extracts (AF50 0.03 mg/mL), and C. roseus showed higher antifeedant activity to the 2(nd) to 5(th) larvae and adult of B. longissima (AF50 0.34, 0.33, 0.11, 0.43, and 0.20 mg/mL, respectively). The semi-field trial indicated that all extracts used in this study might reduce the pest population. Extracts of C. argenea and M. micrantha showed higher activities than that of C. roseus and R communis, and the decrease in population was 75.56% and 80.00% (without Abbott's correction) after seven days of treatment, respectively, at a concentration of 20 mg/mL. Therefore, these active botanical extracts may possess potential for use in control of B. longissima. PMID:22962939

  5. Four Botanical Extracts are Toxic to the Hispine Beetle, Brontispa longissima, in Laboratory and Semi—field Trials

    PubMed Central

    Chaojun, Lv; Baozhu, Zhong; Guohua, Zhong; Qunfang, Weng; Shaohua, Chen; Meiying, Hu; Xiaodong, Sun; Weiquan, Qin

    2012-01-01

    The potential of botanical extracts such as Celosia argenea L. (Caryophyllales: Amaranthaceae), Ricinus communis L. (Malpighiales: Euphorbiaceae), Mikania micrantha Humboldt, Bonpland & Kunth (Astrales: Asteraceae), and Catharanthus roseus (L.) G. Don (Gentianales: Apocynaceae) for the control of Brontispa longissima Gestro was evaluated in a bioassay and semi—field trial. Dose—response bioassay showed no significant difference in oral—toxicity among the extracts of C. argenea, M. micrantha, and C. roseus to larvae and adult of B. longissima. All extracts tested decreased the hatchability of B. longissima eggs. In particular, the extract of M. micrantha showed higher activity than others at the concentration of 5 mg/mL. In an antifeedant bioassay, the extract of C. argenea showed higher activity against the 1st larvae than that of other extracts (AF50 0.03 mg/mL), and C. roseus showed higher antifeedant activity to the 2nd to 5th larvae and adult of B. longissima (AF50 0.34, 0.33, 0.11, 0.43, and 0.20 mg/mL, respectively). The semi—field trial indicated that all extracts used in this study might reduce the pest population. Extracts of C. argenea and M. micrantha showed higher activities than that of C. roseus and R communis, and the decrease in population was 75.56% and 80.00% (without Abbott's correction) after seven days of treatment, respectively, at a concentration of 20 mg/mL. Therefore, these active botanical extracts may possess potential for use in control of B. longissima. PMID:22962939

  6. Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity.

    PubMed

    Stavrinides, Anna; Tatsis, Evangelos C; Caputi, Lorenzo; Foureau, Emilien; Stevenson, Clare E M; Lawson, David M; Courdavault, Vincent; O'Connor, Sarah E

    2016-01-01

    Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold. PMID:27418042

  7. Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception.

    PubMed

    Kessler, Sharon A; Lindner, Heike; Jones, Daniel S; Grossniklaus, Ueli

    2015-01-01

    The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception. PMID:25490905

  8. Predicting the substrates of cloned plant O-methyltransferases.

    PubMed

    Schröder, Gudrun; Wehinger, Elke; Schröder, Joachim

    2002-01-01

    Plant O-methyltransferases (OMTs) have important roles in secondary metabolite biosynthesis. Sequencing projects and homology-based cloning strategies yield sequences for proteins with similarities to known OMTs, but the identification of the physiological substrates is not trivial. We investigated with a cDNA cloned from Catharanthus roseus the possibilities for predicting the substrates of OMTs, using the information from previous work and two newly identified motifs that were based on information from the crystal structures of two plant OMTs. The results, confirmed by functional analysis of the recombinant protein, indicated that a careful analysis of the deduced protein sequence can provide clues for predicting the substrates of cloned OMTs. PMID:11754938

  9. Cytotoxicity of Vinca minor.

    PubMed

    Khanavi, Mahnaz; Pourmoslemi, Shabnam; Farahanikia, Behnaz; Hadjiakhoondi, Abbas; Ostad, Seyed Nasser

    2010-01-01

    Vinca minor L. (Apocynaceae) is a medicinal plant that has long been used to treat cerebral and memory disorders in European folk medicine. Furthermore, it contains more than 50 alkaloids, some of them having bisindole structure such as the antineoplastic alkaloids present in Catharanthus roseus (L.) G. Don (Apocynaceae). In this study, the plant's alkaloid extract was divided into three fractions and the cytotoxic effects on cell proliferation of HT-29, Caco-2, T47D, and NIH/3T3 cell lines were examined. All alkaloid fractions showed a dose-dependent cytotoxic effect on the cell lines. IC(50) values confirmed that the growth and proliferation of NIH/3T3 cells were less affected in comparison to other cell lines. PMID:20645762

  10. Vinca alkaloids.

    PubMed

    Moudi, Maryam; Go, Rusea; Yien, Christina Yong Seok; Nazre, Mohd

    2013-11-01

    Vinca alkaloids are a subset of drugs obtained from the Madagascar periwinkle plant. They are naturally extracted from the pink periwinkle plant, Catharanthus roseus G. Don and have a hypoglycemic as well as cytotoxic effects. They have been used to treat diabetes, high blood pressure and have been used as disinfectants. The vinca alkaloids are also important for being cancer fighters. There are four major vinca alkaloids in clinical use: Vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VDS). VCR, VBL and VRL have been approved for use in the United States. Vinflunine is also a new synthetic vinca alkaloid, which has been approved in Europe for the treatment of second-line transitional cell carcinoma of the urothelium is being developed for other malignancies. Vinca alkaloids are the second-most-used class of cancer drugs and will stay among the original cancer therapies. Different researches and studies for new vinca alkaloid applications will be carried out in this regard. PMID:24404355

  11. Vinca Alkaloids

    PubMed Central

    Moudi, Maryam; Go, Rusea; Yien, Christina Yong Seok; Nazre, Mohd.

    2013-01-01

    Vinca alkaloids are a subset of drugs obtained from the Madagascar periwinkle plant. They are naturally extracted from the pink periwinkle plant, Catharanthus roseus G. Don and have a hypoglycemic as well as cytotoxic effects. They have been used to treat diabetes, high blood pressure and have been used as disinfectants. The vinca alkaloids are also important for being cancer fighters. There are four major vinca alkaloids in clinical use: Vinblastine (VBL), vinorelbine (VRL), vincristine (VCR) and vindesine (VDS). VCR, VBL and VRL have been approved for use in the United States. Vinflunine is also a new synthetic vinca alkaloid, which has been approved in Europe for the treatment of second-line transitional cell carcinoma of the urothelium is being developed for other malignancies. Vinca alkaloids are the second-most-used class of cancer drugs and will stay among the original cancer therapies. Different researches and studies for new vinca alkaloid applications will be carried out in this regard. PMID:24404355

  12. Structural investigation of heteroyohimbine alkaloid synthesis reveals active site elements that control stereoselectivity

    PubMed Central

    Stavrinides, Anna; Tatsis, Evangelos C.; Caputi, Lorenzo; Foureau, Emilien; Stevenson, Clare E. M.; Lawson, David M.; Courdavault, Vincent; O'Connor, Sarah E.

    2016-01-01

    Plants produce an enormous array of biologically active metabolites, often with stereochemical variations on the same molecular scaffold. These changes in stereochemistry dramatically impact biological activity. Notably, the stereoisomers of the heteroyohimbine alkaloids show diverse pharmacological activities. We reported a medium chain dehydrogenase/reductase (MDR) from Catharanthus roseus that catalyses formation of a heteroyohimbine isomer. Here we report the discovery of additional heteroyohimbine synthases (HYSs), one of which produces a mixture of diastereomers. The crystal structures for three HYSs have been solved, providing insight into the mechanism of reactivity and stereoselectivity, with mutation of one loop transforming product specificity. Localization and gene silencing experiments provide a basis for understanding the function of these enzymes in vivo. This work sets the stage to explore how MDRs evolved to generate structural and biological diversity in specialized plant metabolism and opens the possibility for metabolic engineering of new compounds based on this scaffold. PMID:27418042

  13. Functional analysis of related CrRLK1L receptor-like kinases in pollen tube reception

    PubMed Central

    Kessler, Sharon A; Lindner, Heike; Jones, Daniel S; Grossniklaus, Ueli

    2015-01-01

    The Catharanthus roseus Receptor-Like Kinase 1-like (CrRLK1L) family of 17 receptor-like kinases (RLKs) has been implicated in a variety of signaling pathways in Arabidopsis, ranging from pollen tube (PT) reception and tip growth to hormonal responses. The extracellular domains of these RLKs have malectin-like domains predicted to bind carbohydrate moieties. Domain swap analysis showed that the extracellular domains of the three members analyzed (FER, ANX1, HERK1) are not interchangeable, suggesting distinct upstream components, such as ligands and/or co-factors. In contrast, their intercellular domains are functionally equivalent for PT reception, indicating that they have common downstream targets in their signaling pathways. The kinase domain is necessary for FER function, but kinase activity itself is not, indicating that other kinases may be involved in signal transduction during PT reception. PMID:25490905

  14. Oxidative stress and production of bioactive monoterpene indole alkaloids: biotechnological implications.

    PubMed

    Matsuura, Hélio Nitta; Rau, Mariana Ritter; Fett-Neto, Arthur Germano

    2014-02-01

    Monoterpene indole alkaloids (MIAs) encompass plant natural products with important pharmacological relevance. They include the anti-tumoral MIAs found in Catharanthus roseus and Camptotheca acuminata. The often low yields of bioactive alkaloids in plants has prompted research to identify the factors regulating MIA production. Oxidative stress is a general response associated with biotic and abiotic stresses leading to several secondary responses, including elicitation of MIA production. These changes in secondary metabolism may take place directly or via second messengers, such as Ca(2+) and reactive oxygen species (ROS). H2O2 is the main ROS that participates in MIA biosynthesis. This review analyzes the links between oxidative stress, elicitation of bioactive MIA production and their potential roles in antioxidant defense, as well as exploring the implications to developing biotechnological strategies relevant for alkaloid supply. PMID:24062135

  15. Understanding CrRLK1L Function: Cell Walls and Growth Control.

    PubMed

    Nissen, Karen S; Willats, William G T; Malinovsky, Frederikke G

    2016-06-01

    To develop successfully in an ever-changing environment, it is essential for plants to monitor and control their growth. Therefore, cell expansion is carefully regulated to establish correct cell shape and size. In this review, we explore the role of the Catharanthus roseus receptor-like kinase (CrRLK1L) subfamily as regulators of cell expansion. Recently, the downstream signalling events of individual CrRLK1L pathways were discovered, implicating known modulators of cell expansion, such as reactive oxygen species (ROS) production, Ca(2+) dynamics, and exocytosis of cell wall material. Based on these intriguing new insights, we propose a model for a common pathway of CrRLK1L signalling that enables spatial and temporal control of cell wall extensibility throughout the plant. PMID:26778775

  16. 10 day flight performance of the plant generic bioprocessing apparatus (PGBA) plant growth facility aboard STS-77

    NASA Astrophysics Data System (ADS)

    Hoehn, Alex; Chamberlain, Dale J.; Forsyth, Sasha W.; Hanna, David S.; Scovazzo, Paul; Horner, Michael B.; Stodieck, Louis S.; Todd, Paul; Heyenga, A. Gerard; Kliss, Mark H.; Bula, Raymond; Yetka, Robert

    1997-01-01

    PGBA, a plant growth facility developed for space flight biotechnology research, successfully grew a total of 30 plants in a closed, multi-crop chamber for 10 days aboard the Space Shuttle Endeavor (STS-77). Artemisia annua, Catharanthus roseus, Pinus taeda, Spinacia oleracea and Trifolium repens were the five species studied during this mission. The primary mission objectives were to study the effects of microgravity for commercial and pharmaceutical production purposes. PGBA is a payload that represents a consortium of interests including BioServe Space Technologies (payload sponsor), NASA Ames Research Center (Controlled Ecological Life Support System, CELSS, Flight Program), Wisconsin Center for Space Automation and Robotics (WCSAR), and industrial affiliates (spaceflight effects on plants and formation of plant products such as pharmaceuticals). Although BioServe is responsible for the flight hardware development and integration of PGBA, NASA Ames, WSCAR and industrial affiliates provide significant hardware subsystems and technical biological expertise support.

  17. An abundant 'Candidatus Phytoplasma solani' tuf b strain is associated with grapevine, stinging nettle and Hyalesthes obsoletus.

