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Sample records for 3-hydroxylase f3h dihydroflavonol

  1. Identification of flavonoid 3'-hydroxylase in the yellow flower of Delphinium zalil.

    PubMed

    Miyahara, Taira; Hamada, Arisa; Okamoto, Mitsutoshi; Hirose, Yukio; Sakaguchi, Kimitoshi; Hatano, Shoji; Ozeki, Yoshihiro

    2016-09-01

    The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors. PMID:27478933

  2. Light response and potential interacting proteins of a grape flavonoid 3'-hydroxylase gene promoter.

    PubMed

    Sun, Run-Ze; Pan, Qiu-Hong; Duan, Chang-Qing; Wang, Jun

    2015-12-01

    Flavonoid 3'-hydroxylase (F3'H), a member of cytochrome P450 protein family, introduces B-ring hydroxyl group in the 3' position of the flavonoid. In this study, the cDNA sequence of a F3'H gene (VviF3'H), which contains an open reading frame of 1530 bp encoding a polypeptide of 509 amino acids, was cloned and characterized from Vitis vinifera L. cv. Cabernet Sauvignon. VviF3'H showed high homology to known F3'H genes, especially F3'Hs from the V. vinifera reference genome (Pinot Noir) and lotus. Expression profiling analysis using real-time PCR revealed that VviF3'H was ubiquitously expressed in all tested tissues including berries, leaves, flowers, roots, stems and tendrils, suggesting its important physiological role in plant growth and development. Moreover, the transcript level of VviF3'H gene in grape berries was relatively higher at early developmental stages and gradually decreased during véraison, and then increased in the mature phase. In addition, the promoter of VviF3'H was isolated by using TAIL-PCR. Yeast one-hybrid screening of the Cabernet Sauvignon cDNA library and subsequent in vivo/vitro validations revealed the interaction between VviF3'H promoter and several transcription factors, including members of HD-Zip, NAC, MYB and EIN families. A transcriptional regulation mechanism of VviF3'H expression is proposed for the first time. PMID:26433636

  3. Molecular cloning and characterization of a flavanone 3-Hydroxylase gene from Artemisia annua L.

    PubMed

    Xiong, Shuo; Tian, Na; Long, Jinhua; Chen, Yuhong; Qin, Yu; Feng, Jinyu; Xiao, Wenjun; Liu, Shuoqian

    2016-08-01

    Flavonoids were found to synergize anti-malaria and anti-cancer compounds in Artemisia annua, a very important economic crop in China. In order to discover the regulation mechanism of flavonoids in Artemisia annua, the full length cDNA of flavanone 3-hydroxylase (F3H) were isolated from Artemisia annua for the first time by using RACE (rapid amplification of cDNA ends). The completed open read frame of AaF3H was 1095 bp and it encoded a 364-amino acid protein with a predicted molecular mass of 41.18 kDa and a pI of 5.67. The recombinant protein of AaF3H was expressed in E. coli BL21(DE3) as His-tagged protein, purified by Ni-NTA agrose affinity chromatography, and functionally characterized in vitro. The results showed that the His-tagged protein (AaF3H) catalyzed naringenin to dihydrokaempferol in the present of Fe(2+). The Km for naringenin was 218.03 μM. The optimum pH for AaF3H reaction was determined to be pH 8.5, and the optimum temperature was determined to be 35 °C. The AaF3H transcripts were found to be accumulated in the cultivar with higher level of flavonoids than that with lower level of flavonoids, which implied that AaF3H was a potential target for regulation of flavonoids biosynthesis in Artemisia annua through metabolic engineering. PMID:27070290

  4. Regulation of flavanone 3-hydroxylase gene involved in the flavonoid biosynthesis pathway in response to UV-B radiation and drought stress in the desert plant, Reaumuria soongorica.

    PubMed

    Liu, Meiling; Li, Xinrong; Liu, Yubing; Cao, Bo

    2013-12-01

    Flavonoid are known to have various functions in growth, development, reproduction, and also involved in diverse stress responses in plants. However, little is known about the roles of the key enzymes in the flavonoid biosynthetic pathway in response to environmental stress, such as UV-B radiation and drought. To understand this problem, we investigated the participation of flavanone 3-hydroxylase gene (F3H), a key enzyme in flavonoid biosynthetic pathway under UV-B radiation and drought stress in the desert plant Reaumuria soongorica. A novel cDNA sequence, named as RsF3H, was isolated from R. soongorica. The deduced amino acids showed high identities to other F3Hs. A phylogenetic analysis indicated that RsF3H appeared to be most homologous to F3H from Malus domestica (MdF3H). RsF3H protein structure contained all five conserved motifs for 2-oxoglutarate-dependent dioxygenases (2-ODDs) and an Arg-X-Ser motif, all of which were also found in other F3Hs. Quantitative real-time RT-PCR analysis showed that there was a rapid increase in gene expression of RsF3H under stress. Both UV-B radiation and drought stress induced an increase in RsF3H enzyme activity and the accumulation of the products in the flavonoid biosynthetic pathway (total flavonoid and anthocyanin). The antioxidant ability (inhibition of lipid oxidation) of total flavonoid was enhanced during this study. The results suggested that one explanation of the stress tolerance of R. soongorica may be a combination of an increase in RsF3H gene expression, RsF3H enzyme activity and the anti-oxidative ability of the metabolic end products in the flavonoid biosynthetic pathway in response to UV-B radiation and drought. PMID:24121417

  5. Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase.

    PubMed Central

    Zabala, Gracia; Vodkin, Lila

    2003-01-01

    Three loci (I, R, and T) control pigmentation of the seed coats in Glycine max and are genetically distinct from those controlling flower color. The T locus also controls color of the trichome hairs. We report the identification and isolation of a flavonoid 3' hydroxylase gene from G. max (GmF3'H) and the linkage of this gene to the T locus. This GmF3'H gene was highly expressed in early stages of seed coat development and was expressed at very low levels or not at all in other tissues. Evidence that the GmF3'H gene is linked to the T locus came from the occurrence of multiple RFLPs in lines with varying alleles of the T locus, as well as in a population of plants segregating at that locus. GmF3'H genomic and cDNA sequence analysis of color mutant lines with varying t alleles revealed a frameshift mutation in one of the alleles. In another line derived from a mutable genetic stock, the abundance of the mRNAs for GmF3'H was dramatically reduced. Isolation of the GmF3'H gene and its identification as the T locus will enable investigation of the pleiotropic effects of the T locus on cell wall integrity and its involvement in the regulation of the multiple branches of the flavonoid pathway in soybean. PMID:12586717

  6. Molecular cloning and identification of a flavanone 3-hydroxylase gene from Lycium chinense, and its overexpression enhances drought stress in tobacco.

    PubMed

    Song, Xinyu; Diao, Jinjin; Ji, Jing; Wang, Gang; Guan, Chunfeng; Jin, Chao; Wang, Yurong

    2016-01-01

    Flavonoids, as plant secondary metabolites, are widespread throughout the plant kingdom and involved in many physiological and biochemical processes. Drought resistance is attributed to flavonoids with respect to protective functions in the cell wall and membranes. The flavanone 3-hydroxylase (F3H) gene which encodes flavanone 3-hydroxylase, is essential in flavonoids biosynthetic pathway. Lycium chinense (L. chinense) is a deciduous woody perennial halophyte that grows under a large variety of environmental conditions and survives under extreme drought stress. A novel cDNA sequence coding a F3H gene in Lycium chinense (LcF3H, GenBank: KJ636468.1) was isolated. The open reading frame of LcF3H comprised 1101 bp encoding a polypeptide of 366 amino acids with a molecular weight of about 42 kDa and an isoelectric point of 5.32. The deduced LcF3H protein showed high identities with other plant F3Hs, and the conserved motifs were found in LcF3H at similar positions like other F3Hs. The recombinant protein converted naringen into dihydrokaempferol in vitro. Since studies have shown that amongst flavonoids, flavan-3-ols (catechin and epicatechin) have direct free radical scavenging activity to maintain the normal physiological function of cells in vivo, these data support the possible relationship between the oxidative damage and the regulation of LcF3H gene expression in L. chinense under drought stress. In order to better understand the biotechnological potential of LcF3H, gene overexpression was conducted in tobacco. The content of flavan-3-ols and the tolerance to drought stress were increased in LcF3H overexpressing tobacco. Analysis of transgenic tobacco lines also showed that antioxidant enzyme activities were increased meanwhile the malondialdehyde (MDA) content and the content of H2O2 were reduced comparing to nontransformed tobacco plants. Furthermore, the photosynthesis rate was less decreased in the transgenetic plants. These results suggest that LcF3H

  7. Differential expression of flavonoid 3'-hydroxylase during fruit development establishes the different B-ring hydroxylation patterns of flavonoids in Fragaria × ananassa and Fragaria vesca.

    PubMed

    Thill, Jana; Miosic, Silvija; Gotame, Tek Prasad; Mikulic-Petkovsek, Maja; Gosch, Christian; Veberic, Robert; Preuss, Anja; Schwab, Wilfried; Stampar, Franci; Stich, Karl; Halbwirth, Heidi

    2013-11-01

    Flavonoid 3'-hydroxylase (F3'H) was studied for the first time in different Fragaria species. The cDNA clones isolated from unripe and ripe fruits of Fragaria x ananassa (garden strawberry) and Fragaria vesca (wild strawberry) showed high similarity (99% at the amino acid level) to the publically available F. vesca genome sequence and no significant differences could be identified between species and developmental stages of the fruits. In contrast, the genomic F3'H clones showed differences in the non-coding regions and 5'-flanking elements. The recombinant F3'Hs were functionally active and showed high specificity for naringenin, dihydrokaempferol, and kaempferol, whereas apigenin was only a minor substrate. During fruit development, a clear difference in the F3'H expression was observed between F. × ananassa and F. vesca. While a drastic decline of F3'H expression occurred during fruit ripening in F. × ananassa, F3'H in F. vesca was highly expressed in all stages. This was reflected by the anthocyanin composition, which showed a prevalence of pelargonidin in ripe fruits of F. × ananassa, whereas F. vesca had a high content of cyanidin. Screening of 17 berry species for their anthocyanin and flavonol composition showed that the prevalence of monohydroxylated anthocyanins makes garden strawberry unique among all other fruit species indicating that selection of bright red color during strawberry breeding, which consumers typically associate with freshness and ripeness, has selected phenotypes with a special biochemical background. PMID:23623754

  8. Bone matrix hypermineralization in prolyl-3 hydroxylase 1 deficient mice.

    PubMed

    Fratzl-Zelman, Nadja; Bächinger, Hans-Peter; Vranka, Janice A; Roschger, Paul; Klaushofer, Klaus; Rauch, Frank

    2016-04-01

    Lack of prolyl 3-hydroxylase 1 (P3H1) due to mutations in P3H1 results in severe forms of recessive osteogenesis imperfecta. In the present study, we investigated the bone tissue characteristics of P3H1 null mice. Histomorphometric analyses of cancellous bone in the proximal tibia and lumbar vertebra in 1-month and 3-month old mice demonstrated that P3H1 deficient mice had low trabecular bone volume and low mineral apposition rate, but normal osteoid maturation time and normal osteoblast and osteoclast surfaces. Quantitative backscattered electron imaging revealed that the bone mineralization density distribution was shifted towards higher values, indicating hypermineralization of bone matrix. It thus appears that P3H1 deficiency leads to decreased deposition of extracellular matrix by osteoblasts and increased incorporation of mineral into the matrix. PMID:26808442

  9. Identification of the flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase genes from Antarctic moss and their regulation during abiotic stress.