    PubMed

    Aryan, A; Brader, G; Mörtel, J; Pastar, M; Riedle-Bauer, M

    2014-10-01

    Bois noir (BN) associated with 'Candidatus Phytoplasma solani' (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of 'Ca. P. solani' in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated 'Ca. P. solani' strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. 'Candidatus Phytoplasma convolvuli' (bindweed yellows) was ascertained in nettle and bindweed samples. PMID:25309042

  18. An exploration of the potential mechanisms and translational potential of five medicinal plants for applications in Alzheimer’s disease

    PubMed Central

    Shakir, Taner; Coulibaly, Ahmed Y; Kehoe, Patrick G

    2013-01-01

    Alzheimer’s disease (AD) is the most common type of dementia, and represents a vast worldwide socio-economic burden, and in the absence of a current cure, effective therapeutic strategies are still needed. Cholinergic and cerebral blood flow deficits, excessive levels of oxidative stress, neuroinflammation and glutamate excitatory mechanisms are all believed to contribute to the development and progression of the disease. Scoparia dulcis, Catharanthus roseus, Sesamum indicum, Erythrina senegalensis and Vigna unguiculata represent five plants that have been used as traditional medicines for the treatment of AD in certain cultures. Review of the scientific literature was conducted to explore the properties of these plants that might be beneficial and explain what would be perceived by many to be largely anecdotal evidence of their benefit. All plants were found to possess varying levels of anti-oxidant capability. Scoparia dulcis was also found to potentiate nerve growth factor-like effects upon cell lines. Catharanthus roseus appears to inhibit acetylcholinesterase with relatively high potency, while Sesamum indicum demonstrated the strongest antioxidant ability. Comparisons with currently used plant derived therapeutics illustrate how these plants may be likely to have some therapeutic benefits in AD. The evidence presented also highlights how appropriate dietary supplementation with some of these plants in various cultural settings might have effects analogous or complementary to the so-called protective Mediterranean diet. However, prior to embarking on making any formal recommendations to this end, further rigorous evaluation is needed to better elucidate the breadth and potential toxicological aspects of medicinal properties harboured by these plants. This would be vital to ensuring a more informed and safe delivery of preparations of these plants if they were to be considered as a form of dietary supplementation and where appropriate, how these might

  19. An exploration of the potential mechanisms and translational potential of five medicinal plants for applications in Alzheimer's disease.

    PubMed

    Shakir, Taner; Coulibaly, Ahmed Y; Kehoe, Patrick G

    2013-01-01

    Alzheimer's disease (AD) is the most common type of dementia, and represents a vast worldwide socio-economic burden, and in the absence of a current cure, effective therapeutic strategies are still needed. Cholinergic and cerebral blood flow deficits, excessive levels of oxidative stress, neuroinflammation and glutamate excitatory mechanisms are all believed to contribute to the development and progression of the disease. Scoparia dulcis, Catharanthus roseus, Sesamum indicum, Erythrina senegalensis and Vigna unguiculata represent five plants that have been used as traditional medicines for the treatment of AD in certain cultures. Review of the scientific literature was conducted to explore the properties of these plants that might be beneficial and explain what would be perceived by many to be largely anecdotal evidence of their benefit. All plants were found to possess varying levels of anti-oxidant capability. Scoparia dulcis was also found to potentiate nerve growth factor-like effects upon cell lines. Catharanthus roseus appears to inhibit acetylcholinesterase with relatively high potency, while Sesamum indicum demonstrated the strongest antioxidant ability. Comparisons with currently used plant derived therapeutics illustrate how these plants may be likely to have some therapeutic benefits in AD. The evidence presented also highlights how appropriate dietary supplementation with some of these plants in various cultural settings might have effects analogous or complementary to the so-called protective Mediterranean diet. However, prior to embarking on making any formal recommendations to this end, further rigorous evaluation is needed to better elucidate the breadth and potential toxicological aspects of medicinal properties harboured by these plants. This would be vital to ensuring a more informed and safe delivery of preparations of these plants if they were to be considered as a form of dietary supplementation and where appropriate, how these might interact

  20. Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

    PubMed Central

    dos Santos, Bruna Silveira Lamanes; Gomes, Arthur Filipe Sousa; Franciscon, Emanuele Giuliane; de Oliveira, Jean Maikon; Baffi, Milla Alves

    2015-01-01

    Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors. PMID:26413077

  1. Medicago truncatula mutants with an increase in mesophyll calcium oxalate accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants invest a considerable amount of resources and energy into the formation of calcium oxalate crystals. A number of roles for crystal formation in plant growth and development have been assigned based on the prevalence of crystals, their spatial distribution, and the variety of crystal shapes. ...

  2. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability.

    PubMed

    Lei, Rong; Qiao, Wenjie; Hu, Fan; Jiang, Hongshan; Zhu, Shuifang

    2015-01-01

    Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. •The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays. PMID:26150968

  3. Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

    PubMed

    Islam, Md Sayeedul; Niwa, Yasuo; Takagi, Shingo

    2009-06-01

    Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells. PMID:19380350

  4. A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability☆

    PubMed Central

    Lei, Rong; Qiao, Wenjie; Hu, Fan; Jiang, Hongshan; Zhu, Shuifang

    2014-01-01

    Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. • The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer. • There are more choices for membrane pore size, depending on the application. • It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol–gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays. PMID:26150968

  5. Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle

    PubMed Central

    Roepke, Jonathan; Salim, Vonny; Wu, Maggie; Thamm, Antje M. K.; Murata, Jun; Ploss, Kerstin; Boland, Wilhelm; De Luca, Vincenzo

    2010-01-01

    The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them. PMID:20696903

  6. Vinca drug components accumulate exclusively in leaf exudates of Madagascar periwinkle.

    PubMed

    Roepke, Jonathan; Salim, Vonny; Wu, Maggie; Thamm, Antje M K; Murata, Jun; Ploss, Kerstin; Boland, Wilhelm; De Luca, Vincenzo

    2010-08-24

    The monoterpenoid indole alkaloids (MIAs) of Madagascar periwinkle (Catharanthus roseus) continue to be the most important source of natural drugs in chemotherapy treatments for a range of human cancers. These anticancer drugs are derived from the coupling of catharanthine and vindoline to yield powerful dimeric MIAs that prevent cell division. However the precise mechanisms for their assembly within plants remain obscure. Here we report that the complex development-, environment-, organ-, and cell-specific controls involved in expression of MIA pathways are coupled to secretory mechanisms that keep catharanthine and vindoline separated from each other in living plants. Although the entire production of catharanthine and vindoline occurs in young developing leaves, catharanthine accumulates in leaf wax exudates of leaves, whereas vindoline is found within leaf cells. The spatial separation of these two MIAs provides a biological explanation for the low levels of dimeric anticancer drugs found in the plant that result in their high cost of commercial production. The ability of catharanthine to inhibit the growth of fungal zoospores at physiological concentrations found on the leaf surface of Catharanthus leaves, as well as its insect toxicity, provide an additional biological role for its secretion. We anticipate that this discovery will trigger a broad search for plants that secrete alkaloids, the biological mechanisms involved in their secretion to the plant surface, and the ecological roles played by them. PMID:20696903

  7. Molecular interaction of selected phytochemicals under the charged environment of Plasmodium falciparum chloroquine resistance transporter (PfCRT) model.

    PubMed

    Patel, Saumya K; Khedkar, Vijay M; Jha, Prakash C; Jasrai, Yogesh T; Pandya, Himanshu A; George, Linz-Buoy; Highland, Hyacinth N; Skelton, Adam A

    2016-01-01

    Phytochemicals of Catharanthus roseus Linn. and Tylophora indica have been known for their inhibition of malarial parasite, Plasmodium falciparum in cell culture. Resistance to chloroquine (CQ), a widely used antimalarial drug, is due to the CQ resistance transporter (CRT) system. The present study deals with computational modeling of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein and development of charged environment to mimic a condition of resistance. The model of PfCRT was developed using Protein homology/analogy engine (PHYRE ver 0.2) and was validated based on the results obtained using PSI-PRED. Subsequently, molecular interactions of selected phytochemicals extracted from C. roseus Linn. and T. indica were studied using multiple-iterated genetic algorithm-based docking protocol in order to investigate the translocation of these legends across the PfCRT protein. Further, molecular dynamics studies exhibiting interaction energy estimates of these compounds within the active site of the protein showed that compounds are more selective toward PfCRT. Clusters of conformations with the free energy of binding were estimated which clearly demonstrated the potential channel and by this means the translocation across the PfCRT is anticipated. PMID:25783783

  8. Intraspecific differentiation of Hancornia speciosa revealed by simple sequence repeat and random amplified polymorphic DNA markers.

    PubMed

    Nogueira, C A; Stafuzza, N B; Ribeiro, T P; Prado, A D L; Menezes, I P P; Peixoto, N; Gonçalves, P J; Almeida, L M

    2015-01-01

    Hancornia speciosa, popularly known as mangabeira, is a fruit tree native to the Brazilian Cerrado that shows great economic potential, due to its multiple uses. Intraspecific classification of this species is difficult because it shows high morphological diversity. An early study of the species reported that there are six botanic varieties that differ morphologically mainly in the shapes of their leaves and flowers. Except to note the wide morphological variation and economic potential of this species, few studies have been published about the genetic diversity of mangabeira. Knowledge of the genetic variability of this species among populations would be useful for genetic conservation and breeding programs. Therefore, we tested the transferability of 12 simple sequence repeats from expressed sequence tags (EST-SSRs) from Catharanthus roseus to H. speciosa and used 10 random amplified polymorphic DNA markers to evaluate the genetic variability among botanical varieties of H. speciosa. We obtained a high transferability frequency of EST-SSR markers from C. roseus to H. speciosa (75%). However, EST-SSR markers showed low heterozygosity and locus variability (two or three alleles by locus), which suggest low genetic diversity in the mangabeira samples. The Jaccard dissimilarity index and an examination of geographic distances indicated a non-spatial structuring of the genetic variability. Our markers were unable to distinguish H. speciosa botanical varieties. PMID:26662392

  9. Engineering the MEP pathway enhanced ajmalicine biosynthesis.

    PubMed

    Chang, Kai; Qiu, Fei; Chen, Min; Zeng, Lingjiang; Liu, Xiaoqiang; Yang, Chunxian; Lan, Xiaozhong; Wang, Qiang; Liao, Zhihua

    2014-01-01

    The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes encoding DXR and MECS from Taxus species and STR from Catharanthus roseus were used to genetically modify the ajmalicine biosynthetic pathway in hairy root cultures of C. roseus. As expected, the STR-overexpressed root cultures showed twofold higher accumulation of ajmalicine than the control. It was important to discover that overexpression of the single DXR or MECS gene from the MEP pathway also remarkably enhanced ajmalicine biosynthesis in transgenic hairy root cultures, and this suggested that engineering the MEP pathway by overexpression of DXR or MECS promoted the metabolic flux into ajmalicine biosynthesis. The transgenic hairy root cultures with co-overexpression of DXR and STR or MECS and STR had higher levels of ajmalicine than those with overexpression of a single gene alone such as DXR, MECS, and STR. It could be concluded that transgenic hairy root cultures harboring both DXR/MECS and STR possessed an increased flux in the terpenoid indole alkaloid biosynthetic pathway that enhanced ajmalicine yield, which was more efficient than cultures harboring only one of the three genes. PMID:24237015

  10. Isolation of Mesophyll Protoplasts from Mediterranean Woody Plants for the Study of DNA Integrity under Abiotic Stress.

    PubMed

    Kuzminsky, Elena; Meschini, Roberta; Terzoli, Serena; Pavani, Liliana; Silvestri, Cristian; Choury, Zineb; Scarascia-Mugnozza, Giuseppe

    2016-01-01

    Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The single cell gel electrophoresis (SCGE) assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L.) has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L.) was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with SCGE assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions. PMID:27574524

  11. Comparative conventional- and quantum dot-labeling strategies for LPS binding site detection in Arabidopsis thaliana mesophyll protoplasts

    PubMed Central

    Mgcina, Londiwe S.; Dubery, Ian A.; Piater, Lizelle A.

    2015-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is recognized as a microbe-associated molecular pattern (MAMP) and not only induces an innate immune response in plants, but also stimulates the development of characteristic defense responses. However, identification and characterization of a cell surface LPS-receptor/binding site, as described in mammals, remains elusive in plants. As an amphiphilic, macromolecular lipoglycan, intact LPS potentially contains three MAMP-active regions, represented by the O-polysaccharide chain, the core and the lipid A. Binding site studies with intact labeled LPS were conducted in Arabidopsis thaliana protoplasts and quantified using flow cytometry fluorescence changes. Quantum dots (Qdots), which allow non-covalent, hydrophobic labeling were used as a novel strategy in this study and compared to covalent, hydrophilic labeling with Alexa 488. Affinity for LPS-binding sites was clearly demonstrated by concentration-, temperature-, and time-dependent increases in protoplast fluorescence following treatment with the labeled LPS. Moreover, this induced fluorescence increase was convincingly reduced following pre-treatment with excess unlabeled LPS, thereby indicating reversibility of LPS binding. Inhibition of the binding process is also reported using endo- and exocytosis inhibitors. Here, we present evidence for the anticipated presence of LPS-specific binding sites in Arabidopsis protoplasts, and furthermore propose Qdots as a more sensitive LPS-labeling strategy in comparison to the conventional Alexa 488 hydrazide label for binding studies. PMID:26029233

  12. Isolation of Mesophyll Protoplasts from Mediterranean Woody Plants for the Study of DNA Integrity under Abiotic Stress

    PubMed Central

    Kuzminsky, Elena; Meschini, Roberta; Terzoli, Serena; Pavani, Liliana; Silvestri, Cristian; Choury, Zineb; Scarascia-Mugnozza, Giuseppe

    2016-01-01

    Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The single cell gel electrophoresis (SCGE) assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L.) has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L.) was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with SCGE assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions. PMID:27574524

  13. Ethnomedicines used in Trinidad and Tobago for urinary problems and diabetes mellitus

    PubMed Central

    Lans, Cheryl A

    2006-01-01

    Background This paper is based on ethnobotanical interviews conducted from 1996–2000 in Trinidad and Tobago with thirty male and female respondents. Methods A non-experimental validation was conducted on the plants used for urinary problems and diabetes mellitus: This is a preliminary step to establish that the plants used are safe or effective, to help direct clinical trials, and to inform Caribbean physicians of the plants' known properties to avoid counter-prescribing. Results The following plants are used to treat diabetes: Antigonon leptopus, Bidens alba, Bidens pilosa, Bixa orellana, Bontia daphnoides, Carica papaya, Catharanthus roseus, Cocos nucifera, Gomphrena globosa, Laportea aestuans, Momordica charantia, Morus alba, Phyllanthus urinaria and Spiranthes acaulis. Apium graviolens is used as a heart tonic and for low blood pressure. Bixa orellana, Bontia daphnoides, Cuscuta americana and Gomphrena globosa are used for jaundice. The following plants are used for hypertension: Aloe vera, Annona muricata, Artocarpus altilis, Bixa orellana, Bidens alba, Bidens pilosa, Bonta daphnoides, Carica papaya, Cecropia peltata, Citrus paradisi, Cola nitida, Crescentia cujete, Gomphrena globosa, Hibiscus sabdariffa, Kalanchoe pinnata, Morus alba, Nopalea cochinellifera, Ocimum campechianum, Passiflora quadrangularis, Persea americana and Tamarindus indicus. The plants used for kidney problems are Theobroma cacao, Chamaesyce hirta, Flemingia strobilifera, Peperomia rotundifolia, Petiveria alliacea, Nopalea cochinellifera, Apium graveolens, Cynodon dactylon, Eleusine indica, Gomphrena globosa, Pityrogramma calomelanos and Vetiveria zizanioides. Plants are also used for gall stones and for cooling. Conclusion Chamaesyce hirta, Cissus verticillata, Kalanchoe pinnata, Peperomia spp., Portulaca oleraceae, Scoparia dulcis, and Zea mays have sufficient evidence to support their traditional use for urinary problems, "cooling" and high cholesterol. Eggplant extract as a

  14. First report of Catharanthus mosaic virus in Mandevilla in the United States

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012-2013 twelve Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, ...

  15. Anthocyanin and Potential Therapeutic Traits in Clitoria, Desmodium, Corchorus, Catharanthus, and Hibiscus Species

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The USDA, ARS, Plant Genetic Resources Conservation Unit curates several important nutraceutical and medicinal plant species. Anthocyanins are responsible for flower, leaf, seed coat color in plants, and are antioxidants as well. However, little is known about anthocyanin content in Clitoria terna...

  16. Methylation of sulfhydryl groups: a new function for a family of small molecule plant O-methyltransferases

    PubMed Central

    Coiner, Heather; Schröder, Gudrun; Wehinger, Elke; Liu, Chang-Jun; Noel, Joseph P.; Schwab, Wilfried; Schröder, Joachim

    2010-01-01

    Summary In plants, type I and II S-adenosyl-L-methionine-dependent O-methyltransferases (OMTs) catalyze most hydroxyl group methylations of small molecules. A homology-based RT-PCR strategy using Catharanthus roseus (Madagascar periwinkle) RNA previously identified six new type I plant OMT family members. We now describe the molecular and biochemical characterization of a seventh protein. It shares 56–58% identity with caffeic acid OMTs (COMTs), but it failed to methylate COMT substrates, and had no activity with flavonoids. However, the in vitro incubations revealed unusually high background levels without added substrates. A search for the responsible component revealed that the enzyme methylated dithiothreitol (DTT), the reducing agent added for enzyme stabilization. Unexpectedly, product analysis revealed that the methylation occurred on a sulfhydryl moiety, not on a hydroxyl group. Analysis of 34 compounds indicated a broad substrate range, with a preference for small hydrophobic molecules. Benzene thiol (Km 220 μM) and furfuryl thiol (Km 60 μM) were the best substrates (6–7-fold better than DTT). Small isosteric hydrophobic substrates with hydroxyl groups, like phenol and guaiacol, were also methylated, but the activities were at least 5-fold lower than with thiols. The enzyme was named C. roseus S-methyltransferase 1 (CrSMT1). Models based on the COMT crystal structure suggest that S-methylation is mechanistically identical to O-methylation. CrSMT1 so far is the only recognized example of an S-methyltransferase in this protein family. Its properties indicate that a few changes in key residues are sufficient to convert an OMT into a S-methyltransferase (SMT). Future functional investigations of plant methyltransferases should consider the possibility that the enzymes may direct methylation at sulfhydryl groups. PMID:16623883

  17. CrBPF1 overexpression alters transcript levels of terpenoid indole alkaloid biosynthetic and regulatory genes.

    PubMed

    Li, Chun Yao; Leopold, Alex L; Sander, Guy W; Shanks, Jacqueline V; Zhao, Le; Gibson, Susan I

    2015-01-01

    Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Toward this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression of CrBPF1 causes increases in the transcript levels for 11 out of 13 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a "fine-tune" regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression. PMID:26483828

  18. Iridoid Synthase Activity Is Common among the Plant Progesterone 5β-Reductase Family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2014-09-19

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, among which the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterisation of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone, and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2 and CrP5βR4, could also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In depth functional analysis by subcellular protein localisation, gene expression analysis, in situ hybridisation and virus-induced gene silencing, indicates that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. Finally, we cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that 'IS activity' is intrinsic to angiosperm P5βR proteins and has evolved early during evolution. PMID:25239067

  19. Iridoid synthase activity is common among the plant progesterone 5β-reductase family.

    PubMed

    Munkert, Jennifer; Pollier, Jacob; Miettinen, Karel; Van Moerkercke, Alex; Payne, Richard; Müller-Uri, Frieder; Burlat, Vincent; O'Connor, Sarah E; Memelink, Johan; Kreis, Wolfgang; Goossens, Alain

    2015-01-01

    Catharanthus roseus, the Madagascar periwinkle, synthesizes bioactive monoterpenoid indole alkaloids, including the anti-cancer drugs vinblastine and vincristine. The monoterpenoid branch of the alkaloid pathway leads to the secoiridoid secologanin and involves the enzyme iridoid synthase (IS), a member of the progesterone 5β-reductase (P5βR) family. IS reduces 8-oxogeranial to iridodial. Through transcriptome mining, we show that IS belongs to a family of six C. roseus P5βR genes. Characterization of recombinant CrP5βR proteins demonstrates that all but CrP5βR3 can reduce progesterone and thus can be classified as P5βRs. Three of them, namely CrP5βR1, CrP5βR2, and CrP5βR4, can also reduce 8-oxogeranial, pointing to a possible redundancy with IS (corresponding to CrP5βR5) in secoiridoid synthesis. In-depth functional analysis by subcellular protein localization, gene expression analysis, in situ hybridization, and virus-induced gene silencing indicate that besides IS, CrP5βR4 may also participate in secoiridoid biosynthesis. We cloned a set of P5βR genes from angiosperm plant species not known to produce iridoids and demonstrate that the corresponding recombinant proteins are also capable of using 8-oxogeranial as a substrate. This suggests that IS activity is intrinsic to angiosperm P5βR proteins and has evolved early during evolution. PMID:25578278

  20. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids.

    PubMed

    Levac, Dylan; Cázares, Paulo; Yu, Fang; De Luca, Vincenzo

    2016-04-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  1. A Picrinine N-Methyltransferase Belongs to a New Family of γ-Tocopherol-Like Methyltransferases Found in Medicinal Plants That Make Biologically Active Monoterpenoid Indole Alkaloids1[OPEN

    PubMed Central

    Levac, Dylan; Cázares, Paulo; Yu, Fang

    2016-01-01

    Members of the Apocynaceae plant family produce a large number of monoterpenoid indole alkaloids (MIAs) with different substitution patterns that are responsible for their various biological activities. A novel N-methyltransferase involved in the vindoline pathway in Catharanthus roseus showing distinct similarity to γ-tocopherol C-methyltransferases was used in a bioinformatic screen of transcriptomes from Vinca minor, Rauvolfia serpentina, and C. roseus to identify 10 γ-tocopherol-like N-methyltransferases from a large annotated transcriptome database of different MIA-producing plant species (www.phytometasyn.ca). The biochemical function of two members of this group cloned from V. minor (VmPiNMT) and R. serpentina (RsPiNMT) have been characterized by screening their biochemical activities against potential MIA substrates harvested from the leaf surfaces of MIA-accumulating plants. The approach was validated by identifying the MIA picrinine from leaf surfaces of Amsonia hubrichtii as a substrate of VmPiNMT and RsPiNMT. Recombinant proteins were shown to have high substrate specificity and affinity for picrinine, converting it to N-methylpicrinine (ervincine). Developmental studies with V. minor and R. serpentina showed that RsPiNMT and VmPiNMT gene expression and biochemical activities were highest in younger leaf tissues. The assembly of at least 150 known N-methylated MIAs within members of the Apocynaceae family may have occurred as a result of the evolution of the γ-tocopherol-like N-methyltransferase family from γ-tocopherol methyltransferases. PMID:26848097

  2. Development of Transcriptomic Resources for Interrogating the Biosynthesis of Monoterpene Indole Alkaloids in Medicinal Plant Species

    PubMed Central

    Góngora-Castillo, Elsa; Childs, Kevin L.; Fedewa, Greg; Hamilton, John P.; Liscombe, David K.; Magallanes-Lundback, Maria; Mandadi, Kranthi K.; Nims, Ezekiel; Runguphan, Weerawat; Vaillancourt, Brieanne; Varbanova-Herde, Marina; DellaPenna, Dean; McKnight, Thomas D.; O’Connor, Sarah; Buell, C. Robin

    2012-01-01

    The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for

  3. Development of transcriptomic resources for interrogating the biosynthesis of monoterpene indole alkaloids in medicinal plant species.

    PubMed

    Góngora-Castillo, Elsa; Childs, Kevin L; Fedewa, Greg; Hamilton, John P; Liscombe, David K; Magallanes-Lundback, Maria; Mandadi, Kranthi K; Nims, Ezekiel; Runguphan, Weerawat; Vaillancourt, Brieanne; Varbanova-Herde, Marina; Dellapenna, Dean; McKnight, Thomas D; O'Connor, Sarah; Buell, C Robin

    2012-01-01

    The natural diversity of plant metabolism has long been a source for human medicines. One group of plant-derived compounds, the monoterpene indole alkaloids (MIAs), includes well-documented therapeutic agents used in the treatment of cancer (vinblastine, vincristine, camptothecin), hypertension (reserpine, ajmalicine), malaria (quinine), and as analgesics (7-hydroxymitragynine). Our understanding of the biochemical pathways that synthesize these commercially relevant compounds is incomplete due in part to a lack of molecular, genetic, and genomic resources for the identification of the genes involved in these specialized metabolic pathways. To address these limitations, we generated large-scale transcriptome sequence and expression profiles for three species of Asterids that produce medicinally important MIAs: Camptotheca acuminata, Catharanthus roseus, and Rauvolfia serpentina. Using next generation sequencing technology, we sampled the transcriptomes of these species across a diverse set of developmental tissues, and in the case of C. roseus, in cultured cells and roots following elicitor treatment. Through an iterative assembly process, we generated robust transcriptome assemblies for all three species with a substantial number of the assembled transcripts being full or near-full length. The majority of transcripts had a related sequence in either UniRef100, the Arabidopsis thaliana predicted proteome, or the Pfam protein domain database; however, we also identified transcripts that lacked similarity with entries in either database and thereby lack a known function. Representation of known genes within the MIA biosynthetic pathway was robust. As a diverse set of tissues and treatments were surveyed, expression abundances of transcripts in the three species could be estimated to reveal transcripts associated with development and response to elicitor treatment. Together, these transcriptomes and expression abundance matrices provide a rich resource for

  4. Water and heat balance during flight in the rose-colored starling (Sturnus roseus).

    PubMed

    Engel, Sophia; Biebach, Herbert; Visser, G Henk

    2006-01-01

    Water imbalance during flight is considered to be a potentially limiting factor for flight ranges in migrating birds, but empirical data are scarce. We studied flights under controlled ambient conditions with rose-colored starlings in a wind tunnel. In one experiment, we measured water fluxes with stable isotopes at a range of flight speeds (9-14 m s(-1)) at constant temperature (15 degrees C). In a second experiment, we measured evaporation rates at variable ambient temperatures (Ta = 5 deg -27 deg C) but constant speed (12 m s(-1)). During all flights, the birds experienced a net water loss. On average, water influx was 0.98 g h(-1) (SD = 0.16; n = 8), and water efflux was 1.29 g h(-1) (SD = 0.14; n = 8), irrespective of flight speed. Evaporation was related to temperature in a biphasic pattern. At temperatures below 18.2 degrees C, net evaporation was constant at 0.36 g h(-1) (SD = 0.18; n = 10), rising at higher temperatures with a slope of 0.11 per degree to about 1.5 g h(-1) at 27 degrees C. We calculated the relative proportion of dry and evaporative heat loss during flight. Evaporative heat loss at Ta < 18.2 deg C was 14% of total heat production during flight, and dry heat loss accounted for 84%. At higher temperatures, evaporative heat loss increased linearly with T(a) to about 25% at 27 degrees C. Our data suggest that for prolonged flights, rose-colored starlings should adopt behavioral water-saving strategies and that they cannot complete their annual migration without stopovers to replenish their water reserves. PMID:16826502

  5. Strictosidine activation in Apocynaceae: towards a "nuclear time bomb"?

    PubMed Central

    2010-01-01

    Background The first two enzymatic steps of monoterpene indole alkaloid (MIA) biosynthetic pathway are catalysed by strictosidine synthase (STR) that condensates tryptamine and secologanin to form strictosidine and by strictosidine β-D-glucosidase (SGD) that subsequently hydrolyses the glucose moiety of strictosidine. The resulting unstable aglycon is rapidly converted into a highly reactive dialdehyde, from which more than 2,000 MIAs are derived. Many studies were conducted to elucidate the biosynthesis and regulation of pharmacologically valuable MIAs such as vinblastine and vincristine in Catharanthus roseus or ajmaline in Rauvolfia serpentina. However, very few reports focused on the MIA physiological functions. Results In this study we showed that a strictosidine pool existed in planta and that the strictosidine deglucosylation product(s) was (were) specifically responsible for in vitro protein cross-linking and precipitation suggesting a potential role for strictosidine activation in plant defence. The spatial feasibility of such an activation process was evaluated in planta. On the one hand, in situ hybridisation studies showed that CrSTR and CrSGD were coexpressed in the epidermal first barrier of C. roseus aerial organs. However, a combination of GFP-imaging, bimolecular fluorescence complementation and electromobility shift-zymogram experiments revealed that STR from both C. roseus and R. serpentina were localised to the vacuole whereas SGD from both species were shown to accumulate as highly stable supramolecular aggregates within the nucleus. Deletion and fusion studies allowed us to identify and to demonstrate the functionality of CrSTR and CrSGD targeting sequences. Conclusions A spatial model was drawn to explain the role of the subcellular sequestration of STR and SGD to control the MIA metabolic flux under normal physiological conditions. The model also illustrates the possible mechanism of massive activation of the strictosidine vacuolar pool

  6. Glycaemic effects of traditional European plant treatments for diabetes. Studies in normal and streptozotocin diabetic mice.