    PubMed

    Liu, Shenghao; Ju, Jianfang; Xia, Guangmin

    2014-06-10

    Flavonoids are ubiquitous plant secondary metabolites, and their hydroxylation pattern determines their color, stability, and antioxidant capacity. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3',5'H). However, they are still not well documented in lower plants such as bryophytes. We report the identification of gene encoding F3'H, F3',5'H from Antarctic moss Pohlia nutans and their transcriptional regulation under different stress conditions. Totally, sixteen cDNAs were isolated from P. nutans by RT-PCR and RACE techniques, all of which were predicted to code for F3'Hs or F3',5'Hs based on their annotations of Blast results. Amino acid alignment showed that they possessed the featured conserved domains of flavonoid hydroxylase, including proline-rich "hinge" region, EXXR motif, oxygen binding pocket motif, heme binding domain and substrate recognition sites. Phylogenetic analysis indicated that moss F3'Hs and F3',5'Hs were highly conserved and have independent evolution from the monocots, dicots and ferns. Meanwhile, real-time PCR analysis revealed that the expression profiling of flavonoid hydroxylase genes was influenced by diverse abiotic stresses including cold, salinity, drought or UV-B radiation and plant hormone abscisic acid (ABA) or jasmonic acid (JA) treatment. Since 3',4',5'-hydroxylated flavonoid-derivatives may serve a multitude of functions, including antioxidant activity and UV filters, the evolution and expression profile of flavonoid hydroxylase probably reflect the adaptive value of Antarctic moss in the acclimation of polar environment. PMID:24631264

  10. Flower colour modification of chrysanthemum by suppression of F3'H and overexpression of the exogenous Senecio cruentus F3'5'H gene.

    PubMed

    He, Huang; Ke, Hu; Keting, Han; Qiaoyan, Xiang; Silan, Dai

    2013-01-01

    Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium 'LPi' and C × morifolium 'LPu', that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3'H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3'H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3'H using RNAi and overexpressed the Senecio cruentus F3'5'H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3'H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3'5'H only exhibited F3'H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum. PMID:24250783

  11. Expression analysis of dihydroflavonol 4-reductase genes in Petunia hybrida.

    PubMed

    Chu, Y X; Chen, H R; Wu, A Z; Cai, R; Pan, J S

    2015-01-01

    Dihydroflavonol 4-reductase (DFR) genes from Rosa chinensis (Asn type) and Calibrachoa hybrida (Asp type), driven by a CaMV 35S promoter, were integrated into the petunia (Petunia hybrida) cultivar 9702. Exogenous DFR gene expression characteristics were similar to flower-color changes, and effects on anthocyanin concentration were observed in both types of DFR gene transformants. Expression analysis showed that exogenous DFR genes were expressed in all of the tissues, but the expression levels were significantly different. However, both of them exhibited a high expression level in petals that were starting to open. The introgression of DFR genes may significantly change DFR enzyme activity. Anthocyanin ultra-performance liquid chromatography results showed that anthocyanin concentrations changed according to DFR enzyme activity. Therefore, the change in flower color was probably the result of a DFR enzyme change. Pelargonidin 3-O-glucoside was found in two different transgenic petunias, indicating that both CaDFR and RoDFR could catalyze dihydrokaempferol. Our results also suggest that transgenic petunias with DFR gene of Asp type could biosynthesize pelargonidin 3-O-glucoside. PMID:25966276

  12. Genes encoding p-coumarate 3-hydroxylase (C3H) and methods of use

    DOEpatents

    Chapple, Clinton C. S.; Franke, Rochus; Ruegger, Max O.

    2006-07-04

    The present invention is directed to a method for altering secondary metabolism in plants, specifically phenylpropanoid metabolism. The present invention is further directed to a mutant p-coumarate 3-hydroxylase gene, referred to herein as the ref8 gene, its protein product which can be used to prepare gene constructs and transgenic plants. The gene constructs and transgenic plants are further aspects of the present invention.

  13. LIGNIFICATION IN TRANSGENICS DEFICIENT IN P-COUMARATE 3-HYDROXYLASE (C3H) AND THE ASSOCIATED HYDROXYCINNAMOYL TRANSFERASE (HCT)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied in angiosperms. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), and its associated hydroxycinnamyl transferase (HCT),...

  14. Isolation of Dihydroflavonol 4-Reductase cDNA Clones from Angelonia x angustifolia and Heterologous Expression as GST Fusion Protein in Escherichia coli

    PubMed Central

    Gosch, Christian; Nagesh, Karthik Mudigere; Thill, Jana; Miosic, Silvija; Plaschil, Sylvia; Milosevic, Malvina; Olbricht, Klaus; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at −80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast. PMID:25238248

  15. High Myopia Caused by a Mutation in LEPREL1, Encoding Prolyl 3-Hydroxylase 2

    PubMed Central

    Mordechai, Shikma; Gradstein, Libe; Pasanen, Annika; Ofir, Rivka; El Amour, Khalil; Levy, Jaime; Belfair, Nadav; Lifshitz, Tova; Joshua, Sara; Narkis, Ginat; Elbedour, Khalil; Myllyharju, Johanna; Birk, Ohad S.

    2011-01-01

    Autosomal-recessive high-grade axial myopia was diagnosed in Bedouin Israeli consanguineous kindred. Some affected individuals also had variable expressivity of early-onset cataracts, peripheral vitreo-retinal degeneration, and secondary sight loss due to severe retinal detachments. Through genome-wide linkage analysis, the disease-associated gene was mapped to ∼1.7 Mb on chromosome 3q28 (the maximum LOD score was 11.5 at θ = 0 for marker D3S1314). Sequencing of the entire coding regions and intron-exon boundaries of the six genes within the defined locus identified a single mutation (c.1523G>T) in exon 10 of LEPREL1, encoding prolyl 3-hydroxylase 2 (P3H2), a 2-oxoglutarate-dependent dioxygenase that hydroxylates collagens. The mutation affects a glycine that is conserved within P3H isozymes. Analysis of wild-type and p.Gly508Val (c.1523G>T) mutant recombinant P3H2 polypeptides expressed in insect cells showed that the mutation led to complete inactivation of P3H2. PMID:21885030

  16. Overexpression of a tomato flavanone 3-hydroxylase-like protein gene improves chilling tolerance in tobacco.

    PubMed

    Meng, Chen; Zhang, Song; Deng, Yong-Sheng; Wang, Guo-Dong; Kong, Fan-Ying

    2015-11-01

    Flavonoids are secondary metabolites found in plants with a wide range of biological functions, such as stress protection. This study investigated the functions of a tomato (Solanum lycopersicum) flavanone 3-hydroxylase-like protein gene SlF3HL by using transgenic tobacco. The expression of the gene was up-regulated under chilling (4 °C), heat (42 °C), salt (NaCl) and oxidative (H2O2) stresses. The transgenic plants that displayed high SlF3HL mRNA and protein levels showed higher flavonoid content than the WT plants. Moreover, the expression of three flavonoid biosynthesis-related structural genes, namely, chalcone synthase (CHS), chalcone isomerase (CHI) and flavonol synthase (FLS) was also higher in the transgenic plants than in the WT plants. Under chilling stress, the transgenic plants showed not only faster seed germination, better survival and growth, but also lower malondialdehyde (MDA) accumulation, relative electrical conductivity (REC) and H2O2 and O2(·-) levels compared with WT plants. These results suggested that SlF3HL stimulated flavonoid biosynthesis in response to chilling stress. PMID:26372946

  17. Molecular Cloning and Characterization of Two Genes Encoding Dihydroflavonol-4-Reductase from Populus trichocarpa

    PubMed Central

    Jia, Zhichun; Yang, Li; Sun, Yimin; Xiao, Xunyan; Song, Feng; Luo, Keming

    2012-01-01

    Dihydroflavonol 4-reductase (DFR, EC 1.1.1.219) is a rate-limited enzyme in the biosynthesis of anthocyanins and condensed tannins (proanthocyanidins) that catalyzes the reduction of dihydroflavonols to leucoanthocyanins. In this study, two full-length transcripts encoding for PtrDFR1 and PtrDFR2 were isolated from Populus trichocarpa. Sequence alignment of the two PtrDFRs with other known DFRs reveals the homology of these genes. The expression profile of PtrDFRs was investigated in various tissues of P. trichocarpa. To determine their functions, two PtrDFRs were overexpressed in tobacco (Nicotiana tabacum) via Agrobacterium-mediated transformation. The associated color change in the flowers was observed in all 35S:PtrDFR1 lines, but not in 35S:PtrDFR2 lines. Compared to the wild-type control, a significantly higher accumulation of anthocyanins was detected in transgenic plants harboring the PtrDFR1. Furthermore, overexpressing PtrDFR1 in Chinese white poplar (P. tomentosa Carr.) resulted in a higher accumulation of both anthocyanins and condensed tannins, whereas constitutively expressing PtrDFR2 only improved condensed tannin accumulation, indicating the potential regulation of condensed tannins by PtrDFR2 in the biosynthetic pathway in poplars. PMID:22363429

  18. Free-Spinning-Tunnel Investigation of a 1/25-Scale Model of the McDonnell F3H-1N Airplane, TED No. NACA AD 3100

    NASA Technical Reports Server (NTRS)

    Lee, Henry A.; Wilkes, L. Faye

    1954-01-01

    An investigation was conducted in the Langley 20-foot free-spinning tunnel on a 1/23-scale model of the McDonnell F3H-1N airplane. The effects of control settings and movements upon the erect and inverted spin and recovery characteristics of the model were determined for the clean condition. Spin-recovery parachute tests were also performed. The results indicated that erect spins obtained on the airplane for the take-off or combat loadings should be satisfactorily terminated if full rudder reversal is accompanied by moving the ailerons to full with the spin (stick full right in a right spin). The spins obtained should be oscillatory in pitch, roll, and yaw. Recoveries from inverted spins should be satisfactory by full reversal of the rudder. A 16.7-foot- diameter tail parachute with a towline length of 30 feet and a drag coefficient of 0.734 should be adequate for emergency recovery from demonstration spins.

  19. Flower Colour Modification of Chrysanthemum by Suppression of F3'H and Overexpression of the Exogenous Senecio cruentus F3'5'H Gene

    PubMed Central

    Keting, Han; Qiaoyan, Xiang; Silan, Dai

    2013-01-01

    Chrysanthemum (Chrysanthemum × morifolium) is one of the most important ornamental plants in the world. They are typically used as cut flowers or potted plants. Chrysanthemum can exhibit red, purple, pink, yellow and white flowers, but lack bright red and blue flowers. In this study, we identified two chrysanthemum cultivars, C × morifolium ‘LPi’ and C × morifolium ‘LPu’, that only accumulate flavonoids in their ligulate flowers. Next, we isolated seven anthocyanin biosynthesis genes, namely CmCHS, CmF3H, CmF3’H, CmDFR, CmANS, CmCHI and Cm3GT in these cultivars. RT-PCR and qRT-PCR analyses showed that CmF3′H was the most important enzyme required for cyanidin biosynthsis. To rebuild the delphinidin pathway, we downregulated CmF3’H using RNAi and overexpressed the Senecio cruentus F3′5′H (PCFH) gene in chrysanthemum. The resultant chrysanthemum demonstrated a significantly increased content of cyanidin and brighter red flower petals but did not accumulate delphinidin. These results indicated that CmF3′H in chrysanthemum is important for anthocyanin accumulation, and Senecio cruentus F3′5′H only exhibited F3′H activity in chrysanthemum but did not rebuild the delphinidin pathway to form blue flower chrysanthemum. PMID:24250783

  20. 4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

    PubMed

    Wang, Z X; Li, S M; Löscher, R; Heide, L

    1997-11-15

    A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism. PMID:9367532

  1. Transgenic potato plants with overexpression of dihydroflavonol reductase can serve as efficient nutrition sources.

    PubMed

    Kostyn, Kamil; Szatkowski, Michal; Kulma, Anna; Kosieradzka, Iwona; Szopa, Jan

    2013-07-10

    Potato (Solanum tuberosum) is considered to be one of the most important crops cultivated in Europe and the entire world. The tubers of the potato are characterized by rich starch and protein contents and high concentrations of antioxidants, such as vitamin C and flavonoids. Notably, the presence of the phenolic antioxidants is of high importance as they have health-related properties. They are known to reduce the incidence of atherosclerosis, prevent certain kinds of cancer, and aid with many other kinds of diseases. The aim of this study was to find the most efficient way to increase the content of phenolic antioxidants in potato tubers through transgenesis. The results showed that the most efficacious way to achieve this goal was the overexpression of the dihydroflavonol reductase gene (DFR). The produced transgenic potato plants served as a nutrition source for laboratory rats; the study has confirmed their nontoxicity and nutritional benefits on the tested animals. PMID:23692339

  2. A Fruit-Specific Putative Dihydroflavonol 4-Reductase Gene Is Differentially Expressed in Strawberry during the Ripening Process1