    PubMed

    Swanston-Flatt, S K; Day, C; Flatt, P R; Gould, B J; Bailey, C J

    1989-02-01

    Twelve plants used for the traditional treatment of diabetes mellitus in northern Europe were studied using normal and streptozotocin diabetic mice to evaluate effects on glucose homeostasis. The plants were administered in the diet (6.25% by weight) and/or as decoctions or infusions in place of drinking water, to coincide with the traditional method of preparation. Treatment for 28 days with preparations of burdock (Arctium lappa), cashew (Anacardium occidentale), dandelion (Taraxacum officinale), elder (Sambucus nigra), fenugreek (Trigonella foenum-graecum), guayusa (Ilex guayusa), hop (Humulus lupulus), nettle (Urtica dioica), cultivated mushroom (Agaricus bisporus), periwinkle (Catharanthus roseus), sage (Salvia officinale), and wild carrot (Daucus carrota) did not affect the parameters of glucose homeostasis examined in normal mice (basal plasma glucose and insulin, glucose tolerance, insulin-induced hypoglycaemia and glycated haemoglobin). After administration of streptozotocin (200 mg/kg) burdock and nettle aggravated the diabetic condition, while cashew, dandelion, elder, fenugreek, hop, periwinkle, sage and wild carrot did not significantly affect the parameters of glucose homeostasis studied (basal glucose and insulin, insulin-induced hypoglycaemia, glycated haemoglobin and pancreatic insulin concentration). Guayusa and mushroom retarded the development of hyperglycaemia in streptozotocin diabetes and reduced the hyperphagia, polydipsia, body weight loss, and glycated haemoglobin. Mushroom also countered the initial reduction in plasma insulin and the reduction in pancreatic insulin concentration, and improved the hypoglycaemic effect of exogenous insulin. These studies suggest the presence of potentially useful antidiabetic agents in guayusa and mushroom. PMID:2743711

  7. Effects of Solarization and Ammonium Amendments on Plant-Parasitic Nematodes

    PubMed Central

    McSorley, R.; McGovern, R. J.

    2000-01-01

    The effects of soil solarization and ammonium bicarbonate or ammonium sulfate against plant-parasitic nematodes on yellow squash (Cucurbita pepo) and on vinca (Catharanthus roseus) were evaluated at two sites. Solarization for 3 weeks in the spring suppressed population levels of Belonolaimus longicaudatus, Criconemella spp., and Dolichodorus heterocephalus throughout the growing season on both crops at both sites. Levels of Meloidogyne incognita were suppressed initially, but population densities increased by the end of the crop in several cases. In one site, numbers of Paratrichodorus minor resurged following solarization to levels that were greater than those present in unsolarized control plots. The effect of solarization was not enhanced by combination with ammonium amendments, but, in one site, application of ammonium bicarbonate or ammonium sulfate resulted in lower numbers of B. longicaudatus than in the unamended control. Additional research and improved efficacy of candidate amendments are required before they can be successfully integrated with solarization for nematode management. Efficacy of solarization against plant-parasitic nematodes was achieved despite a relatively short (3 weeks) solarization period. PMID:19271007

  8. Zoospore density-dependent behaviors of Phytophthora nicotianae are autoregulated by extracellular products.

    PubMed

    Kong, Ping; Hong, Chuanxue

    2010-07-01

    Phytophthora species are destructive fungus-like plant pathogens that use asexual single-celled flagellate zoospores for dispersal and plant infection. Many of the zoospore behaviors are density-dependent although the underlying mechanisms are poorly understood. Here, we use P. nicotianae as a model and demonstrate autoregulation of some zoospore behaviors using signal molecules that zoospores release into the environment. Specifically, zoospore aggregation, plant targeting, and infection required or were enhanced by threshold concentrations of these signal molecules. Below the threshold concentration, zoospores did not aggregate and move toward a cauline leaf of Arabidopsis thaliana (Col-0) and failed to individually attack annual vinca (Catharanthus roseus cv. Little Bright Eye). These processes were reversed when supplemented with zoospore-free fluid (ZFF) prepared from a zoospore suspension above threshold densities but not with calcium chloride at a concentration equivalent to extracellular Ca(2+) in ZFF. These results suggest that Ca(2+) is not a primary signal molecule regulating these communal behaviors. Zoospores coordinated their communal behaviors by releasing, detecting, and responding to signal molecules. This chemical communication mechanism raises the possibility that Phytophthora plant infection may not depend solely on zoospore number in the real world. Single zoospore infection may take place if it is signaled by a common molecule available in the environment which contributes to the destructiveness of these plant pathogens. PMID:20528180

  9. Evaluation of the Larvicidal Efficacy of Five Indigenous Weeds against an Indian Strain of Dengue Vector, Aedes aegypti L. (Diptera: Culicidae)

    PubMed Central

    Sharma, Aarti; Kumar, Sarita; Tripathi, Pushplata

    2016-01-01

    Background and Objectives. Aedes aegypti, dengue fever mosquito, is primarily associated with the transmission of dengue and chikungunya in tropical and subtropical regions of the world. The present investigations were carried out to assess the larvicidal efficiency of five indigenous weeds against Ae. aegypti. Methods. The 1,000 ppm hexane and ethanol extracts prepared from the leaves and stem of five plants (Achyranthes aspera, Cassia occidentalis, Catharanthus roseus, Lantana camara, and Xanthium strumarium) were screened for their larvicidal activity against early fourth instars of dengue vector. The extracts which could cause 80–100% mortality were further investigated for their efficacy. Results. The preliminary screening established the efficacy of hexane extracts as compared to the ethanol extracts. Further investigations revealed the highest larvicidal potential of A. aspera extracts exhibiting LC50 value of 82.555 ppm and 68.133 ppm, respectively. Further, their leaf extracts showed 5–85.9% higher larvicidal activity and stem extracts exhibited 0.23- to 0.85-fold more efficiency than the other four extracts. Conclusion. The present investigations suggest the possible use of A. aspera as an ideal ecofriendly, larvicidal agent for the control of dengue vector, Ae. aegypti. Future studies are, however, required to explore and identify the bioactive component involved and its mode of action. PMID:26941996

  10. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway.

    PubMed

    Cárdenas, Pablo D; Sonawane, Prashant D; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-01-01

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops. PMID:26876023

  11. GAME9 regulates the biosynthesis of steroidal alkaloids and upstream isoprenoids in the plant mevalonate pathway

    PubMed Central

    Cárdenas, Pablo D.; Sonawane, Prashant D.; Pollier, Jacob; Vanden Bossche, Robin; Dewangan, Veena; Weithorn, Efrat; Tal, Lior; Meir, Sagit; Rogachev, Ilana; Malitsky, Sergey; Giri, Ashok P.; Goossens, Alain; Burdman, Saul; Aharoni, Asaph

    2016-01-01

    Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules produced by solanaceous species. They contribute to pathogen defence but are toxic to humans and considered as anti-nutritional compounds. Here we show that GLYCOALKALOID METABOLISM 9 (GAME9), an APETALA2/Ethylene Response Factor, related to regulators of alkaloid production in tobacco and Catharanthus roseus, controls SGA biosynthesis. GAME9 knockdown and overexpression in tomato and potato alters expression of SGAs and upstream mevalonate pathway genes including the cholesterol biosynthesis gene STEROL SIDE CHAIN REDUCTASE 2 (SSR2). Levels of SGAs, C24-alkylsterols and the upstream mevalonate and cholesterol pathways intermediates are modified in these plants. Δ(7)-STEROL-C5(6)-DESATURASE (C5-SD) in the hitherto unresolved cholesterol pathway is a direct target of GAME9. Transactivation and promoter-binding assays show that GAME9 exerts its activity either directly or cooperatively with the SlMYC2 transcription factor as in the case of the C5-SD gene promoter. Our findings provide insight into the regulation of SGA biosynthesis and means for manipulating these metabolites in crops. PMID:26876023

  12. Relative mass defect filtering of mass spectra: a path to discovery of plant specialized metabolites.

    PubMed

    Ekanayaka, E A Prabodha; Celiz, Mary Dawn; Jones, A Daniel

    2015-04-01

    The rapid identification of novel plant metabolites and assignments of newly discovered substances to natural product classes present the main bottlenecks to defining plant specialized phenotypes. Although mass spectrometry provides powerful support for metabolite discovery by measuring molecular masses, ambiguities in elemental formulas often fail to reveal the biosynthetic origins of specialized metabolites detected using liquid chromatography-mass spectrometry. A promising approach for mining liquid chromatography-mass spectrometry metabolite profiling data for specific metabolite classes is achieved by calculating relative mass defects (RMDs) from molecular and fragment ions. This strategy enabled the rapid recognition of an extensive range of terpenoid metabolites in complex plant tissue extracts and is independent of retention time, abundance, and elemental formula. Using RMD filtering and tandem mass spectrometry data analysis, 24 novel elemental formulas corresponding to glycosylated sesquiterpenoid metabolites were identified in extracts of the wild tomato Solanum habrochaites LA1777 trichomes. Extensive isomerism was revealed by ultra-high-performance liquid chromatography, leading to evidence of more than 200 distinct sesquiterpenoid metabolites. RMD filtering led to the recognition of the presence of glycosides of two unusual sesquiterpenoid cores that bear limited similarity to known sesquiterpenes in the genus Solanum. In addition, RMD filtering is readily applied to existing metabolomics databases and correctly classified the annotated terpenoid metabolites in the public metabolome database for Catharanthus roseus. PMID:25659383

  13. Arabidopsis CAP1-mediated ammonium sensing required reactive oxygen species in plant cell growth.

    PubMed

    Bai, Ling; Zhou, Yun; Ma, Xiaonan; Gao, Lijie; Song, Chun-Peng

    2014-06-18

    [Ca (2+)]cyt-associated protein kinase (CAP) gene 1 is a receptor-like kinase that belongs to CrRLK1L (Catharanthus roseus Receptor like kinase) subfamily. CAP1 has been identified as a novel modulator of NH 4(+) in the tonoplast, which regulates root hair growth by maintaining the cytoplasmic Ca (2+) gradients. Different expression pattern of tonoplast intrinsic protein (TIP2;3) in the CAP1 knock out mutant and wild type on Murashige and Skoog (MS) medium suggested that CAP1 influences transport activity to regulate the compartmentalization of NH 4(+) into vacuole. Lower expression level of Oxidative Signal-Inducible1(OXI1) in the cap1-1 root and the abnormal reactive oxygen species (ROS) gradient in root hair of cap1-1 on MS medium indicated that ROS signaling involve in CAP1-regulated root hair growth. Wild-type-like ROS distribution pattern in the cap1-1 root hair can be reestablished in seedlings grown on NH 4(+) deficient medium, which indicated that CAP1 functions as a sensor for NH 4(+) signaling in maintaining tip-focused ROS gradient in root hairs polar growth. PMID:24940875

  14. Enhanced camptothecin production by ethanol addition in the suspension culture of the endophyte, Fusarium solani.

    PubMed

    Venugopalan, Aarthi; Srivastava, Smita

    2015-01-01

    Ethanolic extract of a non-camptothecin producing plant, Catharanthus roseus when added in the suspension culture of the endophyte Fusarium solani known to produce camptothecin, resulted in enhanced production of camptothecin by 10.6-fold in comparison to that in control (2.8 μg/L). Interestingly, addition of pure ethanol (up to 5% v/v) in the suspension culture of F. solani resulted in maximum enhancement in camptothecin production (up to 15.5-fold) from that obtained in control. In the presence of ethanol, a reduced glucose uptake (by ∼ 40%) and simultaneous ethanol consumption (up to 9.43 g/L) was observed during the cultivation period (14 days). Also, the total NAD level and the protein content in the biomass increased by 3.7- and 1.9-fold, respectively, in comparison to that in control. The study indicates a dual role of ethanol, presumably as an elicitor and also as a carbon/energy source, leading to enhanced biomass and camptothecin production. PMID:25603728

  15. Cytotoxic Effect of Erythroxylum suberosum Combined with Radiotherapy in Head and Neck Cancer Cell Lines.

    PubMed

    Macedo, Taysa B C; Elias, Silvia T; Torres, Hianne M; Yamamoto-Silva, Fernanda Paula; Silveira, Dâmaris; Magalhães, Pérola O; Lofrano-Porto, Adriana; Guerra, Eliete N S; Silva, Maria Alves G

    2016-01-01

    The mouth and oropharynx cancer is the 6th most common type of cancer in the world. The treatment may involve surgery, chemotherapy and radiotherapy. More than 50% of drugs against cancer were isolated from natural sources, such as Catharanthus roseus and epipodophyllotoxin, isolated from Podophyllum. The biggest challenge is to maximize the control of the disease, while minimizing morbidity and toxicity to the surrounding normal tissues. The Erythroxylum suberosum is a common plant in the Brazilian Cerrado biome and is popularly known as "cabelo-de-negro". The objective of this study was to evaluate the cytotoxic activity of Erythroxylum suberosum plant extracts of the Brazilian Cerrado biome associated with radiotherapy in human cell lines of oral and hypopharynx carcinomas. Cells were treated with aqueous, ethanolic and hexanic extracts of Erythroxylum suberosum and irradiated at 4 Gy, 6 Gy and 8 Gy. Cytotoxicity was evaluated by MTT assay and the absorbance was measured at 570 nm in a Beckman Counter reader. Cisplatin, standard chemotherapy, was used as positive control. The use of Erythroxylum suberosum extracts showed a possible radiosensitizing effect in vitro for head and neck cancer. The cytotoxicity effect in the cell lines was not selective and it is very similar to the effect of standard chemotherapy. The aqueous extract of Erythroxylum suberosum, combined with radiotherapy was the most cytotoxic extract to oral and hypopharynx carcinomas. PMID:27007356

  16. Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila.

    PubMed

    Cui, Lijie; Ni, Xiaoling; Ji, Qian; Teng, Xiaojuan; Yang, Yanru; Wu, Chao; Zekria, David; Zhang, Dasheng; Kai, Guoyin

    2015-01-01

    Camptothecin (CPT) belongs to a group of monoterpenoidindole alkaloids (TIAs) and its derivatives such as irinothecan and topothecan have been widely used worldwide for the treatment of cancer, giving rise to rapidly increasing market demands. Genes from Catharanthus roseus encoding strictosidine synthase (STR) and geraniol 10-hydroxylase (G10H), were separately and simultaneously introduced into Ophiorrhiza pumila hairy roots. Overexpression of individual G10H (G lines) significantly improved CPT production with respect to non-transgenic hairy root cultures (NC line) and single STR overexpressing lines (S lines), indicating that G10H plays a more important role in stimulating CPT accumulation than STR in O. pumila. Furthermore, co-overexpression of G10H and STR genes (SG Lines) caused a 56% increase on the yields of CPT compared to NC line and single gene transgenic lines, showed that simultaneous introduction of G10H and STR can produce a synergistic effect on CPT biosynthesis in O. pumila. The MTT assay results indicated that CPT extracted from different lines showed similar anti-tumor activity, suggesting that transgenic O. pumila hairy root lines could be an alternative approach to obtain CPT. To our knowledge, this is the first report on the enhancement of CPT production in O. pumila employing a metabolic engineering strategy. PMID:25648209

  17. Engineering of a Nepetalactol-Producing Platform Strain of Saccharomyces cerevisiae for the Production of Plant Seco-Iridoids.

    PubMed

    Campbell, Alex; Bauchart, Philippe; Gold, Nicholas D; Zhu, Yun; De Luca, Vincenzo; Martin, Vincent J J

    2016-05-20

    The monoterpene indole alkaloids (MIAs) are a valuable family of chemicals that include the anticancer drugs vinblastine and vincristine. These compounds are of global significance-appearing on the World Health Organization's list of model essential medicines-but remain exorbitantly priced due to low in planta levels. Chemical synthesis and genetic manipulation of MIA producing plants such as Catharanthus roseus have so far failed to find a solution to this problem. Synthetic biology holds a potential answer, by building the pathway into more tractable organisms such as Saccharomyces cerevisiae. Recent work has taken the first steps in this direction by producing small amounts of the intermediate strictosidine in yeast. In order to help improve on these titers, we aimed to optimize the early biosynthetic steps of the MIA pathway to the metabolite nepetalactol. We combined a number of strategies to create a base strain producing 11.4 mg/L of the precursor geraniol. We also show production of the critical intermediate 10-hydroxygeraniol and demonstrate nepetalactol production in vitro. Lastly we demonstrate that activity of the iridoid synthase toward the intermediates geraniol and 10-hydroxygeraniol results in the synthesis of the nonproductive intermediates citronellol and 10-hydroxycitronellol. This discovery has serious implications for the reconstruction of the MIA in heterologous organisms. PMID:26981892

  18. Conserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms.

    PubMed

    Galindo-Trigo, Sergio; Gray, Julie E; Smith, Lisa M

    2016-01-01

    Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant-pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants. PMID:27621737

  19. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    NASA Astrophysics Data System (ADS)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  20. Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging.

    PubMed

    Christensen, Nynne Meyn; Nicolaisen, Mogens; Hansen, Michael; Schulz, Alexander

    2004-11-01

    Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining. PMID:15553243

  1. Effects of solarization and ammonium amendments on plant-parasitic nematodes.

    PubMed

    McSorley, R; McGovern, R J

    2000-12-01

    The effects of soil solarization and ammonium bicarbonate or ammonium sulfate against plant-parasitic nematodes on yellow squash (Cucurbita pepo) and on vinca (Catharanthus roseus) were evaluated at two sites. Solarization for 3 weeks in the spring suppressed population levels of Belonolaimus longicaudatus, Criconemella spp., and Dolichodorus heterocephalus throughout the growing season on both crops at both sites. Levels of Meloidogyne incognita were suppressed initially, but population densities increased by the end of the crop in several cases. In one site, numbers of Paratrichodorus minor resurged following solarization to levels that were greater than those present in unsolarized control plots. The effect of solarization was not enhanced by combination with ammonium amendments, but, in one site, application of ammonium bicarbonate or ammonium sulfate resulted in lower numbers of B. longicaudatus than in the unamended control. Additional research and improved efficacy of candidate amendments are required before they can be successfully integrated with solarization for nematode management. Efficacy of solarization against plant-parasitic nematodes was achieved despite a relatively short (3 weeks) solarization period. PMID:19271007

  2. Glucosylation of aroma chemicals and hydroxy fatty acids.

    PubMed

    Huang, Fong-Chin; Hinkelmann, Jens; Schwab, Wilfried

    2015-12-20

    To explore the utility of glycosyltransferases as novel biocatalysts, we isolated the glycosyltransferase genes CaUGT2 and SbUGTA1 from Catharanthus roseus and Starmerella bombicola, respectively and heterologously expressed them in Escherichia coli. The purified recombinant proteins were assayed with a variety of small molecule substrates. Carvacrol and its phenol isomer thymol are efficiently glucosylated by CaUGT2. The Vmax/Km ratios show that CaUGT2 exhibits the highest specificity towards carvacrol, followed by thymol, geraniol, eugenol, vanillin, menthol, and tyrosol. In contrast, SbUGTA1 accepts ω-hydroxy fatty acids and 1-alkanols as substrates. The Vmax/Km ratios indicate that SbUGTA1 exhibits the highest specificity towards 16-hydroxy palmitic acid, followed by octanol, decanol, and hexadecanol. In biotransformation experiments 23, 88 and 99% of octanol, 16-hydroxy palmitic acid, and decanol, respectively is converted into the corresponding β-glucosides by E. coli cells expressing SbUGTA1 whereas those cells expressing CaUGT2 glucosylate 18, 61, 77 and 97% of applied eugenol, thymol, vanillin, and carvacrol, respectively. To optimize the biotransformation rate, the effects of the concentration of IPTG, glucose, and substrate on the production of glucosides were tested. Taken together, this procedure is a simple operation, environmentally friendly, and is useful for the preparation of glycosides as additives for food and cosmetics. PMID:26481830

  3. A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle[C][W][OPEN

    PubMed Central

    Asada, Keisuke; Salim, Vonny; Masada-Atsumi, Sayaka; Edmunds, Elizabeth; Nagatoshi, Mai; Terasaka, Kazuyoshi; Mizukami, Hajime; De Luca, Vincenzo

    2013-01-01

    Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-SUGAR GLYCOSYLTRANSFERASE6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species. PMID:24104568

  4. Beneficial behavior of nitric oxide in copper-treated medicinal plants.

    PubMed

    Liu, Shiliang; Yang, Rongjie; Pan, Yuanzhi; Ren, Bo; Chen, Qibing; Li, Xi; Xiong, Xi; Tao, Jianjun; Cheng, Qingsu; Ma, Mingdong

    2016-08-15

    Despite numerous reports implicating nitric oxide (NO) in the environmental-stress responses of plants, the specific metabolic and ionic mechanisms of NO-mediated adaptation to metal stress remain unclear. Here, the impacts of copper (Cu) and NO donor (SNP, 50μM) alone or in combination on the well-known medicinal plant Catharanthus roseus L. were investigated. Our results showed that Cu markedly increased Cu(2+) accumulation, decreased NO production, and disrupted mineral equilibrium and proton pumps, thereby stimulating a burst of ROS; in addition, SNP ameliorates the negative toxicity of Cu, and cPTIO reverses this action. Furthermore, the accumulations of ROS and NO resulted in reciprocal changes. Interestingly, nearly all of the investigated amino acids and the total phenolic content in the roots were promoted by the SNP treatment but were depleted by the Cu+SNP treatment, which is consistent with the self-evident increases in phenylalanine ammonia-lyase activity and total soluble phenol content induced by SNP. Unexpectedly, leaf vincristine and vinblastine as well as the total alkaloid content (ca. 1.5-fold) were decreased by Cu but markedly increased by SNP (+38% and +49% of the control levels). This study provides the first evidence of the beneficial behavior of NO, rather than other compounds, in depleting Cu toxicity by regulating mineral absorption, reestablishing ATPase activities, and stimulating secondary metabolites. PMID:27131454

  5. In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways.

    PubMed

    Rijhwani, S K; Ho, C H; Shanks, J V

    1999-01-01

    Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates. PMID:10935751

  6. Boosting Sensitivity in Liquid Chromatography–Fourier Transform Ion Cyclotron Resonance–Tandem Mass Spectrometry for Product Ion Analysis of Monoterpene Indole Alkaloids

    PubMed Central

    Nakabayashi, Ryo; Tsugawa, Hiroshi; Kitajima, Mariko; Takayama, Hiromitsu; Saito, Kazuki

    2015-01-01

    In metabolomics, the analysis of product ions in tandem mass spectrometry (MS/MS) is noteworthy to chemically assign structural information. However, the development of relevant analytical methods are less advanced. Here, we developed a method to boost sensitivity in liquid chromatography–Fourier transform ion cyclotron resonance–tandem mass spectrometry analysis (MS/MS boost analysis). To verify the MS/MS boost analysis, both quercetin and uniformly labeled 13C quercetin were analyzed, revealing that the origin of the product ions is not the instrument, but the analyzed compounds resulting in sensitive product ions. Next, we applied this method to the analysis of monoterpene indole alkaloids (MIAs). The comparative analyses of MIAs having indole basic skeleton (ajmalicine, catharanthine, hirsuteine, and hirsutine) and oxindole skeleton (formosanine, isoformosanine, pteropodine, isopteropodine, rhynchophylline, isorhynchophylline, and mitraphylline) identified 86 and 73 common monoisotopic ions, respectively. The comparative analyses of the three pairs of stereoisomers showed more than 170 common monoisotopic ions in each pair. This method was also applied to the targeted analysis of MIAs in Catharanthus roseus and Uncaria rhynchophylla to profile indole and oxindole compounds using the product ions. This analysis is suitable for chemically assigning features of the metabolite groups, which contributes to targeted metabolome analysis. PMID:26734034

  7. Processing methods for differential analysis of LC/MS profile data

    PubMed Central

    Katajamaa, Mikko; Orešič, Matej

    2005-01-01

    Background Liquid chromatography coupled to mass spectrometry (LC/MS) has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. Results We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. Conclusion The software is freely available under the GNU General Public License and it can be obtained from the project web page at: . PMID:16026613

  8. Conserved Roles of CrRLK1L Receptor-Like Kinases in Cell Expansion and Reproduction from Algae to Angiosperms

    PubMed Central

    Galindo-Trigo, Sergio; Gray, Julie E.; Smith, Lisa M.