    PubMed Central

    Moyano, Enriqueta; Portero-Robles, Ignacio; Medina-Escobar, Nieves; Valpuesta, Victoriano; Muñoz-Blanco, Juan; Luis Caballero, José

    1998-01-01

    A cDNA clone encoding a putative dihydroflavonol 4-reductase gene has been isolated from a strawberry (Fragaria × ananassa cv Chandler) DNA subtractive library. Northern analysis showed that the corresponding gene is predominantly expressed in fruit, where it is first detected during elongation (green stages) and then declines and sharply increases when the initial fruit ripening events occur, at the time of initiation of anthocyanin accumulation. The transcript can be induced in unripe green fruit by removing the achenes, and this induction can be partially inhibited by treatment of de-achened fruit with naphthylacetic acid, indicating that the expression of this gene is under hormonal control. We propose that the putative dihydroflavonol 4-reductase gene in strawberry plays a main role in the biosynthesis of anthocyanin during color development at the late stages of fruit ripening; during the first stages the expression of this gene could be related to the accumulation of condensed tannins. PMID:9625725

  3. Dihydroflavonol BB-1, an extract of natural plant Blumea balsamifera, abrogates TRAIL resistance in leukemia cells.

    PubMed

    Hasegawa, Hiroo; Yamada, Yasuaki; Komiyama, Kanki; Hayashi, Masahiko; Ishibashi, Masami; Yoshida, Tatsushi; Sakai, Toshiyuki; Koyano, Takashi; Kam, Toh-Seok; Murata, Ken; Sugahara, Kazuyuki; Tsuruda, Kazuto; Akamatsu, Norihiko; Tsukasaki, Kunihiro; Masuda, Masato; Takasu, Nobuyuki; Kamihira, Shimeru

    2006-01-15

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells and, hence, has emerged as a novel anticancer agent. Previously, we showed that although most adult T-cell leukemia/lymphoma (ATLL) cells express the TRAIL death receptor DR4 (TRAIL-R1) or DR5 (TRAIL-R2), they are resistant to TRAIL. Thus, in this study, we tried to find natural products that can overcome TRAIL resistance. Among more than 150 materials screened, a dihydroflavonol that was extracted from Blumea balsamifera (BB-1) exhibited the most striking synergism with TRAIL. Treatment of the TRAIL-resistant ATLL cell line KOB, with a combination of BB-1 and TRAIL, resulted in apparent apoptosis that was not observed on treatment with either agent alone. Furthermore, pretreatment with BB-1 followed by TRAIL further augmented the synergism. BB-1 increased the level of TRAIL-R2 promoter activity and surface protein expression in a p53-independent manner. TRAIL-R2 siRNA inhibited the synergism, indicating that sensitization was caused by the increase of TRAIL-R2 expression. More interestingly, similar effects were observed in other leukemia cell lines by exactly the same mechanisms. These results suggest that combined treatment with BB-1 and TRAIL may be a new strategy for cancer therapy. PMID:16195335

  4. Tyrosine 3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing.

    PubMed Central

    Schalling, M; Hökfelt, T; Wallace, B; Goldstein, M; Filer, D; Yamin, C; Schlesinger, D H

    1986-01-01

    Rat brain and adrenal gland were analyzed by hybridization histochemistry using an RNA probe complementary to mRNA for tyrosine 3-hydroxylase (TyrOHase; tyrosine 3-monooxygenase, EC 1.14.16.2), by immunohistochemistry using TyrOHase antiserum, and by retrograde tracing using the fluorescent compound Fast blue. Cell bodies in the ventral mesencephalon contained mRNA for TyrOHase, and these cells were also TyrOHase immunoreactive. After injection of Fast blue into the striatum, such double-labeled cells in addition contained the retrograde tracer, showing that these cells send axonal projections to the injection site. These results show that hybridization histochemistry can be used to identify transmitter-specific neuron populations and that their projections can be established. Images PMID:2874560

  5. The Balance of Expression of Dihydroflavonol 4-reductase and Flavonol Synthase Regulates Flavonoid Biosynthesis and Red Foliage Coloration in Crabapples

    PubMed Central

    Tian, Ji; Han, Zhen-yun; Zhang, Jie; Hu, YuJing; Song, Tingting; Yao, Yuncong

    2015-01-01

    Red leaf color is an attractive trait of Malus families, including crabapple (Malus spp.); however, little is known about the molecular mechanisms that regulate the coloration. Dihydroflavonols are intermediates in the production of both colored anthocyanins and colorless flavonols, and this current study focused on the gene expression balance involved in the relative accumulation of these compounds in crabapple leaves. Levels of anthocyanins and the transcript abundances of the anthocyanin biosynthetic gene, dihydroflavonol 4-reductase (McDFR) and the flavonol biosynthetic gene, flavonol synthase (McFLS), were assessed during the leaf development in two crabapple cultivars, ‘Royalty’ and ‘Flame’. The concentrations of anthocyanins and flavonols correlated with leaf color and we propose that the expression of McDFR and McFLS influences their accumulation. Further studies showed that overexpression of McDFR, or silencing of McFLS, increased anthocyanin production, resulting in red-leaf and red fruit peel phenotypes. Conversely, elevated flavonol production and green phenotypes in crabapple leaves and apple peel were observed when McFLS was overexpressed or McDFR was silenced. These results suggest that the relative activities of McDFR and McFLS are important determinants of the red color of crabapple leaves, via the regulation of the metabolic fate of substrates that these enzymes have in common. PMID:26192267

  6. A new monoterpene glucoside and complete assignments of dihydroflavonols of Pulicaria jaubertii: potential cytotoxic and blood pressure lowering activity.

    PubMed

    Ragab, Ehab A; Raafat, Mohamed

    2016-06-01

    One new monoterpene glucoside and five dihydroflavonols were isolated for the first time from the aerial parts of Pulicaria jaubertii and identified as p-menthane-2-O-β-D-glucopyranoside [1], dihydroquercetin (taxifolin) [2], 7,3'-di-O-methyltaxifolin [3], 3'-O-methyltaxifolin [4], 7-O-methyltaxifolin (padmatin) [5] and 7-O-methyl-dihydrokampferol (7-O-methylaromadenderin) [6]. The structures of these compounds were unambiguously assigned on the basis of NMR spectroscopic data ((1)H, (13)C, DEPT, HSQC, HMBC) and MS analysis. 2D-NMR methods required revision of assignments of H-6 and H-8 for dihydroflavonol compounds. Possible cytotoxic activity as well as blood pressure (BP) lowering activity were tested. The alcoholic extract showed cytotoxic activity against prostate carcinoma (PC-3), breast carcinoma (MCF-7) and hepatocellular carcinoma (HepG-2) human cell lines with IC50 19.1, 20.0 and 24.1 μg, respectively. The higher dose levels of the alcoholic extract significantly reduced normal BP of rats in a dose-dependent manner. PMID:26247309

  7. A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (-)-limonene hydroxylase from a C6- to a C3-hydroxylase.

    PubMed

    Schalk, M; Croteau, R

    2000-10-24

    The essential oils of peppermint and spearmint are distinguished by the position of oxygenation on the constituent p-menthane monoterpenes. Peppermint produces monoterpenes bearing an oxygen at C3, whereas spearmint produces monoterpenes bearing an oxygen at C6. Branching of the monoterpene biosynthetic pathways in these species is determined by two distinct cytochrome P450s that catalyze the regiospecific hydroxylation of (-)-4S-limonene at C3 or C6 exclusively. cDNAs encoding the limonene-3-hydroxylase from peppermint and the limonene-6-hydroxylase from spearmint have been isolated, shown to be 70% identical at the amino acid level, and functionally expressed. A combination of domain swapping and reciprocal site-directed mutagenesis between these two enzymes demonstrated that the exchange of a single residue (F363I) in the spearmint limonene-6-hydroxylase led to complete conversion to the regiospecificity and catalytic efficiency of the peppermint limonene-3-hydroxylase. PMID:11050228

  8. A single amino acid substitution (F363I) converts the regiochemistry of the spearmint (−)-limonene hydroxylase from a C6- to a C3-hydroxylase

    PubMed Central

    Schalk, Michel; Croteau, Rodney

    2000-01-01

    The essential oils of peppermint and spearmint are distinguished by the position of oxygenation on the constituent p-menthane monoterpenes. Peppermint produces monoterpenes bearing an oxygen at C3, whereas spearmint produces monoterpenes bearing an oxygen at C6. Branching of the monoterpene biosynthetic pathways in these species is determined by two distinct cytochrome P450s that catalyze the regiospecific hydroxylation of (−)-4S-limonene at C3 or C6 exclusively. cDNAs encoding the limonene-3-hydroxylase from peppermint and the limonene-6-hydroxylase from spearmint have been isolated, shown to be 70% identical at the amino acid level, and functionally expressed. A combination of domain swapping and reciprocal site-directed mutagenesis between these two enzymes demonstrated that the exchange of a single residue (F363I) in the spearmint limonene-6-hydroxylase led to complete conversion to the regiospecificity and catalytic efficiency of the peppermint limonene-3-hydroxylase. PMID:11050228

  9. Posttranslational Modifications in Type I Collagen from Different Tissues Extracted from Wild Type and Prolyl 3-Hydroxylase 1 Null Mice*

    PubMed Central

    Pokidysheva, Elena; Zientek, Keith D.; Ishikawa, Yoshihiro; Mizuno, Kazunori; Vranka, Janice A.; Montgomery, Nathan T.; Keene, Douglas R.; Kawaguchi, Tatsuya; Okuyama, Kenji; Bächinger, Hans Peter

    2013-01-01

    Type I collagen extracted from tendon, skin, and bone of wild type and prolyl 3-hydroxylase 1 (P3H1) null mice shows distinct patterns of 3-hydroxylation and glycosylation of hydroxylysine residues. The A1 site (Pro-986) in the α1-chain of type I collagen is almost completely 3-hydroxylated in every tissue of the wild type mice. In contrast, no 3-hydroxylation of this proline residue was found in P3H1 null mice. Partial 3-hydroxylation of the A3 site (Pro-707) was present in tendon and bone, but absent in skin in both α-chains of the wild type animals. Type I collagen extracted from bone of P3H1 null mice shows a large reduction in 3-hydroxylation of the A3 site in both α-chains, whereas type I collagen extracted from tendon of P3H1 null mice shows little difference as compared with wild type. These results demonstrate that the A1 site in type I collagen is exclusively 3-hydroxylated by P3H1, and presumably, this enzyme is required for the 3-hydroxylation of the A3 site of both α-chains in bone but not in tendon. The increase in glycosylation of hydroxylysine in P3H1 null mice in bone was found to be due to an increased occupancy of normally glycosylated sites. Despite the severe disorganization of collagen fibrils in adult tissues, the D-period of the fibrils is unchanged. Tendon fibrils of newborn P3H1 null mice are well organized with only a slight increase in diameter. The absence of 3-hydroxyproline and/or the increased glycosylation of hydroxylysine in type I collagen disturbs the lateral growth of the fibrils. PMID:23861401

  10. Purification, characterization, and directed evolution study of a vitamin D{sub 3} hydroxylase from Pseudonocardia autotrophica

    SciTech Connect

    Fujii, Yoshikazu; Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi; Takeda, Koji; Tamura, Noriko; Arisawa, Akira; Tamura, Tomohiro

    2009-07-24

    Vitamin D{sub 3} (VD{sub 3}) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD{sub 3} into its physiologically active forms, namely, 25(OH)VD{sub 3} or 1{alpha},25(OH){sub 2}VD{sub 3}. In this study, we isolated and characterized Vdh (vitamin D{sub 3} hydroxylase), which hydroxylates VD{sub 3} from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V{sub max} values for VD{sub 3} 25-hydroxylation and 25(OH)VD{sub 3} 1{alpha}-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD{sub 3}. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD{sub 3} 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD{sub 3} into 25(OH)VD{sub 3} was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD{sub 3} by a single cytochrome P450.

  11. Lift, Drag, Static Stability, and Buffet Boundaries of a Model of the McDonnell F3H-1N Airplane at Mach Numbers from 0.40 to 1.27, TED No. NACA DE 351

    NASA Technical Reports Server (NTRS)

    Crabill, Norman L.