    2016-01-01

    Receptor-like kinases (RLKs) are regulators of plant development through allowing cells to sense their extracellular environment. They facilitate detection of local endogenous signals, in addition to external biotic and abiotic stimuli. The Catharanthus roseus RLK1-like (CrRLK1L) protein kinase subfamily, which contains FERONIA, plays a central role in regulating fertilization and in cell expansion mechanisms such as cell elongation and tip growth, as well as having indirect links to plant–pathogen interactions. Several components of CrRLK1L signaling pathways have been identified, including an extracellular ligand, coreceptors, and downstream signaling elements. The presence and abundance of the CrRLK1L proteins in the plant kingdom suggest an origin within the Streptophyta lineage, with a notable increase in prevalence in the seeded land plants. Given the function of the sole CrRLK1L protein in a charophycean alga, the possibility of a conserved role in detection and/or regulation of cell wall integrity throughout the Strephtophytes is discussed. Orthologs of signaling pathway components are also present in extant representatives of non-vascular land plants and early vascular land plants including the liverwort Marchantia polymorpha, the moss Physcomitrella patens and the lycophyte Selaginella moellendorffii. Deciphering the roles in development of the CrRLK1L protein kinases in early diverging land plants will provide insights into their ancestral function, furthering our understanding of this diversified subfamily of receptors in higher plants. PMID:27621737

  9. Evaluation of the Larvicidal Efficacy of Five Indigenous Weeds against an Indian Strain of Dengue Vector, Aedes aegypti L. (Diptera: Culicidae).

    PubMed

    Sharma, Aarti; Kumar, Sarita; Tripathi, Pushplata

    2016-01-01

    Background and Objectives. Aedes aegypti, dengue fever mosquito, is primarily associated with the transmission of dengue and chikungunya in tropical and subtropical regions of the world. The present investigations were carried out to assess the larvicidal efficiency of five indigenous weeds against Ae. aegypti. Methods. The 1,000 ppm hexane and ethanol extracts prepared from the leaves and stem of five plants (Achyranthes aspera, Cassia occidentalis, Catharanthus roseus, Lantana camara, and Xanthium strumarium) were screened for their larvicidal activity against early fourth instars of dengue vector. The extracts which could cause 80-100% mortality were further investigated for their efficacy. Results. The preliminary screening established the efficacy of hexane extracts as compared to the ethanol extracts. Further investigations revealed the highest larvicidal potential of A. aspera extracts exhibiting LC50 value of 82.555 ppm and 68.133 ppm, respectively. Further, their leaf extracts showed 5-85.9% higher larvicidal activity and stem extracts exhibited 0.23- to 0.85-fold more efficiency than the other four extracts. Conclusion. The present investigations suggest the possible use of A. aspera as an ideal ecofriendly, larvicidal agent for the control of dengue vector, Ae. aegypti. Future studies are, however, required to explore and identify the bioactive component involved and its mode of action. PMID:26941996

  10. Boosting Sensitivity in Liquid Chromatography-Fourier Transform Ion Cyclotron Resonance-Tandem Mass Spectrometry for Product Ion Analysis of Monoterpene Indole Alkaloids.

    PubMed

    Nakabayashi, Ryo; Tsugawa, Hiroshi; Kitajima, Mariko; Takayama, Hiromitsu; Saito, Kazuki

    2015-01-01

    In metabolomics, the analysis of product ions in tandem mass spectrometry (MS/MS) is noteworthy to chemically assign structural information. However, the development of relevant analytical methods are less advanced. Here, we developed a method to boost sensitivity in liquid chromatography-Fourier transform ion cyclotron resonance-tandem mass spectrometry analysis (MS/MS boost analysis). To verify the MS/MS boost analysis, both quercetin and uniformly labeled (13)C quercetin were analyzed, revealing that the origin of the product ions is not the instrument, but the analyzed compounds resulting in sensitive product ions. Next, we applied this method to the analysis of monoterpene indole alkaloids (MIAs). The comparative analyses of MIAs having indole basic skeleton (ajmalicine, catharanthine, hirsuteine, and hirsutine) and oxindole skeleton (formosanine, isoformosanine, pteropodine, isopteropodine, rhynchophylline, isorhynchophylline, and mitraphylline) identified 86 and 73 common monoisotopic ions, respectively. The comparative analyses of the three pairs of stereoisomers showed more than 170 common monoisotopic ions in each pair. This method was also applied to the targeted analysis of MIAs in Catharanthus roseus and Uncaria rhynchophylla to profile indole and oxindole compounds using the product ions. This analysis is suitable for chemically assigning features of the metabolite groups, which contributes to targeted metabolome analysis. PMID:26734034

  11. Confirmation of conjugation processes during TNT metabolism by axenic plant roots

    SciTech Connect

    Bhadra, R.; Wayment, D.G.; Hughes, J.B.; Shanks, J.V.

    1999-02-01

    This paper examines processes in plants for the formation of fate products of TNT beyond its animated reduction products, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. TNT metabolites were isolated and characterized in combination with temporal analyses of production profiles and {sup 14}C distribution, in microbe-free, axenic root cultures of Catharanthus roseus. Four unique TNT-derived compounds were isolated. Using evidence from {sup 1}H NMR, mass spectroscopy, HPLC, acid hydrolysis, and enzymatic hydrolysis with {beta}-glucuronidase and {beta}-glucosidase, they were established as conjugates formed by reactions of the amine groups of 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. From the mass spectral evidence, at least a six-carbon unit from the plant intracellular milleu was involved in conjugate formation. Mass balance analysis indicated that, by 75 h after TNT amendment of the initial TNT radiolabel, extractable conjugates comprised 22%, bound residues comprised another 29%, 2-amino-4,6-dinitrotoluene was 4%, and the rest remained unidentified. Isolates from TNT-amended roots versus monoamino-dinitrotoluene-amended roots were not identical, suggesting numerous possible outcomes for the plant-based conjugation of 2-amino-2,6-dinitrotoluene or 4-amino-2,6-dinitrotoluene. This study is the first direct evidence for the involvement of the primary reduction products of TNT--2-amino-4,6-dinitrotoluene ad 4-amino--2,6-dinitrotoluene--in conjugation process in plant detoxification of TNT.

  12. Phytochemica: a platform to explore phytochemicals of medicinal plants

    PubMed Central

    Pathania, Shivalika; Ramakrishnan, Sai Mukund; Bagler, Ganesh

    2015-01-01

    Plant-derived molecules (PDMs) are known to be a rich source of diverse scaffolds that could serve as the basis for rational drug design. Structured compilation of phytochemicals from traditional medicinal plants can facilitate prospection for novel PDMs and their analogs as therapeutic agents. Atropa belladonna, Catharanthus roseus, Heliotropium indicum, Picrorhiza kurroa and Podophyllum hexandrum are important Himalayan medicinal plants, reported to have immense therapeutic properties against various diseases. We present Phytochemica, a structured compilation of 963 PDMs from these plants, inclusive of their plant part source, chemical classification, IUPAC names, SMILES notations, physicochemical properties and 3-dimensional structures with associated references. Phytochemica is an exhaustive resource of natural molecules facilitating prospection for therapeutic molecules from medicinally important plants. It also offers refined search option to explore the neighbourhood of chemical space against ZINC database to identify analogs of natural molecules at user-defined cut-off. Availability of phytochemical structured dataset may enable their direct use in in silico drug discovery which will hasten the process of lead identification from natural products under proposed hypothesis, and may overcome urgent need for phytomedicines. Compilation and accessibility of indigenous phytochemicals and their derivatives can be a source of considerable advantage to research institutes as well as industries. Database URL: home.iitj.ac.in/∼bagler/webservers/Phytochemica PMID:26255307

  13. Bioreactor production of secondary metabolites from cell cultures of periwinkle and sandalwood.

    PubMed

    Valluri, Jagan V

    2009-01-01

    A bench-top bioreactor allowing continuous extraction of secondary metabolites is designed for Catharanthus roseus L. (G.) Don (periwinkle) and Santalum album L. (sandalwood) plant cell suspensions. Periwinkle cell cultures are exposed to biotic elicitors (Aspergillus niger, crude chitin) and abiotic elicitors (mannitol, methyl jasmonate) to induce alkaloid production. Whereas most of the biotic elicitors are effective when added on day 15 of culture, the abiotic elicitors are effective when added on day 20. The use of trans-cinnamic acid, an inhibitor of phenylalanine ammonia lyase (PAL) activity, results in significant increase in the alkaloid production of periwinkle cell cultures. Exposure of the cells to mannitol-induced osmotic stress produced marked increment in the total alkaloid production. When biotic and abiotic stress treatments are applied sequentially, an additive effect in alkaloid accumulation is observed. Although no essential oils are detected, secondary metabolites in the form of phenolics are produced by the sandalwood cell cultures in the bioreactor environment. The use of morphologic modification such as organ cultures and transformed cultures is believed to be required for both production and storage of essential oil constituents in sandalwood. The present chapter demonstrates that periwinkle and sandalwood cell suspensions could be developed and successfully cultured in a modified air-lift bioreactor. The exploitation of variant cell strains and biotransformation of added precursors can certainly improve the use of periwinkle and sandalwood cell cultures for the bioproduction of desired compounds. PMID:19521856

  14. Linking drought-resistance mechanisms to drought avoidance in upland rice using a QTL approach: progress and new opportunities to integrate stomatal and mesophyll responses.

    PubMed

    Price, Adam H; Cairns, Jill E; Horton, Peter; Jones, Hamlyn G; Griffiths, Howard

    2002-05-01

    The advent of saturated molecular maps promised rapid progress towards the improvement of crops for genetically complex traits like drought resistance via analysis of quantitative trait loci (QTL). Progress with the identification of QTLs for drought resistance-related traits in rice is summarized here with the emphasis on a mapping population of a cross between drought-resistant varieties Azucena and Bala. Data which have used root morphological traits and indicators of drought avoidance in field-grown plants are reviewed, highlighting problems and uncertainties with the QTL approach. The contribution of root-growth QTLs to drought avoidance appears small in the experiments so far conducted, and the limitations of screening methodologies and the involvement of shoot-related mechanisms of drought resistance are studied. When compared to Azucena, Bala has been observed to have highly sensitive stomata, does not roll its leaves readily, has a greater ability to adjust osmotically, slows growth more rapidly when droughted and has a lower water-use efficiency. It is also a semi-dwarf variety and hence has a different canopy structure. There is a need to clarify the contribution of the shoot to drought resistance from the level of the biochemistry of photosynthesis through stomatal behaviour and leaf anatomy to canopy architecture. Recent advances in studying the physical and biochemical processes related to water use and drought stress offer the opportunity to advance a more holistic understanding of drought resistance. These include the potential use of infrared thermal imaging to study energy balance, integrated and online stable isotope analysis to dissect processes involved in carbon dioxide fixation and water evaporation, and leaf fluorescence to monitor photosynthesis and photochemical quenching. Justification and a strategy for this integrated approach is described, which has relevance to the study of drought resistance in most crops. PMID:11971911

  15. Changes in mesophyll element distribution and phytometabolite contents involved in fluoride tolerance of the arid gypsum-tolerant plant species Atractylis serratuloides Sieber ex Cass. (Asteraceae).

    PubMed

    Boukhris, Asma; Laffont-Schwob, Isabelle; Rabier, Jacques; Salducci, Marie-Dominique; El Kadri, Lefi; Tonetto, Alain; Tatoni, Thierry; Chaieb, Mohamed

    2015-05-01

    Atractylis serratuloides is an abundant native spiny species that grows in the surroundings of superphosphate factories in Tunisia. This plant species is adapted to arid environments and tolerates a high level of fluoride pollution in soils. The aim of this study was to better understand the physiological mechanisms of fluoride tolerance of this species, comparing the fluoride-contaminated sites of Gabes and Skhira with the reference site of Smara. Results demonstrated the involvement of leaf element and phytometabolite balances in the in situ response of A. serrulatoides to fluoride. Calcium, sulphur and magnesium were differently distributed between the sites of Gabes and Smara in all plant organs. No specific tissue fluorine accumulation in root, stem and leaf, even in the most contaminated site at Gabes, was detected by EDAX mapping. Lower anthocyan and flavonol levels but enhanced nitrogen balance index were found in A. serrulatoides leaves from Gabes compared to the two other sites. A. serratuloides appeared as a fluoride excluder and its tolerance involved calcium interactions with fluoride. Moreover, an occurrence of dark septate endophytes and arbuscular mycorhizal fungi in root systems of A. serratuloides was reported for the first time, and these symbioses were present but low at all sites. We suggest the use of this plant species for fluoride-polluted soil stabilization. PMID:25510616

  16. Use of the response of photosynthesis to oxygen to estimate mesophyll conductance to carbon dioxide in water-stressed soybean leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several types of evidence indicate that there is a significant resistance to the movement of carbon dioxide from the substomatal air space to the site of fixation in the chloroplasts and that the resistance may vary with temperature, carbon dioxide concentration and water stress. Methods of estimat...

  17. CO2 enrichment and leaf aging down-regulate both maximum rates of Rubisco carboxylation and mesophyll conductance in SoyFACE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several soybean cultivars were grown under 2 levels of CO2, the ambient level of 370 microbar versus the elevated level of 550 microbar, in SoyFACE (Free Air CO2 Enrichment) in 2007. The responses of CO2 assimilation to CO2, leaf chlorophyll fluorescence, leaf contents of chlorophyll and carotenoids...

  18. Carbon dioxide diffusion across stomata and mesophyll and photo-biochemical processes as affected by growth CO2 and phosphorus nutrition in cotton

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nutrients such as phosphorus availability may exert a major control over plant response to rising atmospheric carbon dioxide concentration (CO2), which is projected to double by the end of 21st century. Elevated CO2 may overcome the diffusional limitation to photosynthesis posed by stomata and mesop...

  19. THE ENDOPHYTE CURTOBACTERIUM FLACCUMFACIENS REDUCES SYMPTOMS CAUSED BY XYLELLA FASTIDIOSA IN CATHARANTHUS ROSEUSAN ENDOPHYTIC BACTERIUM FROM CITRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus variegated chlorosis (CVC) is a disease of sweet orange (Citrus sinensis (L.)) caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that can infect all sweet orange cultivars. Sweet orange trees are sometimes observed to be infected by Xylella fastidiosa without showing seve...

  20. Structures of Iridoid Synthase from Cantharanthus roseus with Bound NAD(+) , NADPH, or NAD(+) /10-Oxogeranial: Reaction Mechanisms.

    PubMed

    Hu, Yumei; Liu, Weidong; Malwal, Satish R; Zheng, Yingying; Feng, Xinxin; Ko, Tzu-Ping; Chen, Chun-Chi; Xu, Zhongxia; Liu, Meixia; Han, Xu; Gao, Jian; Oldfield, Eric; Guo, Rey-Ting

    2015-12-14

    Structures of the iridoid synthase nepetalactol synthase in the presence of NAD(+) , NADPH or NAD(+) /10-oxogeranial were solved. The 10-oxogeranial substrate binds in a transoid-O1-C3 conformation and can be reduced by hydride addition to form the byproduct S-10-oxo-citronellal. Tyr178 Oζ is positioned 2.5 Å from the substrate O1 and provides the second proton required for reaction. Nepetalactol product formation requires rotation about C1-C2 to form the cisoid isomer, leading to formation of the cis-enolate, together with rotation about C4-C5, which enables cyclization and lactol production. The structure is similar to that of progesterone-5β-reductase, with almost identical positioning of NADP, Lys146(147), Tyr178(179), and F342(343), but only Tyr178 and Phe342 appear to be essential for activity. The transoid 10-oxogeranial structure also serves as a model for β-face hydride attack in progesterone 5β-reductases and is of general interest in the context of asymmetric synthesis. PMID:26768532

  1. MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector.

    PubMed

    Yang, Chiao-Yin; Huang, Yu-Hsin; Lin, Chan-Pin; Lin, Yen-Yu; Hsu, Hao-Chun; Wang, Chun-Neng; Liu, Li-Yu Daisy; Shen, Bing-Nan; Lin, Shih-Shun

    2015-08-01

    Leafy flowers are the major symptoms of peanut witches' broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development. PMID:26103992

  2. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth.

    PubMed

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S; Grossniklaus, Ueli

    2015-09-29

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRI(R240C) mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  3. CjbHLH1 homologs regulate sanguinarine biosynthesis in Eschscholzia californica cells.

    PubMed

    Yamada, Yasuyuki; Motomura, Yukiya; Sato, Fumihiko

    2015-05-01

    Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica. PMID:25713177

  4. Molecular docking and pharmacogenomics of vinca alkaloids and their monomeric precursors, vindoline and catharanthine.

    PubMed

    Sertel, Serkan; Fu, Yujie; Zu, Yuangang; Rebacz, Blanka; Konkimalla, Badireenath; Plinkert, Peter K; Krämer, Alwin; Gertsch, Jürg; Efferth, Thomas

    2011-03-15

    Vinblastine and vincristine are dimeric indole alkaloids derived from Catharanthus roseus (formerly: Vinca rosea). Their monomeric precursor molecules are vindoline and catharanthine. While vinblastine and vincristine are well-known mitotic spindle poisons, not much is known about vindoline and catharanthine. Vindoline and catharanthine showed weak cytotoxicity, while vinblastine, vincristine, and the semisynthetic vindesine and vinorelbine revealed high cytotoxicity towards cancer cells. This may reflect a general biological principle of poisonous plants. Highly toxic compounds are not only active towards predators, but also towards plant tissues. Hence, plants need mechanisms to protect themselves from their own poisons. One evolutionary strategy to solve this problem is to generate less toxic precursors, which are dimerized to toxic end products when needed. As shown by in silico molecular docking and biochemical approaches, vinblastine, vincristine and vinorelbine bound with high affinity to α/β-tubulin and inhibited tubulin polymerization, whereas the effects of vindoline and catharanthine were weak. Similarly, vinblastine produced high fractions of mono- and multipolar mitotic spindles, while vindoline and catharanthine did only weakly affect bipolar mitotic spindle formation. Here, we show that vinblastine contributes to cell death by interference with spindle polarity. P-glycoprotein-overexpressing multidrug-resistant CEM/VCR1000 cells were highly resistant towards vincristine and cross-resistant to vinblastine, vindesine, and vinorelbine, but not or only weakly cross-resistant to vindoline and catharanthine. In addition to tubulin as primary target, microarray-based mRNA signatures of responsiveness of these compounds have been identified by COMPARE and signaling pathway profiling. PMID:21219884

  5. Receptor-like cytoplasmic kinase MARIS functions downstream of CrRLK1L-dependent signaling during tip growth

    PubMed Central

    Boisson-Dernier, Aurélien; Franck, Christina Maria; Lituiev, Dmytro S.; Grossniklaus, Ueli

    2015-01-01

    Growing plant cells need to rigorously coordinate external signals with internal processes. For instance, the maintenance of cell wall (CW) integrity requires the coordination of CW sensing with CW remodeling and biosynthesis to avoid growth arrest or integrity loss. Despite the involvement of receptor-like kinases (RLKs) of the Catharanthus roseus RLK1-like (CrRLK1L) subfamily and the reactive oxygen species-producing NADPH oxidases, it remains largely unknown how this coordination is achieved. ANXUR1 (ANX1) and ANX2, two redundant members of the CrRLK1L subfamily, are required for tip growth of the pollen tube (PT), and their closest homolog, FERONIA, controls root-hair tip growth. Previously, we showed that ANX1 overexpression mildly inhibits PT growth by oversecretion of CW material, whereas pollen tubes of anx1 anx2 double mutants burst spontaneously after germination. Here, we report the identification of suppressor mutants with improved fertility caused by the rescue of anx1 anx2 pollen tube bursting. Mapping of one these mutants revealed an R240C nonsynonymous substitution in the activation loop of a receptor-like cytoplasmic kinase (RLCK), which we named MARIS (MRI). We show that MRI is a plasma membrane-localized member of the RLCK-VIII subfamily and is preferentially expressed in both PTs and root hairs. Interestingly, mri-knockout mutants display spontaneous PT and root-hair bursting. Moreover, expression of the MRIR240C mutant, but not its wild-type form, partially rescues the bursting phenotypes of anx1 anx2 PTs and fer root hairs but strongly inhibits wild-type tip growth. Thus, our findings identify a novel positive component of the CrRLK1L-dependent signaling cascade that coordinates CW integrity and tip growth. PMID:26378127

  6. Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases*

    PubMed Central

    Torrens-Spence, Michael P.; Liu, Pingyang; Ding, Haizhen; Harich, Kim; Gillaspy, Glenda; Li, Jianyong

    2013-01-01

    Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site

  7. Useful ethnophytomedicinal recipes of angiosperms used against diabetes in South East Asian Countries (India, Pakistan & Sri Lanka).

    PubMed

    Marwat, Sarfaraz Khan; Rehman, Fazalur; Khan, Ejaz Ahmad; Khakwani, Abdul Aziz; Ullah, Imdad; Khan, Kaleem Ullah; Khan, Inam Ullah

    2014-09-01

    This paper is based on data recorded from various literatures pertaining to ethnophytomedicinal recipes used against diabetes in South East Asia (India, Pakistan and Srilanka). Traditional plant treatments have been used throughout the world for the therapy of diabetes mellitus. In total 419 useful phytorecipes of 270 plant species belonging to 74 Angiospermic families were collected. From the review it was revealed that plants showing hypoglycemic potential mainly belong to the families, Cucurbitaceae (16 spp.), Euphorbiaceae (15 spp.), Caesalpiniaceae and Papilionaceae (13 spp. each), Moraceae (11 spp.), Acanthaceae (10 spp.), Mimosaceae (09 spp.), Asteraceae, Malvaceae and Poaceae (08 spp. each), Hippocrateaceae, Rutaceae and Zingiberaceae (07 spp. each), Apocynaceae, Asclepiadaceae and Verbenaceae (06 spp. each), Apiaceae, Convolvulaceae, Lamiaceae, Myrtaceae, Solanaceae (05 spp.each). The most active plants are Syzigium cumini (14 recipes), Phyllanthus emblica (09 recipes), Centella asiatica and Momordica charantia (08 recipes each), Azadirachta indica (07 recipes), Aegle marmelos, Catharanthus roseus, Ficus benghalensis, Ficus racemosa, Gymnema sylvestre (06 recipes each), Allium cepa, A. sativum, Andrographis paniculata, Curcuma longa (05 recipes each), Citrullus colocynthis, Justicia adhatoda, Nelumbo nucifera, Tinospora cordifolia, Trigonella foenum-graecum, Ziziphus mauritiana and Wattakaka volubilis (4 recipes each). These traditional recipes include extracts, leaves, powders, flour, seeds, vegetables, fruits and herbal mixtures. Data inventory consists of botanical name, recipe, vernacular name, English name. Some of the plants of the above data with experimentally confirmed antidiabetic properties have also been recorded. More investigations must be carried out to evaluate the mechanism of action of diabetic medicinal plants. Toxicity of these plants should also be explained. Scientific validation of these recipes may help in discovering new drugs from

  8. Antiproliferative and phytochemical analyses of leaf extracts of ten Apocynaceae species

    PubMed Central

    Wong, Siu Kuin; Lim, Yau Yan; Abdullah, Noor Rain; Nordin, Fariza Juliana

    2011-01-01

    Background: The anticancer properties of Apocynaceae species are well known in barks and roots but less so in leaves. Materials and Methods: In this study, leaf extracts of 10 Apocynaceae species were assessed for antiproliferative (APF) activities using the sulforhodamine B assay. Their extracts were also analyzed for total alkaloid content (TAC), total phenolic content (TPC), and radical scavenging activity (RSA) using the Dragendorff precipitation, Folin–Ciocalteu, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, respectively. Results: Leaf extracts of Alstonia angustiloba, Calotropis gigantea, Catharanthus roseus, Nerium oleander, Plumeria obtusa, and Vallaris glabra displayed positive APF activities. Extracts of Allamanda cathartica, Cerbera odollam, Dyera costulata, and Kopsia fruticosa did not show any APF activity. Dichloromethane (DCM) extract of C. gigantea, and DCM and DCM:MeOH extracts of V. glabra showed strong APF activities against all six human cancer cell lines. Against breast cancer cells of MCF-7 and MDA-MB-231, DCM extracts of C. gigantea and N. oleander were stronger than or comparable to standard drugs of xanthorrhizol, curcumin, and tamoxifen. All four extracts of N. oleander were effective against MCF-7 cells. Extracts of Kopsia fruticosa had the highest TAC while those of Dyera costulata had the highest TPC and RSA. Extracts of C. gigantea and V. glabra inhibited the growth of all six cancer cell lines while all extracts of N. oleander were effective against MCF-7 cells. Conclusion: Extracts of C. gigantea, V. glabra, and N. oleander therefore showed great promise as potential candidates for anticancer drugs. The wide-spectrum APF activities of these three species are reported for the first time and their bioactive compounds warrant further investigation. PMID:21772753

  9. Screening molecules for control of citrus Huanglongbing (HLB) using an optimized regeneration system for 'Candidatus Liberibacter asiaticus' infected periwinkle (Catharunthus roseus) cuttings

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Citrus huanglongbing (HLB) ( also known as citrus greening) is one of the most destructive diseases of citrus worldwide. The disease is associated with three different species of Candidatus Liberibacter, of which, ‘Ca. L. asiaticus’ (Las) is the most widely-distributed. An improved system using HLB-...

  10. Comparison of plasma vitamin A and E, copper and zinc levels in free-ranging and captive greater flamingos (Phoenicopterus roseus) and their relation to pododermatitis.

    PubMed

    Wyss, F; Wolf, P; Wenker, C; Hoby, S; Schumacher, V; Béchet, A; Robert, N; Liesegang, A

    2014-12-01

    Pododermatitis is a worldwide problem in captive flamingos. Studies in domestic poultry showed that nutrition is a possible influencing factor for pododermatitis. Vitamin A and E, copper and zinc levels were analysed in two different diets (diet 1 = in-house mix and diet 2 = commercial diet) and in plasma of captive greater flamingos fed these diets and compared to those of free-ranging greater flamingos. Results were analysed with respect to type and severity of foot lesions of the individuals from the different groups. Juvenile and subadult/adult captive flamingos on diet 1 showed various types and severities of foot lesions, whereas no foot lesions were found at the time of blood sampling in juvenile captive flamingos on diet 2. Juvenile captive flamingos on diet 1 had significantly lower plasma zinc levels than juvenile captive flamingos on diet 2 and juvenile free-ranging flamingos; data were also lower than reference ranges for flamingos, poultry and cranes. There were no significant differences in plasma vitamin A, vitamin E, copper or zinc levels between animals with different types of foot lesions or with different severity scores. Shortly after the change to diet 2 (fed to juvenile captive flamingos that did not show any foot lesion), the flooring of the outdoor water pools was covered with fine granular sand. Because both factors (nutrition and flooring) were changed during the same evaluation period, it cannot be concluded which factor contributed in what extent to the reduction of foot lesions. While it is assumed that low plasma zinc levels identified in the group of juvenile captive flamingos on diet 1 were not directly responsible for foot lesions observed in these animals, they may have played a role in altering the skin integrity of the feet and predisposing them to pododermatitis. PMID:24661548

  11. Inconsistency of mesophyll conductance estimate causes the inconsistency for the estimates of maximum rate of Rubisco carboxylation among the linear, rectangular, and non-rectangular hyperbola biochemical models of leaf...