    1956-01-01

    The National Advisory Committee for Aeronautics has conducted a flight test of a model approximating the McDonnell F3H-lN airplane configuration to determine its pitch-up and buffet boundaries, as well as the usual longitudinal stability derivatives obtainable from the pulsed- tail technique. The test was conducted by the freely flying rocket- boosted model technique developed at the Langley Laboratory; results were obtained at Mach numbers from 0.40 to 1.27 at corresponding Reynolds numbers of 2.6 x 10(exp 6) and 9.0 x 10(exp 6). The phenomena of pitch-up, buffet, and maximum lift were encountered at Mach numbers between 0.42 and 0.85. The lift-curve slope and wing-root bending-moment slope increased with increasing angle of attack, whereas the static stability decreased with angle of attack at subsonic speeds and increased at transonic speeds. There was little change in trim at low lift at transonic speeds.

  12. Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia x intermedia.

    PubMed

    Rosati, C; Cadic, A; Duron, M; Renou, J P; Simoneau, P

    1997-10-01

    The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia x intermedia cv. 'Spring Glory'. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals. PMID:9349254

  13. Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides).

    PubMed

    Peters, Darren J; Constabel, C Peter

    2002-12-01

    In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single-copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes. PMID:12472686

  14. Characterization of dihydroflavonol 4-reductase (DFR) genes and their association with cold and freezing stress in Brassica rapa.

    PubMed

    Ahmed, Nasar Uddin; Park, Jong-In; Jung, Hee-Jeong; Yang, Tae-Jin; Hur, Yoonkang; Nou, Ill-Sup

    2014-10-15

    Flavonoids including anthocyanins provide flower and leaf colors, as well as other derivatives that play diverse roles in plant development and interactions with the environment. Dihydroflavonol 4-reductase (DFR) is part of an important step in the flavonoid biosynthetic pathway of anthocyanins. This study characterized 12 DFR genes of Brassica rapa and investigated their association with anthocyanin coloration, as well as cold and freezing stress in several genotypes of B. rapa. Comparison of sequences of these genes with DFR gene sequences from other species revealed a high degree of homology. Constitutive expression of the genes in several pigmented and non-pigmented lines of B. rapa demonstrated correlation with anthocyanin accumulation for BrDFR8 and 9. Conversely, BrDFR2, 4, 8 and 9 only showed very high responses to cold stress in pigmented B. rapa samples. BrDFR1, 3, 5, 6 and 10 responded to cold and freezing stress treatments, regardless of pigmentation. BrDFRs were also shown to be regulated by two transcription factors, BrMYB2-2 and BrTT8, contrasting with anthocyanin accumulation and cold and freezing stress. Thus, the above results suggest that these genes are associated with anthocyanin biosynthesis and cold and freezing stress tolerance and might be useful resources for development of cold and/or freezing stress resistant Brassica crops with desirable colors as well. These findings may also facilitate exploration of the molecular mechanism that regulates anthocyanin biosynthesis and its response to abiotic stresses. PMID:25108127

  15. Molecular Cloning and Characterization of Three Genes Encoding Dihydroflavonol-4-Reductase from Ginkgo biloba in Anthocyanin Biosynthetic Pathway

    PubMed Central

    Hua, Cheng; Linling, Li; Shuiyuan, Cheng; Fuliang, Cao; Feng, Xu; Honghui, Yuan; Conghua, Wu

    2013-01-01

    Dihydroflavonol-4-reductase (DFR, EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Three DFR cDNA clones (designated GbDFRs) were isolated from the gymnosperm Ginkgo biloba. The deduced GbDFR proteins showed high identities to other plant DFRs, which form three distinct DFR families. Southern blot analysis showed that the three GbDFRs each belong to a different DFR family. Phylogenetic tree analysis revealed that the GbDFRs share the same ancestor as other DFRs. The expression of the three recombinant GbDFRs in Escherichia coli showed that their actual protein sizes were in agreement with predictions from the cDNA sequences. The recombinant proteins were purified and their activity was analyzed; both GbDFR1 and GbDFR3 could catalyze dihydroquercetin conversion to leucocyanidin, while GbDFR2 catalyzed dihydrokaempferol conversion to leucopelargonidin. qRT-PCR showed that the GbDFRs were expressed in a tissue-specific manner, and transcript accumulation for the three genes was highest in young leaves and stamens. These transcription patterns were in good agreement with the pattern of anthocyanin accumulation in G.biloba. The expression profiles suggested that GbDFR1 and GbDFR2 are mainly involved in responses to plant hormones, environmental stress and damage. During the annual growth cycle, the GbDFRs were significantly correlated with anthocyanin accumulation in leaves. A fitted linear curve showed the best model for relating GbDFR2 and GbDFR3 with anthocyanin accumulation in leaves. GbDFR1 appears to be involved in environmental stress response, while GbDFR3 likely has primary functions in the synthesis of anthocyanins. These data revealed unexpected properties and differences in three DFR proteins from a single species. PMID:23991027

  16. Molecular and Biochemical Analysis of Two cDNA Clones Encoding Dihydroflavonol-4-Reductase from Medicago truncatula1

    PubMed Central

    Xie, De-Yu; Jackson, Lisa A.; Cooper, John D.; Ferreira, Daneel; Paiva, Nancy L.

    2004-01-01

    Dihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids important to plant survival and human nutrition. Two DFR cDNA clones (MtDFR1 and MtDFR2) were isolated from the model legume Medicago truncatula cv Jemalong. Both clones were functionally expressed in Escherichia coli, confirming that both encode active DFR proteins that readily reduce taxifolin (dihydroquercetin) to leucocyanidin. M. truncatula leaf anthocyanins were shown to be cyanidin-glucoside derivatives, and the seed coat proanthocyanidins are known catechin and epicatechin derivatives, all biosynthesized from leucocyanidin. Despite high amino acid similarity (79% identical), the recombinant DFR proteins exhibited differing pH and temperature profiles and differing relative substrate preferences. Although no pelargonidin derivatives were identified in M. truncatula, MtDFR1 readily reduced dihydrokaempferol, consistent with the presence of an asparagine residue at a location known to determine substrate specificity in other DFRs, whereas MtDFR2 contained an aspartate residue at the same site and was only marginally active on dihydrokaempferol. Both recombinant DFR proteins very efficiently reduced 5-deoxydihydroflavonol substrates fustin and dihydrorobinetin, substances not previously reported as constituents of M. truncatula. Transcript accumulation for both genes was highest in young seeds and flowers, consistent with accumulation of condensed tannins and leucoanthocyanidins in these tissues. MtDFR1 transcript levels in developing leaves closely paralleled leaf anthocyanin accumulation. Overexpression of MtDFR1 in transgenic tobacco (Nicotiana tabacum) resulted in visible increases in anthocyanin accumulation in flowers, whereas MtDFR2 did not. The data reveal unexpected properties and differences in two DFR proteins from a single species. PMID:14976232

  17. Disequilibrium of Flavonol Synthase and Dihydroflavonol-4-Reductase Expression Associated Tightly to White vs. Red Color Flower Formation in Plants

    PubMed Central

    Luo, Ping; Ning, Guogui; Wang, Zhen; Shen, Yuxiao; Jin, Huanan; Li, Penghui; Huang, Shasha; Zhao, Jian; Bao, Manzhu

    2016-01-01

    Flower color is the main character throughout the plant kingdom. Though substantial information exists regarding the structural and regulatory genes involved in anthocyanin and flavonol biosynthesis, little is known that what make a diverse white vs. red color flower in natural species. Here, the contents of pigments in seven species from varied phylogenetic location in plants with red and white flowers were determined. Flavonols could be detected in red and white flowers, but anthocyanins were almost undetectable in the white cultivar. Comparisons of expression patterns of gene related to the flavonoid biosynthesis indicated that disequilibrium expression of flavonol synthase (FLS) and dihydroflavonol-4-reductase (DFR) genes determined the accumulation of flavonols and anothcyanins in both red and white flowers of seven species. To further investigate the role of such common regulatory patterns in determining flower color, FLS genes were isolated from Rosa rugosa (RrFLS1), Prunus persica (PpFLS), and Petunia hybrida (PhFLS), and DFR genes were isolated from Rosa rugosa (RrDFR1) and Petunia hybrida (PhDFR). Heterologous expression of the FLS genes within tobacco host plants demonstrated conservation of function, with the transgenes promoting flavonol biosynthesis and inhibiting anthocyanin accumulation, so resulting in white flowers. Conversely, overexpression of DFR genes in tobacco displayed down-regulation of the endogenous NtFLS gene, and the promotion of anthocyanin synthesis. On this basis, we propose a model in which FLS and DFR gene-products compete for common substrates in order to direct the biosynthesis of flavonols and anthocyanins, respectively, thereby determining white vs. red coloration of flowers. PMID:26793227

  18. Post-translationally Abnormal Collagens of Prolyl 3-Hydroxylase-2 Null Mice Offer a Pathobiological Mechanism for the High Myopia Linked to Human LEPREL1 Mutations*

    PubMed Central

    Hudson, David M.; Joeng, Kyu Sang; Werther, Rachel; Rajagopal, Abbhirami; Weis, MaryAnn; Lee, Brendan H.; Eyre, David R.

    2015-01-01

    Myopia, the leading cause of visual impairment worldwide, results from an increase in the axial length of the eyeball. Mutations in LEPREL1, the gene encoding prolyl 3-hydroxylase-2 (P3H2), have recently been identified in individuals with recessively inherited nonsyndromic severe myopia. P3H2 is a member of a family of genes that includes three isoenzymes of prolyl 3-hydroxylase (P3H), P3H1, P3H2, and P3H3. Fundamentally, it is understood that P3H1 is responsible for converting proline to 3-hydroxyproline. This limited additional knowledge also suggests that each isoenzyme has evolved different collagen sequence-preferred substrate specificities. In this study, differences in prolyl 3-hydroxylation were screened in eye tissues from P3h2-null (P3h2n/n) and wild-type mice to seek tissue-specific effects due the lack of P3H2 activity on post-translational collagen chemistry that could explain myopia. The mice were viable and had no gross musculoskeletal phenotypes. Tissues from sclera and cornea (type I collagen) and lens capsule (type IV collagen) were dissected from mouse eyes, and multiple sites of prolyl 3-hydroxylation were identified by mass spectrometry. The level of prolyl 3-hydroxylation at multiple substrate sites from type I collagen chains was high in sclera, similar to tendon. Almost every known site of prolyl 3-hydroxylation in types I and IV collagen from P3h2n/n mouse eye tissues was significantly under-hydroxylated compared with their wild-type littermates. We conclude that altered collagen prolyl 3-hydroxylation is caused by loss of P3H2. We hypothesize that this leads to structural abnormalities in multiple eye tissues, but particularly sclera, causing progressive myopia. PMID:25645914

  19. Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylates free L-proline to cis-3-hydroxy-L-proline.

    PubMed Central

    Mori, H; Shibasaki, T; Yano, K; Ozaki, A

    1997-01-01

    Proline 3-hydroxylase was purified from Streptomyces sp. strain TH1, and its structural gene was cloned. The purified enzyme hydroxylated free L-proline to cis-3-hydroxy-L-proline and showed properties of a 2-oxoglutarate-dependent dioxygenase (H. Mori, T. Shibasaki, Y. Uosaki, K. Ochiai, and A. Ozaki, Appl. Environ. Microbiol, 62:1903-1907, 1996). The molecular mass of the purified enzyme was 35 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 4.3. The optimal pH and temperature were 7.0 and 35 degrees C, respectively. The K(m) values were 0.56 and 0.11 mM for L-proline and 2-oxoglutarate, respectively. The Kcat value of hydroxylation was 3.2 s-1. Determined N-terminal and internal amino acid sequences of the purified protein were not found in the SwissProt protein database. A DNA fragment of 74 bp was amplified by PCR with degenerate primers based on the determined N-terminal amino acid sequence. With this fragment as a template, a digoxigenin-labeled N-terminal probe was synthesized by PCR. A 6.5-kbp chromosome fragment was cloned by colony hybridization with the labeled probe. The determined DNA sequence of the cloned fragment revealed a 870-bp open reading frame (ORF 3), encoding a protein of 290 amino acids with a calculated molecular weight of 33,158. No sequence homolog was found in EMBL, GenBank, and DDBJ databases. ORF 3 was expressed in Escherichia coli DH1. Recombinants showed hydroxylating activity five times higher than that of the original bacterium, Streptomyces sp. strain TH1. It was concluded that the ORF 3 encodes functional proline 3-hydroxylase. PMID:9294421

  20. Dihydroflavonol 4-Reductase Genes Encode Enzymes with Contrasting Substrate Specificity and Show Divergent Gene Expression Profiles in Fragaria Species

    PubMed Central

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3’H activity late in fruit development of F.×ananassa. PMID:25393679

  1. Co-downregulation of the hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase and coumarate 3-hydroxylase significantly increases cellulose content in transgenic alfalfa (Medicago sativa L.).

    PubMed

    Tong, Zongyong; Li, Heng; Zhang, Rongxue; Ma, Lei; Dong, Jiangli; Wang, Tao

    2015-10-01

    Lignin is a component of the cell wall that is essential for growth, development, structure and pathogen resistance in plants, but high lignin is an obstacle to the conversion of cellulose to ethanol for biofuel. Genetically modifying lignin and cellulose contents can be a good approach to overcoming that obstacle. Alfalfa (Medicago sativa L.) is rich in lignocellulose biomass and used as a model plant for the genetic modification of lignin in this study. Two key enzymes in the lignin biosynthesis pathway-hydroxycinnamoyl -CoA:shikimate hydroxycinnamoyl transferase (HCT) and coumarate 3-hydroxylase (C3H)-were co-downregulated. Compared to wild-type plants, the lignin content in the modified strain was reduced by 38%, cellulose was increased by 86.1%, enzyme saccharification efficiency was increased by 10.9%, and cell wall digestibility was increased by 13.0%. The modified alfalfa exhibited a dwarf phenotype, but normal above ground biomass. This approach provides a new strategy for reducing lignin and increasing cellulose contents and creates a new genetically modified crop with enhanced value for biofuel. PMID:26398807

  2. Cloning and characterization of Lonicera japonica p-coumaroyl ester 3-hydroxylase which is involved in the biosynthesis of chlorogenic acid.

    PubMed

    Pu, Gaobin; Wang, Peng; Zhou, Bingqian; Liu, Zhenhua; Xiang, Fengning

    2013-01-01

    Lonicera japonica is used in Chinese medicine as a source of antioxidants, primarily flavonoids, and a phenolic acid chlorogenic acid (CGA). Here we report the isolation and characterization of the full-length cDNA of LjC3H, a gene encoding p-coumaroyl ester 3-hydroxylase, an enzyme involved in CGA synthesis. Phylogenetic analysis indicated that is protein belongs to the CYP98A subfamily, and homology modeling revealed that its structure resembles that of other cytochrome P450 family proteins. Southern blot analysis indicated that more than one copy of sequences homologous to LjC3H is present in the L. japonica genome. Heterologous expression of LjC3H cDNA in Escherichia coli allowed an in vitro assay of LjC3H to be performed. This experiment revealed that the enzyme favors p-coumaroylshikimate over p-coumaroylquinate as substrate. LjC3H transcript abundance was increased both by treatment of the leaves with methyl jasmonate and by exposure to UV-B radiation. The CGA levels in the leaves of L. japonica were positively correlated with LjC3H transcript abundance. PMID:23832359

  3. Functional Characterization of Dihydroflavonol-4-Reductase in Anthocyanin Biosynthesis of Purple Sweet Potato Underlies the Direct Evidence of Anthocyanins Function against Abiotic Stresses

    PubMed Central

    Wang, Hongxia; Fan, Weijuan; Li, Hong; Yang, Jun; Huang, Jirong; Zhang, Peng

    2013-01-01

    Dihydroflavonol-4-reductase (DFR) is a key enzyme in the catalysis of the stereospecific reduction of dihydroflavonols to leucoanthocyanidins in anthocyanin biosynthesis. In the purple sweet potato (Ipomoea batatas Lam.) cv. Ayamurasaki, expression of the IbDFR gene was strongly associated with anthocyanin accumulation in leaves, stems and roots. Overexpression of the IbDFR in Arabidopsis tt3 mutants fully complemented the pigmentation phenotype of the seed coat, cotyledon and hypocotyl. Downregulation of IbDFR expression in transgenic sweet potato (DFRi) using an RNAi approach dramatically reduced anthocyanin accumulation in young leaves, stems and storage roots. In contrast, the increase of flavonols quercetin-3-O-hexose-hexoside and quercetin-3-O-glucoside in the leaves and roots of DFRi plants is significant. Therefore, the metabolic pathway channeled greater flavonol influx in the DFRi plants when their anthocyanin and proanthocyanidin accumulation were decreased. These plants also displayed reduced antioxidant capacity compared to the wild type. After 24 h of cold treatment and 2 h recovery, the wild-type plants were almost fully restored to the initial phenotype compared to the slower recovery of DFRi plants, in which the levels of electrolyte leakage and hydrogen peroxide accumulation were dramatically increased. These results provide direct evidence of anthocyanins function in the protection against oxidative stress in the sweet potato. The molecular characterization of the IbDFR gene in the sweet potato not only confirms its important roles in flavonoid metabolism but also supports the protective function of anthocyanins of enhanced scavenging of reactive oxygen radicals in plants under stressful conditions. PMID:24223813

  4. Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin

    PubMed Central

    Castellarin, Simone D; Di Gaspero, Gabriele; Marconi, Raffaella; Nonis, Alberto; Peterlunger, Enrico; Paillard, Sophie; Adam-Blondon, Anne-Francoise; Testolin, Raffaele

    2006-01-01

    Background Structural genes of the phenyl-propanoid pathway which encode flavonoid 3'- and 3',5'-hydroxylases (F3'H and F3'5'H) have long been invoked to explain the biosynthesis of cyanidin- and delphinidin-based anthocyanin pigments in the so-called red cultivars of grapevine. The relative proportion of the two types of anthocyanins is largely under genetic control and determines the colour variation among red/purple/blue berry grape varieties and their corresponding wines. Results Gene fragments of VvF3'H and VvF3'5'H, that were isolated from Vitis vinifera 'Cabernet Sauvignon' using degenerate primers designed on plant homologous genes, translated into 313 and 239 amino acid protein fragments, respectively, with up to 76% and 82% identity to plant CYP75 cytochrome P450 monooxygenases. Putative function was assigned on the basis of sequence homology, expression profiling and its correlation with metabolite accumulation at ten different ripening stages. At the onset of colour transition, transcriptional induction of VvF3'H and VvF3'5'H was temporally coordinated with the beginning of anthocyanin biosynthesis, the expression being 2-fold and 50-fold higher, respectively, in red berries versus green berries. The peak of VvF3'5'H expression was observed two weeks later concomitantly with the increase of the ratio of delphinidin-/cyanidin-derivatives. The analysis of structural genomics revealed that two copies of VvF3'H are physically linked on linkage group no. 17 and several copies of VvF3'5'H are tightly clustered and embedded into a segmental duplication on linkage group no. 6, unveiling a high complexity when compared to other plant flavonoid hydroxylase genes known so far, mostly in ornamentals. Conclusion We have shown that genes encoding flavonoid 3'- and 3',5'-hydroxylases are expressed in any tissues of the grape plant that accumulate flavonoids and, particularly, in skin of ripening red berries that synthesise mostly anthocyanins. The correlation between

  5. Salicylic acid-induced changes in physiological parameters and genes of the flavonoid biosynthesis pathway in Artemisia vulgaris and Dendranthema nankingense during aphid feeding.

    PubMed

    Sun, Y; Xia, X L; Jiang, J F; Chen, S M; Chen, F D; Lv, G S

    2016-01-01

    Phloem-feeding aphids cause serious damage to plants. The mechanisms of plant-aphid interactions are only partially understood and involve multiple pathways, including phytohormones. In order to investigate whether salicylic acid (SA) is involved and how it plays a part in the defense response to the aphid Macrosiphoniella sanbourni, physiological changes and gene expression profiles in response to aphid inoculation with or without SA pretreatment were compared between the aphid-resistant Artemisia vulgaris 'Variegata' and the susceptible chrysanthemum, Dendranthema nankingense. Changes in levels of reactive oxygen species, malondialdehyde (MDA), and flavonoids, and in the expression of genes involved in flavonoid biosynthesis, including PAL (phenylalanine ammonia-lyase), CHS (chalcone synthase), CHI (chalcone isomerase), F3H (flavanone 3-hydroxylase), F3'H (flavanone 3'-hydroxylase), and DFR (dihydroflavonol reductase), were investigated. Levels of hydrogen peroxide, superoxide anions, MDA, and flavonoids, and their related gene expression, increased after aphid infestation and SA pretreatment followed by aphid infestation; the aphid-resistant A. vulgaris exhibited a more rapid response than the aphid-susceptible D. nankingense to SA treatment and aphid infestation. Taken together, our results suggest that SA could be used to increase aphid resistance in the chrysanthemum. PMID:26909993

  6. Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

    DOE PAGESBeta

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; Rottmann, William H.; Kuhn, Sean A.; Foster, Cliff E.; Ziebell, Angela; Turner, Geoffrey B.; Decker, Stephen R.; Hinchee, Maud A. W.; et al

    2015-08-27

    In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

  7. Relationship between gene expression and the accumulation of catechin during spring and autumn in tea plants (Camellia sinensis L.)

    PubMed Central

    Liu, Min; Tian, Heng-lu; Wu, Jian-Hua; Cang, Ren-Rong; Wang, Run-Xian; Qi, Xiao-Hua; Xu, Qiang; Chen, Xue-Hao

    2015-01-01

    The tea plant (Camellia sinensis L.) is an important commercial crop with remarkably high catechin concentrations. Tea is popular worldwide given the plant's health benefits. Catechins are the main astringent substance in tea and are synthesized mainly via the phenylpropanoid pathway. In this study, eight cultivars of tea plants harvested both in spring and autumn were used to investigate differences in catechin concentrations by using high-performance liquid chromatography. The expression levels of genes associated with catechin biosynthesis were investigated using reverse transcription-quantitative polymerase chain reaction. The results indicated that the total catechin (TC) concentrations were significantly higher in tea plants harvested in autumn than in those harvested in spring, based on higher concentrations of epigallocatechin (EGC) in autumn tea (P<0.01). The expression of the genes phenylalanine ammonia-lyase (PAL), flavanone 3-hydroxylase (F3H), flavonoid 3′,5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS) is closely related to the TC content of tea plants in both spring and autumn. Positive correlations between PAL, cinnamate 4-hydroxylase (C4H), F3H, and DFR expression and EGC accumulation in autumn tea were identified, with correlation coefficients of 0.710, 0.763, 0.884, and 0.707, respectively. A negative correlation between ANS expression level and EGC concentrations in tea plants harvested in spring was noted (r=−0.732). Additionally, negative correlations between F3H and ANS expression levels and the catechin content were identified in spring tea, whereas the correlations were positive in autumn tea. Significant differences in the F3H and ANS expression levels between spring and autumn tea indicate that F3H and ANS are potentially key genes affecting catechin accumulation in tea plants. PMID:26504566

  8. Zvyaginite, NaZnNb2Ti[Si2O7]2O(OH,F)3(H2O)4 + x ( x < 1), a new mineral of the epistolite group from the Lovozero Alkaline Pluton, Kola Peninsula, Russia

    NASA Astrophysics Data System (ADS)

    Pekov, I. V.; Lykova, I. S.; Chukanov, N. V.; Yapaskurt, V. O.; Belakovskiy, D. I.; Zolotarev, A. A.; Zubkova, N. V.