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The responses of CO2 assimilation to [CO2] (A/Ci) were investigated at two developmental stages (R5 and R6) and in several soybean cultivars grown under two levels of [CO2], the ambient level of 370 µbar versus the elevated level of 550 µbar. The A/Ci data were analyzed and compared using various cu...

  12. Building a model: developing genomic resources for common milkweed (Asclepias syriaca) with low coverage genome sequencing

    PubMed Central

    2011-01-01

    Background Milkweeds (Asclepias L.) have been extensively investigated in diverse areas of evolutionary biology and ecology; however, there are few genetic resources available to facilitate and compliment these studies. This study explored how low coverage genome sequencing of the common milkweed (Asclepias syriaca L.) could be useful in characterizing the genome of a plant without prior genomic information and for development of genomic resources as a step toward further developing A. syriaca as a model in ecology and evolution. Results A 0.5× genome of A. syriaca was produced using Illumina sequencing. A virtually complete chloroplast genome of 158,598 bp was assembled, revealing few repeats and loss of three genes: accD, clpP, and ycf1. A nearly complete rDNA cistron (18S-5.8S-26S; 7,541 bp) and 5S rDNA (120 bp) sequence were obtained. Assessment of polymorphism revealed that the rDNA cistron and 5S rDNA had 0.3% and 26.7% polymorphic sites, respectively. A partial mitochondrial genome sequence (130,764 bp), with identical gene content to tobacco, was also assembled. An initial characterization of repeat content indicated that Ty1/copia-like retroelements are the most common repeat type in the milkweed genome. At least one A. syriaca microread hit 88% of Catharanthus roseus (Apocynaceae) unigenes (median coverage of 0.29×) and 66% of single copy orthologs (COSII) in asterids (median coverage of 0.14×). From this partial characterization of the A. syriaca genome, markers for population genetics (microsatellites) and phylogenetics (low-copy nuclear genes) studies were developed. Conclusions The results highlight the promise of next generation sequencing for development of genomic resources for any organism. Low coverage genome sequencing allows characterization of the high copy fraction of the genome and exploration of the low copy fraction of the genome, which facilitate the development of molecular tools for further study of a target species and its relatives

  13. Antiplasmodial potential of medicinal plant extracts from Malaiyur and Javadhu hills of South India.

    PubMed

    Kamaraj, Chinnaperumal; Kaushik, Naveen Kumar; Mohanakrishnan, Dinesh; Elango, Gandhi; Bagavan, Asokan; Zahir, Abdul Abduz; Rahuman, Abdul Abdul; Sahal, Dinkar

    2012-08-01

    The emergence and spread of Plasmodium falciparum with resistance to chloroquine (CQ), the safest and cheapest anti-malarial drug, coupled with the increasing cost of alternative drugs especially in developing countries have necessitated the urgent need to tap the potential of plants for novel anti-malarials. The present study investigates the anti-malarial activity of the methanolic extracts of 13 medicinal plants from the Malaiyur and Javadhu hills of South India against blood stage CQ-sensitive (3D7) and CQ-resistant (INDO) strains of P. falciparum in culture using the fluorescence-based SYBR Green I assay. Sorbitol-synchronized parasites were incubated under normal culture conditions at 2% hematocrit and 1% parasitemia in the absence or presence of increasing concentrations of plant extracts. CQ and artemisinin were used as positive controls, while 0.4% DMSO was used as the negative control. The cytotoxic effects of extracts on host cells were assessed by functional assay using HeLa cells cultured in RPMI containing 10% fetal bovine serum, 0.21% sodium bicarbonate and 50 μg/mL gentamycin (complete medium). Plant extracts (bark methanol extracts of Annona squamosa (IC(50), 30 μg/mL), leaf extracts of Ocimum gratissimum (IC(50), 32 μg/mL), Ocimum tenuiflorum (IC(50), 31 μg/mL), Solanum torvum (IC(50), 31 μg/mL) and Justicia procumbens (IC(50), 63 μg/mL), showed moderate activity. The leaf extracts of Aristolochia indica (IC(50), 10 μg/mL), Cassia auriculata (IC(50), 14 μg/mL), Chrysanthemum indicum (IC(50), 20 μg/mL) and Dolichos biflorus (IC(50), 20 μg/mL) showed promising activity and low activity was observed in the flower methanol extracts of A. indica , leaf methanol extract of Catharanthus roseus, and Gymnema sylvestre (IC(50), >100 μg/mL). These four extracts exhibited promising IC(50) (μg/mL) of 17, 24, 19 and 24 respectively also against the CQ resistant INDO strain of P. falciparum. The high TC(50) in mammalian cell cytotoxicity assay and

  14. Ethnomedicines used in Trinidad and Tobago for reproductive problems

    PubMed Central

    Lans, Cheryl

    2007-01-01

    Background Throughout history women have tried to control or enhance their fertility using herbal remedies, with various levels of societal support. Caribbean folk medicine has been influenced by European folk medicine, either through the early Spanish and French settlers or through the continuous immigration of Spanish-speaking peoples from Venezuela. Some folk uses are ancient and were documented by Galen and Pliny the Elder. Methods Thirty respondents, ten of whom were male were interviewed from September 1996 to September 2000. The respondents were obtained by snowball sampling, and were found in thirteen different sites, 12 in Trinidad (Paramin, Talparo, Sangre Grande, Mayaro, Carapichaima, Kernahan, Newlands, Todd's Road, Arima, Guayaguayare, Santa Cruz, Port of Spain and Siparia) and one in Tobago (Mason Hall). Snowball sampling was used because there was no other means of identifying respondents and to cover the entire islands. The validation of the remedies was conducted with a non-experimental method. Results Plants are used for specific problems of both genders. Clusea rosea, Urena sinuata and Catharanthus roseus are used for unspecified male problems. Richeria grandis and Parinari campestris are used for erectile dysfunction. Ageratum conyzoides, Scoparia dulcis, Cucurbita pepo, Cucurbita maxima, Gomphrena globosa and Justicia pectoralis are used for prostate problems. The following plants are used for childbirth and infertility: Mimosa pudica, Ruta graveolens, Abelmoschus moschatus, Chamaesyce hirta, Cola nitida, Ambrosia cumanenesis, Pilea microphylla, Eryngium foetidum, Aristolochia rugosa, Aristolochia trilobata, Coleus aromaticus, Laportea aestuans and Vetiveria zizanioides. The following plants are used for menstrual pain and unspecified female complaints: Achyranthes indica, Artemisia absinthium, Brownea latifolia, Eleutherine bulbosa, Hibiscus rosa-sinensis, Eupatorium macrophyllum, Justicia secunda, Parthenium hysterophorus, Wedelia trilobata

  15. Zoospore interspecific signaling promotes plant infection by Phytophthora

    PubMed Central

    2010-01-01

    Background Oomycetes attack a huge variety of economically and ecologically important plants. These pathogens release, detect and respond to signal molecules to coordinate their communal behaviors including the infection process. When signal molecules are present at or above threshold level, single zoospores can infect plants. However, at the beginning of a growing season population densities of individual species are likely below those required to reach a quorum and produce threshold levels of signal molecules to trigger infection. It is unclear whether these molecules are shared among related species and what their chemistries are. Results Zoospore-free fluids (ZFF) from Phytophthora capsici, P. hydropathica, P. nicotianae (ZFFnic), P. sojae (ZFFsoj) and Pythium aphanidermatum were cross tested for stimulating plant infection in three pathosystems. All ZFFs tested significantly increased infection of Catharanthus roseus by P. nicotianae. Similar cross activities were observed in infection of Lupinus polyphyllus and Glycine max by P. sojae. Only ZFFnic and ZFFsoj cross induced zoospore aggregation at a density of 2 × 103 ml-1. Pure autoinducer-2 (AI-2), a component in ZFF, caused zoospore lysis of P. nicotianae before encystment and did not stimulate plant infection at concentrations from 0.01 to 1000 μM. P. capsici transformants with a transiently silenced AI-2 synthase gene, ribose phosphate isomerase (RPI), infected Capsicum annuum seedlings at the same inoculum concentration as the wild type. Acyl-homoserine lactones (AHLs) were not detected in any ZFFs. After freeze-thaw treatments, ZFF remained active in promoting plant infection but not zoospore aggregation. Heat treatment by boiling for 5 min also did not affect the infection-stimulating property of ZFFnic. Conclusion Oomycetes produce and use different molecules to regulate zoospore aggregation and plant infection. We found that some of these signal molecules could act in an inter-specific manner

  16. Nonphytotoxic Aluminum-Peat Complexes Suppress Phytophthora parasitica.

    PubMed

    Fichtner, E J; Hesterberg, D L; Shew, H D

    2001-11-01

    ABSTRACT Amendment of peat-based potting media with Al(2)(SO(4))(3) suppresses damping-off of Vinca (Catharanthus roseus) caused by Phytophthora parasitica. The species of aluminum (Al) responsible for disease suppression have not been identified. The objective of this study was to determine the effects of amount and pH of Al(2)(SO(4))(3) amendment solutions on survival of P. parasitica. In separate experiments, peat was amended with Al(2)(SO(4))(3) solutions adjusted to pH 4 or 6 at either 0.0158 or 0.0079 g of Al per gram of peat. Amended peat was placed in Büchner funnels maintained at -2.5 kPa matric potential. Peat was infested with P. parasitica by placing zero, two, or five colonized Vinca leaf disks in each funnel, and 15 Vinca seeds were placed in each funnel. After 24 h, the matric potential was brought to 0 kPa to induce zoospore release and returned to -2.5 kPa after 24 h. Pathogen populations and stand counts were assessed after 2-week incubation. Al amendment solutions at both pH 4 and 6 reduced pathogen populations at 0.0158 g of Al per gram of peat. Solutions at pH 4 reduced pathogen populations by more than 90% at both inoculum levels; amendment solutions at pH 6 reduced populations by 95% at the low inoculum level and 65% at the high inoculum level. The prevalence of Al(OH)(2)(+) in peat amended with Al(2)(SO(4))(3) solution at pH 6 suggests that ions other than Al(3+) may be responsible for pathogen suppression. Based on the difference in chemical conditions of Al-amended peat and suppressive mineral soils, the mechanism of Al-mediated suppression of plant pathogens is speculated to be different in the two systems. Peat containing Al-peat complexes was chemically suppressive to P. parasitica and may confer Al-mediated suppression of plant pathogens with a nonphytotoxic form of Al. PMID:18943446

  17. Potent α-amylase inhibitory activity of Indian Ayurvedic medicinal plants

    PubMed Central

    2011-01-01

    Background Indian medicinal plants used in the Ayurvedic traditional system to treat diabetes are a valuable source of novel anti-diabetic agents. Pancreatic α-amylase inhibitors offer an effective strategy to lower the levels of post-prandial hyperglycemia via control of starch breakdown. In this study, seventeen Indian medicinal plants with known hypoglycemic properties were subjected to sequential solvent extraction and tested for α-amylase inhibition, in order to assess and evaluate their inhibitory potential on PPA (porcine pancreatic α-amylase). Preliminary phytochemical analysis of the lead extracts was performed in order to determine the probable constituents. Methods Analysis of the 126 extracts, obtained from 17 plants (Aloe vera (L.) Burm.f., Adansonia digitata L., Allium sativum L., Casia fistula L., Catharanthus roseus (L.) G. Don., Cinnamomum verum Persl., Coccinia grandis (L.) Voigt., Linum usitatisumum L., Mangifera indica L., Morus alba L., Nerium oleander L., Ocimum tenuiflorum L., Piper nigrum L., Terminalia chebula Retz., Tinospora cordifolia (Willd.) Miers., Trigonella foenum-graceum L., Zingiber officinale Rosc.) for PPA inhibition was initially performed qualitatively by starch-iodine colour assay. The lead extracts were further quantified with respect to PPA inhibition using the chromogenic DNSA (3, 5-dinitrosalicylic acid) method. Phytochemical constituents of the extracts exhibiting≥ 50% inhibition were analysed qualitatively as well as by GC-MS (Gas chromatography-Mass spectrometry). Results Of the 126 extracts obtained from 17 plants, 17 extracts exhibited PPA inhibitory potential to varying degrees (10%-60.5%) while 4 extracts showed low inhibition (< 10%). However, strong porcine pancreatic amylase inhibitory activity (> 50%) was obtained with 3 isopropanol extracts. All these 3 extracts exhibited concentration dependent inhibition with IC50 values, viz., seeds of Linum usitatisumum (540 μgml-1), leaves of Morus alba (1440

  18. Differential Localization of Antioxidants in Maize Leaves.

    PubMed Central

    Doulis, A. G.; Debian, N.; Kingston-Smith, A. H.; Foyer, C. H.

    1997-01-01

    The aim of this work was to determine the compartmentation of antioxidants between the bundle-sheath and mesophyll cells of maize (Zea mays L.) leaves. Rapid fractionation of the mesophyll compartment was used to minimize modifications in the antioxidant status and composition due to extraction procedures. The purity of the mesophyll isolates was assessed via the distribution of enzyme and metabolite ma