    2014-12-01

    A new mineral, zvyaginite, a member of the epistolite group, has been found at Mt. Malyi Punkaruaiv, Lovozero Alkaline Complex, Kola Peninsula, Russia. It occurs in a hydrothermally altered peralkaline pegmatite and is associated with ussingite, microcline, aegirine, sphalerite, vigrishinite, and sauconite. Zvyaginite forms rectangular or irregular-shaped lamellae up to 0.1 × 1 × 2 cm in size when flattened [001]. The mineral is translucent to transparent and colorless, pearly-white, yellowish brownish, pale pink, or violet pink. The luster is nacreous on crystal faces and greasy on broken surfaces. Its Mohs' hardness is 2.5-3. Zvyaginite is brittle. The cleavage parallel to {001} is perfect. D meas = 2.88(3), D calc = 2.94 g/cm3. The mineral is optically biaxial (-), α = 1.626(5), β = 1.714(3), γ = 1.740(5), 2 V meas = 45(15)°, 2 V calc = 55°. The IR spectrum is given. Chemical composition is as follows (wt %; average of five point analyses; H2O was determined using the modified Penfield method): 4.74 Na2O, 0.22 K2O, 0.77 CaO, 1.36 MnO, 0.24 FeO, 9.61 ZnO, 0.19 Al2O3, 29.42 SiO2, 12.33 TiO2, 27.22 Nb2O5, 1.94 F, 12.65 H2O, -0.82 -O = F2, for a total of 99.87. The empirical formula calculated on the basis of Si + Al = 4 is: Na1.24K0.04Ca0.11Mn0.16Fe0.03Zn0.96Nb1.66Ti1.25(Si3.97Al0.03)Σ4O15.07(OH)2.10F0.83(H2O)4.64. The simplified formula is: NaZnNb2Ti[Si2O7]2O(OH,F)3(H2O)4 + x ( x < 1), Zvyaginite is triclinic, , a = 8.975(3), b = 8.979(3), c = 12.135(4) Å, α = 74.328(9)°, β = 80.651(8)°, γ = 73.959(8)°, V = 900.8(6) Å3, Z = 2. The strongest reflections in the X-ray powder pattern ( d, Å- I[ hkl]) are: 11.72-100[001], 5.83-40[002], 5.28-53[-1-11, 112], 4.289-86[200, 021], 3.896-36[-1-12, -201, 003, 022, 113], 2.916-57[310, 132, 004], 2.862-72[130, 312]. The model of the crystal structure was obtained on a single crystal, R = 0.159. Zvyaginite and epistolite are similar in the structure of the NbTiSiO motif, but differ from each other in composition

  9. Development of SNP markers for genes of the phenylpropanoid pathway and their association to kernel and malting traits in barley

    PubMed Central

    2013-01-01

    Background Flavonoids are an important class of secondary compounds in angiosperms. Next to certain biological functions in plants, they play a role in the brewing process and have an effect on taste, color and aroma of beer. The aim of this study was to reveal the haplotype diversity of candidate genes involved in the phenylpropanoid biosynthesis pathway in cultivated barley varieties (Hordeum vulgare L.) and to determine associations to kernel and malting quality parameters. Results Five genes encoding phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthase (CHS), flavanone 3-hydroxylase (F3H) and dihydroflavonol reductase (DFR) of the phenylpropanoid biosynthesis pathway were partially resequenced in 16 diverse barley reference genotypes. Their localization in the barley genome, their genetic structure, and their genetic variation e.g. single nucleotide polymorphism (SNP) and Insertion/Deletion (InDel) patterns were revealed. In total, 130 SNPs and seven InDels were detected. Of these, 21 polymorphisms were converted into high-throughput pyrosequencing markers. The resulting SNP and haplotype patterns were used to calculate associations with kernel and malting quality parameters. Conclusions SNP patterns were found to be highly variable for the investigated genes. The developed high-throughput markers are applicable for assessing the genetic variability and for the determination of haplotype patterns in a set of barley accessions. The candidate genes PAL, C4H and F3H were shown to be associated to several malting properties like glassiness (PAL), viscosity (C4H) or to final attenuation (F3H). PMID:24088365

  10. Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis leads to improved sugar release

    SciTech Connect

    Sykes, Robert W.; Gjersing, Erica L.; Foutz, Kirk; Rottmann, William H.; Kuhn, Sean A.; Foster, Cliff E.; Ziebell, Angela; Turner, Geoffrey B.; Decker, Stephen R.; Hinchee, Maud A. W.; Davis, Mark F.

    2015-08-27

    In this study, lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance.

  11. Production of red-flowered plants by genetic engineering of multiple flavonoid biosynthetic genes.

    PubMed

    Nakatsuka, Takashi; Abe, Yoshiko; Kakizaki, Yuko; Yamamura, Saburo; Nishihara, Masahiro

    2007-11-01

    Orange- to red-colored flowers are difficult to produce by conventional breeding techniques in some floricultural plants. This is due to the deficiency in the formation of pelargonidin, which confers orange to red colors, in their flowers. Previous researchers have reported that brick-red colored flowers can be produced by introducing a foreign dihydroflavonol 4-reductase (DFR) with different substrate specificity in Petunia hybrida, which does not accumulate pelargonidin pigments naturally. However, because these experiments used dihydrokaempferol (DHK)-accumulated mutants as transformation hosts, this strategy cannot be applied directly to other floricultural plants. Thus in this study, we attempted to produce red-flowered plants by suppressing two endogenous genes and expressing one foreign gene using tobacco as a model plant. We used a chimeric RNAi construct for suppression of two genes (flavonol synthase [FLS] and flavonoid 3'-hydroxylase [F3'H]) and expression of the gerbera DFR gene in order to accumulate pelargonidin pigments in tobacco flowers. We successfully produced red-flowered tobacco plants containing high amounts of additional pelargonidin as confirmed by HPLC analysis. The flavonol content was reduced in the transgenic plants as expected, although complete inhibition was not achieved. Expression analysis also showed that reduction of the two-targeted genes and expression of the foreign gene occurred simultaneously. These results demonstrate that flower color modification can be achieved by multiple gene regulation without use of mutants if the vector constructs are designed resourcefully. PMID:17639403

  12. A De novo Transcriptomic Approach to Identify Flavonoids and Anthocyanins "Switch-Off" in Olive (Olea europaea L.) Drupes at Different Stages of Maturation.

    PubMed

    Iaria, Domenico L; Chiappetta, Adriana; Muzzalupo, Innocenzo

    2015-01-01

    Highlights A de novo transcriptome reconstruction of olive drupes was performed in two genotypesGene expression was monitored during drupe development in two olive cultivarsTranscripts involved in flavonoid and anthocyanin pathways were analyzed in Cassanese and Leucocarpa cultivarsBoth cultivar and developmental stage impact gene expression in Olea europaea fruits. During ripening, the fruits of the olive tree (Olea europaea L.) undergo a progressive chromatic change characterized by the formation of a red-brown "spot" which gradually extends on the epidermis and in the innermost part of the mesocarp. This event finds an exception in the Leucocarpa cultivar, in which we observe a destabilized equilibrium between the metabolisms of chlorophyll and other pigments, particularly the anthocyanins whose switch-off during maturation promotes the white coloration of fruits. Despite its importance, genomic information on the olive tree is still lacking. Different RNA-seq libraries were generated from drupes of "Leucocarpa" and "Cassanese" olive genotypes, sampled at 100 and 130 days after flowering (DAF), and were used in order to identify transcripts involved in the main phenotypic changes of fruits during maturation and their corresponding expression patterns. A total of 103,359 transcripts were obtained and 3792 and 3064 were differentially expressed in "Leucocarpa" and "Cassanese" genotypes, respectively, during 100-130 DAF transition. Among them flavonoid and anthocyanin related transcripts such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol 3'-hydrogenase (F3'H), flavonol 3'5 '-hydrogenase (F3'5'H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose:anthocianidin: flavonoid glucosyltransferase (UFGT) were identified. These results contribute to reducing the current gap in

  13. Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa).

    PubMed

    Zhou, Bo; Li, Yuhua; Xu, Zhiru; Yan, Haifang; Homma, Shinichiro; Kawabata, Saneyuki

    2007-01-01

    Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors. PMID:17426056

  14. Red Anthocyanins and Yellow Carotenoids Form the Color of Orange-Flower Gentian (Gentiana lutea L. var. aurantiaca).

    PubMed

    Berman, Judit; Sheng, Yanmin; Gómez Gómez, Lourdes; Veiga, Tania; Ni, Xiuzhen; Farré, Gemma; Capell, Teresa; Guitián, Javier; Guitián, Pablo; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

    2016-01-01

    Flower color is an important characteristic that determines the commercial value of ornamental plants. Gentian flowers occur in a limited range of colors because this species is not widely cultivated as a cut flower. Gentiana lutea L. var. aurantiaca (abbr, aurantiaca) is characterized by its orange flowers, but the specific pigments responsible for this coloration are unknown. We therefore investigated the carotenoid and flavonoid composition of petals during flower development in the orange-flowered gentian variety of aurantiaca and the yellow-flowered variety of G. lutea L. var. lutea (abbr, lutea). We observed minor varietal differences in the concentration of carotenoids at the early and final stages, but only aurantiaca petals accumulated pelargonidin glycosides, whereas these compounds were not found in lutea petals. We cloned and sequenced the anthocyanin biosynthetic gene fragments from petals, and analyzed the expression of these genes in the petals of both varieties to determine the molecular mechanisms responsible for the differences in petal color. Comparisons of deduced amino acid sequences encoded by the isolated anthocyanin cDNA fragments indicated that chalcone synthase (CHS), chalcone isomerase (CHI), anthocyanidin synthase 1 (ANS1) and ANS2 are identical in both aurantiaca and lutea varieties whereas minor amino acid differences of the deduced flavonone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR) between both varieties were observed. The aurantiaca petals expressed substantially higher levels of transcripts representing CHS, F3H, DFR, ANS and UDP-glucose:flavonoid-3-O-glucosyltransferase genes, compared to lutea petals. Pelargonidin glycoside synthesis in aurantiaca petals therefore appears to reflect the higher steady-state levels of pelargonidin synthesis transcripts. Moreover, possible changes in the substrate specificity of DFR enzymes may represent additional mechanisms for producing red pelargonidin glycosides in petals of

  15. Identification of flavonoids and expression of flavonoid biosynthetic genes in two coloured tree peony flowers.

    PubMed

    Zhao, Daqiu; Tang, Wenhui; Hao, Zhaojun; Tao, Jun

    2015-04-10

    Tree peony (Paeonia suffruticosa Andr.) has been named the "king of flowers" because of its elegant and gorgeous flower colour. Among these colours, the molecular mechanisms of white formation and how white turned to red in P. suffruticosa is little known. In this study, flower colour variables, flavonoid accumulation and expression of flavonoid biosynthetic genes of white ('Xueta') and red ('Caihui') P. suffruticosa were investigated. The results showed that the flower colours of both cultivars were gradually deepened with the development of flowers. Moreover, two anthoxanthin compositions apigenin 7-O-glucoside together with apigenin deoxyheso-hexoside were identified in 'Xueta' and 'Caihui', but one main anthocyanin composition peonidin 3,5-di-O-glucoside (Pn3G5G) was only found in 'Caihui'. Total contents of anthocyanins in 'Caihui' was increased during flower development, and the same trend was presented in anthoxanthins and flavonoids of these two cultivars, but the contents of these two category flavonoid in 'Caihui' were always higher than those in 'Xueta'. Furthermore, nine structural genes in flavonoid biosynthetic pathway were isolated including the full-length cDNAs of phenylalanine ammonialyase gene (PAL), chalcone synthase gene (CHS) and chalcone isomerase gene (CHI), together with the partial-length cDNAs of flavanone 3-hydroxylase gene (F3H), flavonoid 3'-hydroxylase gene (F3'H), dihydroflavonol 4-reductase gene (DFR), anthocyanidin synthase gene (ANS), UDP-glucose: flavonoid 3-O-glucosyltransferase gene (UF3GT) and UDP-glucose: flavonoid 5-O-glucosyltransferase gene (UF5GT), and PAL, UF3GT and UF5GT were reported in P. suffruticosa for the first time. Their expression patterns showed that transcription levels of downstream genes in 'Caihui' were basically higher than those in 'Xueta', especially PsDFR and PsANS, suggesting that these two genes may play a key role in the anthocyanin biosynthesis which resulted in the shift from white to red in

  16. A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis)

    PubMed Central

    Ohno, Sho; Hosokawa, Munetaka; Hoshino, Atsushi; Kitamura, Yoshikuni; Morita, Yasumasa; Park, Kyeung-II; Nakashima, Akiko; Deguchi, Ayumi; Tatsuzawa, Fumi; Doi, Motoaki; Iida, Shigeru; Yazawa, Susumu

    2011-01-01

    Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in ‘Michael J’ (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription–PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix–loop–helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed. PMID:21765172

  17. Effect of calcium on strawberry fruit flavonoid pathway gene expression and anthocyanin accumulation.

    PubMed

    Xu, Wenping; Peng, Hui; Yang, Tianbao; Whitaker, Bruce; Huang, Luhong; Sun, Jianghao; Chen, Pei

    2014-09-01

    Two diploid woodland strawberry (Fragaria vesca) inbred lines, Ruegen F7-4 (red fruit-bearing) and YW5AF7 (yellow fruit-bearing) were used to study the regulation of anthocyanin biosynthesis in fruit. Ruegen F7-4 fruit had similar total phenolics and anthocyanin contents to commercial octoploid (F. × ananassa) cultivar Seascape, while YW5AF7 exhibited relatively low total phenolics content and no anthocyanin accumulation. Foliar spray of CaCl2 boosted fruit total phenolics content, especially anthocyanins, by more than 20% in both Seascape and RF7-4. Expression levels of almost all the flavonoid pathway genes were comparable in Ruegen F7-4 and YW5AF7 green-stage fruit. However, at the turning and ripe stages, key anthocyanin structural genes, including flavanone 3-hydroxylase (F3H1), dihydroflavonol 4-reductase (DFR2), anthocyanidin synthase (ANS1), and UDP-glucosyltransferase (UGT1), were highly expressed in Ruegen F7-4 compared with YW5AF7 fruit. Calcium treatment further stimulated the expression of those genes in Ruegen F7-4 fruit. Anthocyanins isolated from petioles of YW5AF7 and Ruegen F-7 had the same HPLC-DAD profile, which differed from that of Ruegen F-7 fruit anthocyanins. All the anthocyanin structural genes except FvUGT1 were detected in petioles of YW5AF7 and Ruegen F-7. Taken together, these results indicate that the "yellow" gene in YW5AF7 is a fruit specific regulatory gene(s) for anthocyanin biosynthesis. Calcium can enhance accumulation of anthocyanins and total phenolics in fruit possibly via upregulation of anthocyanin structural genes. Our results also suggest that the anthocyanin biosynthesis machinery in petioles is different from that in fruit. PMID:25036468

  18. Differential Expression of Anthocyanin Biosynthetic Genes in Relation to Anthocyanin Accumulation in the Pericarp of Litchi Chinensis Sonn

    PubMed Central

    Li, Xiao-Jing; Huang, Xu-Ming; Wang, Hui-Cong

    2011-01-01

    Litchi has diverse fruit color phenotypes, yet no research reflects the biochemical background of this diversity. In this study, we evaluated 12 litchi cultivars for chromatic parameters and pigments, and investigated the effects of abscisic acid, forchlorofenron (CPPU), bagging and debagging treatments on fruit coloration in cv. Feizixiao, an unevenly red cultivar. Six genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) were isolated from the pericarp of the fully red litchi cv. Nuomici, and their expression was analyzed in different cultivars and under the above mentioned treatments. Pericarp anthocyanin concentration varied from none to 734 mg m−2 among the 12 litchi cultivars, which were divided into three coloration types, i.e. non-red (‘Kuixingqingpitian’, ‘Xingqiumili’, ‘Yamulong’and ‘Yongxing No. 2′), unevenly red (‘Feizixiao’ and ‘Sanyuehong’) and fully red (‘Meiguili’, ‘Baila’, Baitangying’ ’Guiwei’, ‘Nuomici’ and ‘Guinuo’). The fully red type cultivars had different levels of anthocyanin but with the same composition. The expression of the six genes, especially LcF3H, LcDFR, LcANS and LcUFGT, in the pericarp of non-red cultivars was much weaker as compared to those red cultivars. Their expression, LcDFR and LcUFGT in particular, was positively correlated with anthocyanin concentrations in the pericarp. These results suggest the late genes in the anthocyanin biosynthetic pathway were coordinately expressed during red coloration of litchi fruits. Low expression of these genes resulted in absence or extremely low anthocyanin accumulation in non-red cultivars. Zero-red pericarp from either immature or CPPU treated fruits appeared to be lacking in anthocyanins due to the absence of UFGT expression. Among these six genes, only the expression of UFGT

  19. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum

    PubMed Central

    Huang, Wenjun; Khaldun, A. B. M.; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  20. A R2R3-MYB Transcription Factor Regulates the Flavonol Biosynthetic Pathway in a Traditional Chinese Medicinal Plant, Epimedium sagittatum.

    PubMed

    Huang, Wenjun; Khaldun, A B M; Chen, Jianjun; Zhang, Chanjuan; Lv, Haiyan; Yuan, Ling; Wang, Ying

    2016-01-01

    Flavonols as plant secondary metabolites with vital roles in plant development and defense against UV light, have been demonstrated to be the main bioactive components (BCs) in the genus Epimedium plants, several species of which are used as materials for Herba Epimedii, an important traditional Chinese medicine. The flavonol biosynthetic pathway genes had been already isolated from Epimedium sagittatum, but a R2R3-MYB transcription factor regulating the flavonol synthesis has not been functionally characterized so far in Epimedium plants. In this study, we isolated and characterized the R2R3-MYB transcription factor EsMYBF1 involved in regulation of the flavonol biosynthetic pathway from E. sagittatum. Sequence analysis indicated that EsMYBF1 belongs to the subgroup 7 of R2R3-MYB family which contains the flavonol-specific MYB regulators identified to date. Transient reporter assay showed that EsMYBF1 strongly activated the promoters of EsF3H (flavanone 3-hydroxylase) and EsFLS (flavonol synthase), but not the promoters of EsDFRs (dihydroflavonol 4-reductase) and EsANS (anthocyanidin synthase) in transiently transformed Nicotiana benthamiana leaves. Both yeast two-hybrid assay and transient reporter assay validated EsMYBF1 to be independent of EsTT8, or AtTT8 bHLH regulators of the flavonoid pathway as cofactors. Ectopic expression of EsMYBF1 in transgenic tobacco resulted in the increased flavonol content and the decreased anthocyanin content in flowers. Correspondingly, the structural genes involved in flavonol synthesis were upregulated in the EsMYBF1 overexpression lines, including NtCHS (chalcone synthase), NtCHI (chalcone isomerase), NtF3H and NtFLS, whereas the late biosynthetic genes of the anthocyanin pathway (NtDFR and NtANS) were remarkably downregulated, compared to the controls. These results suggest that EsMYBF1 is a flavonol-specific R2R3-MYB regulator, and involved in regulation of the biosynthesis of the flavonol-derived BCs in E. sagittatum. Thus

  1. Comparative Transcriptome Analysis of Genes Involved in Anthocyanin Biosynthesis in the Red and Yellow Fruits of Sweet Cherry (Prunus avium L.)

    PubMed Central

    Wei, Hairong; Chen, Xin; Zong, Xiaojuan; Shu, Huairui; Gao, Dongsheng; Liu, Qingzhong

    2015-01-01

    Background Fruit color is one of the most important economic traits of the sweet cherry (Prunus avium L.). The red coloration of sweet cherry fruit is mainly attributed to anthocyanins. However, limited information is available regarding the molecular mechanisms underlying anthocyanin biosynthesis and its regulation in sweet cherry. Methodology/Principal Findings In this study, a reference transcriptome of P. avium L. was sequenced and annotated to identify the transcriptional determinants of fruit color. Normalized cDNA libraries from red and yellow fruits were sequenced using the next-generation Illumina/Solexa sequencing platform and de novo assembly. Over 66 million high-quality reads were assembled into 43,128 unigenes using a combined assembly strategy. Then a total of 22,452 unigenes were compared to public databases using homology searches, and 20,095 of these unigenes were annotated in the Nr protein database. Furthermore, transcriptome differences between the four stages of fruit ripening were analyzed using Illumina digital gene expression (DGE) profiling. Biological pathway analysis revealed that 72 unigenes were involved in anthocyanin biosynthesis. The expression patterns of unigenes encoding phenylalanine ammonia-lyase (PAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavanone 3’-hydroxylase (F3’H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS) and UDP glucose: flavonol 3-O-glucosyltransferase (UFGT) during fruit ripening differed between red and yellow fruit. In addition, we identified some transcription factor families (such as MYB, bHLH and WD40) that may control anthocyanin biosynthesis. We confirmed the altered expression levels of eighteen unigenes that encode anthocyanin biosynthetic enzymes and transcription factors using quantitative real-time PCR (qRT-PCR). Conclusions/Significance The obtained sweet cherry transcriptome and DGE profiling data

  2. A De novo Transcriptomic Approach to Identify Flavonoids and Anthocyanins “Switch-Off” in Olive (Olea europaea L.) Drupes at Different Stages of Maturation

    PubMed Central

    Iaria, Domenico L.; Chiappetta, Adriana; Muzzalupo, Innocenzo

    2016-01-01

    Highlights A de novo transcriptome reconstruction of olive drupes was performed in two genotypesGene expression was monitored during drupe development in two olive cultivarsTranscripts involved in flavonoid and anthocyanin pathways were analyzed in Cassanese and Leucocarpa cultivarsBoth cultivar and developmental stage impact gene expression in Olea europaea fruits. During ripening, the fruits of the olive tree (Olea europaea L.) undergo a progressive chromatic change characterized by the formation of a red-brown “spot” which gradually extends on the epidermis and in the innermost part of the mesocarp. This event finds an exception in the Leucocarpa cultivar, in which we observe a destabilized equilibrium between the metabolisms of chlorophyll and other pigments, particularly the anthocyanins whose switch-off during maturation promotes the white coloration of fruits. Despite its importance, genomic information on the olive tree is still lacking. Different RNA-seq libraries were generated from drupes of “Leucocarpa” and “Cassanese” olive genotypes, sampled at 100 and 130 days after flowering (DAF), and were used in order to identify transcripts involved in the main phenotypic changes of fruits during maturation and their corresponding expression patterns. A total of 103,359 transcripts were obtained and 3792 and 3064 were differentially expressed in “Leucocarpa” and “Cassanese” genotypes, respectively, during 100–130 DAF transition. Among them flavonoid and anthocyanin related transcripts such as phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol 3′-hydrogenase (F3′H), flavonol 3′5 ′-hydrogenase (F3′5′H), flavonol synthase (FLS), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), UDP-glucose:anthocianidin: flavonoid glucosyltransferase (UFGT) were identified. These results contribute

  3. 'Le Rouge et le Noir': A decline in flavone formation correlates with the rare color of black dahlia (Dahlia variabilis hort.) flowers

    PubMed Central

    2012-01-01

    Background More than 20,000 cultivars of garden dahlia (Dahlia variabilis hort.) are available showing flower colour from white, yellow and orange to every imaginable hue of red and purple tones. Thereof, only a handful of cultivars are so-called black dahlias showing distinct black-red tints. Flower colour in dahlia is a result of the accumulation of red anthocyanins, yellow anthochlors (6’-deoxychalcones and 4-deoxyaurones) and colourless flavones and flavonols, which act as copigments. White and yellow coloration occurs only if the pathway leading to anthocyanins is incomplete. Not in all cultivars the same step of the anthocyanin pathway is affected, but the lack of dihydroflavonol 4-reductase activity is frequently observed and this seems to be based on the suppression of the transcription factor DvIVS. The hitherto unknown molecular background for black colour in dahlia is here presented. Results Black cultivars accumulate high amounts of anthocyanins, but show drastically reduced flavone contents. High activities were observed for all enzymes from the anthocyanin pathway whereas FNS II activity could not be detected or only to a low extent in 13 of 14 cultivars. cDNA clones and genomic clones of FNS II were isolated. Independently from the colour type, heterologous expression of the cDNA clones resulted in functionally active enzymes. FNS II possesses one intron of varying length. Quantitative Real-time PCR showed that FNS II expression in black cultivars is low compared to other cultivars. No differences between black and red cultivars were observed in the expression of transcription factors IVS and possible regulatory genes WDR1, WDR2, MYB1, MYB2, 3RMYB and DEL or the structural genes of the flavonoid pathway. Despite the suppression of FHT expression, flavanone 3-hydroxylase (FHT, synonym F3H) enzyme activity was clearly present in the yellow and white cultivars. Conclusions An increased accumulation of anthocyanins establishes the black flowering

  4. Bovine renal mitochondrial vitamin D3 hydroxylases: regulation of in vitro activities by inhibitors and antioxidants.

    PubMed

    Crivello, J F

    1985-08-01

    The regulation of bovine renal 1 alpha- and 24-hydroxylase activities was examined in primary bovine proximal tubule cell cultures. Maximal 1 alpha- and 24-hydroxylase activities in primary bovine proximal tubule cultures ranged from 1.5-1.8 and 2.0-2.7 pmol/min X 10(6) cells, respectively. The apparent Km was 795 nM for 1 alpha-hydroxylase activity and 1130 nM for 24-hydroxylase activity. 1 alpha- and 24-hydroxylase activities decreased in primary culture after cell plating. Activities decreased both as a function of cell number and as a function of the culture dish. 1 alpha-Hydroxylase activity decayed with a t1/2 of 37 h, while 24-hydroxylase activity decayed with a t1/2 of 45 h. Decreasing cell densities, at which cells were plated, increased the t1/2 for the decay of both activities [t1/2 = 21 h at 5,000 cells/cm vs. t1/2 = 37 h at 25,000 cells/cm for 1 alpha-hydroxylase (P greater than 0.001); t1/2 = 33 h at 5,000 cells/cm vs. t1/2 = 45 h at 25,000 cells/cm for 24-hydroxylase, (P greater than 0.0001)]. Direct addition of 0.25 mM metyrapone inhibited 1 alpha-hydroxylase activity by 33% and 24-hydroxylase activity by 51%. Long term incubation of cell cultures with 0.25 mM metyrapone resulted in a slowing in the loss of both hydroxylase activities, but did not stop the decay. 1 alpha-Hydroxylase activity in 4-day metyrapone-treated cultures was 35% higher than in 4-day untreated cultures. 24-Hydroxylase activity was increased 42% in treated cultures vs. that in untreated cell cultures. Both 1 alpha- and 24-hydroxylase activities were inhibited by direct addition of antioxidants. 1 alpha-Hydroxylase activity was directly inhibited 74% by the addition of 0.1 mM butylated hydroxyanisole (BHA), 69% by the addition of 0.1 mM butylated hydroxytoluene (BHT), and 56% by the addition of 0.05 mM benzyl sulfoxide (BS). 24-Hydroxylase activity was also directly inhibited 72% by 0.1 mM BHA, 55% by 0.1 mM BHT, and 73% 0.05 mM BS. There was no significant difference between the inhibition of either hydroxylase by each antioxidant. Antioxidant mixtures increased the inhibition of hydroxylase activities above that with single antioxidant. The addition of 0.1 mM BHA and 0.05 mM BS to cultures resulted in 100% inhibition of 24-hydroxylase activity and 95% inhibition of 1 alpha-hydroxylase activity. The results were very similar when 0.01 mM BS and 0.1 mM BHT were added to cultures, i.e. 100% and 91% inhibition of 24- and 1 alpha-hydroxylase activities.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:4017943

  5. Targeted analysis of polyphenol metabolism during development of hop (Humulus lupulus L.) cones following treatment with prohexadione-calcium.

    PubMed

    Kavalier, Adam R; Ma, Chunhui; Figueroa, Mario; Kincaid, Dwight; Matthews, Paul D; Kennelly, Edward J

    2014-02-15

    Hops (Humulus lupulus), a main ingredient in beer, are valued as a source of bitter flavour and biologically active polyphenols. We treated immature hop cones with prohexadione-calcium (Pro-Ca), a flavanone-3-hydroxylase (F3H) inhibitor, to perturb the flavonoid pathway, and conducted a targeted analysis of 29 compounds from the phenolic acid, flavonoid, and terpenophenolic pathways, using photodiode array (UHPLC-PDA), and time of flight mass spectrometry (LC-TOF-MS) analysis. Hop cones were analysed over four stages of development spanning 22days following Pro-Ca treatment. The content of terpenophenolics as well as metabolic precursors upstream from F3H significantly increased, and levels of flavonoid products downstream from F3H decreased. The methods developed here serve to compliment flavour analysis of polyphenol rich foods, and our results suggest ways to improve upon traditional agricultural methods to produce hops with altered chemical profiles. PMID:24128475

  6. Methylfolate modulates potassium evoked neuro-secretion: evidence for a role at the pteridine cofactor level of tyrosine 3-hydroxylase.

    PubMed

    Lucock, M D; Green, M; Levene, M I

    1995-06-01

    We have previously shown that 5-methyltetrahydrofolate influences neuro-secretion. The present study more precisely characterises the processes involved and considers one probable site of action. Focusing on the tyrosine-noradrenalin axis in cerebellum we showed 5-methyltetrahydrofolate causes a significant reduction in the apparent K+ evoked secretion of noradrenalin to only 12.9% of control release. Evidence supports the idea that this could actually be due to increased synthesis leading to; depletion of reserves, possibly through leakage, exocytotic inhibition via activation of presynaptic receptors or end product inhibition by noradrenalin at the pteridine cofactor level of tyrosine hydroxylase: a) concomitant decreased measurement of perfusate and intracellular tyrosine with released noradrenalin following 5-methyltetrahydrofolate treatment supports the idea of increased transmitter turn over; b) kinetic studies indicate that at saturating concentrations of tyrosine and in the presence of an inhibitor of L-DOPA decarboxylase, 5-methyltetrahydrofolate partially duplicates the rate limiting behaviour of a synthetic pteridine cofactor--DL,2-amino-4-hydroxy-6,7,dimethyltetrahydropteridine. We debate whether, in vivo, CSF 5-methyltetrahydrofolate might interact at the tetrahydrobiopterin cofactor level of tyrosine hydroxylase and other aromatic amino-acid hydroxylases. PMID:7566370

  7. Molecular characterization of mutations in white-flowered torenia plants

    PubMed Central

    2014-01-01

    Background Torenia (Torenia fournieri Lind.) is a model plant increasingly exploited in studies in various disciplines, including plant engineering, biochemistry, physiology, and ecology. Additionally, cultivars with different flower colors have been bred and made commercially available. Flower color in torenia is mainly attributed to the accumulation of anthocyanins, but the molecular mechanisms inducing flower color mutations in torenia have not been well elucidated. In this study, we therefore attempted to identify the cause of white coloration in torenia by comparing the white-flowered cultivar Crown White (CrW) with Crown Violet (CrV), a violet-flowered variety. Results In an expression analysis, no flavanone 3-hydroxylase (TfF3H) transcript accumulation was detected in CrW petals. Sequence analyses revealed that a novel long terminal repeat (LTR)-type retrotransposable element, designated as TORE1 (Torenia retrotransposon 1), is inserted into the 5′-upstream region of the TfF3H gene in CrW. A transient expression assay using torenia F3H promoters with or without TORE1 insertion showed that the TORE1 insertion substantially suppressed F3H promoter activity, suggesting that this insertion is responsible for the absence of F3H transcripts in white petals. Furthermore, a transformation experiment demonstrated that the introduction of a foreign gentian F3H cDNA, GtF3H, into CrW was able to recover pink-flower pigmentation, indicating that F3H deficiency is indeed the cause of the colorless flower phenotype in CrW. Detailed sequence analysis also identified deletion mutations in flavonoid 3′-hydroxylase (TfF3′H) and flavonoid 3′,5′- hydroxylase (TfF3′5′H) genes, but these were not directly responsible for white coloration in this cultivar. Conclusions Taken together, a novel retrotransposable element, TORE1, inserted into the F3H 5′-upstream region is the cause of deficient F3H transcripts in white-flowered torenia, thereby leading to reduced petal

  8. A new rainbow: angular scattering of the F + H2(v(i) = 0, j(i) = 0) --> FH(v(f) = 3, j(f) = 3) + H reaction.

    PubMed

    Xiahou, Chengkui; Connor, J N L

    2009-12-31

    The angular scattering of a state-to-state chemical reaction contains fundamental information on its dynamics. Often the angular distributions are highly structured and the physical interpretation of this structure is an important and difficult problem. Here, we report a surprising finding for the benchmark F + H(2) --> FH + H reaction, when the product molecule FH is in a vibrational state with quantum number = 3 and a rotational state with quantum number = 3. We demonstrate that the differential cross section (DCS) is an example of (attractive) rainbow scattering, being characterized by an Airy function and its derivative. The rainbow reveals its presence in the DCS by interference with the repulsive (or nearside) scattering producing characteristic diffraction oscillations. The rainbow is broad, which explains why it has not been recognized in the many earlier theoretical and experimental investigations of this reaction. There is an angular region in the DCS where the rainbow dominates, but with the unusual property that the DCS is less intense than in adjoining angular regions. The reaction investigated is F + H(2)(v(i) = 0, j(i) = 0, m(i) = 0) --> FH(v(f) = 3, j(f) = 3, m(f) = 0) + H, where v(i), j(i), m(i) and v(f), j(f), m(f) are initial and final vibrational, rotational and helicity quantum numbers, respectively. The relative translational energy is 0.119 eV. We use rigorous semiclassical (asymptotic) techniques that provide physical insight as well as a mathematically sound and numerically accurate description of the angular scattering. The semiclassical DCS agrees very closely with the exact quantum DCS. The semiclassical scattering amplitude is used to assess the physical effectiveness of the Fuller nearside-farside decomposition for the partial wave series of the F + H(2) reaction, including the effect of one resummation. We also compare the semiclassical and exact quantum nearside, farside, and full local angular momenta and find good agreement. Although our new rainbow has unusual and unexpected properties, similar rainbows are predicted to occur in the DCSs of many state-to-state chemical reactions, since the semiclassical analysis is generic and not specific to the present F + H(2) example. PMID:19908879

  9. A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia.

    PubMed

    Kasajima, Ichiro; Sasaki, Katsutomo

    2016-05-01

    The development of new phenotypes is key to the commercial development of the main floricultural species and cultivars. Important new phenotypes include features such as multiple-flowers, color variations, increased flower size, new petal shapes, variegation and distinctive petal margin colourations. Although their commercial use is not yet common, the transgenic technologies provide a potentially rapid means of generating interesting new phenotypes. In this report, we construct 5 vectors which we expected to change the color of the flower anthocyanins, from purple to blue, regulating vacuolar pH. When these constructs were transformed into purple torenia, we unexpectedly recovered some genotypes having slightly margined petals. These transgenic lines expressed a chimeric repressor of the petunia PhPH4 gene under the control of Cauliflower mosaic virus 35 S RNA promoter. PhPH4 is an R2R3-type MYB transcription factor. The transgenic lines lacked pigmentation in the petal margin cells both on the adaxial and abaxial surfaces. Expressions of Flavanone 3-hydroxylase (F3H), Flavonoid 3'-hydroxylase (F3'H) and Flavonoid 3'5'-hydroxylase (F3'5'H) genes were reduced in the margins of these transgenic lines, suggesting an inhibitory effect of PhPH4 repressor on anthocyanin synthesis. PMID:27089475

  10. Chlorogenic acid biosynthesis: characterization of a light-induced microsomal 5-O-(4-coumaroyl)-D-quinate/shikimate 3'-hydroxylase from carrot (Daucus carota L. ) cell suspension cultures

    SciTech Connect

    Kuehnl, T.K.; Koch, U.; Heller, W.; Wellmann, E.

    1987-10-01

    Microsomal preparations from carrot (Daucus carota L.) cell suspension cultures catalyze the formation of trans-5-O-caffeoyl-D-quinate (chlorogenate) from trans-5-O-(4-coumaroyl)-D-quinate. trans-5-O-(4-Coumaroyl)shikimate is converted to about the same extent to trans-5-O-caffeoylshikimate. trans-4-O-(4-Coumaroyl)-D-quinate, trans-3-O-(4-coumaroyl)-D-quinate, trans-4-coumarate, and cis-5-O-(4-coumaroyl)-D-quinate do not act as substrates. The reaction is strictly dependent on molecular oxygen and on NADPH as reducing cofactor. NADH and ascorbic acid cannot substitute for NADPH. Cytochrome c, Tetcyclacis, and carbon monoxide inhibit the reaction suggesting a cytochrome P-450-dependent mixed-function monooxygenase. Competition experiments as well as induction and inhibition phenomena indicate that there is only one enzyme species which is responsible for the hydroxylation of the 5-O-(4-coumaric) esters of both D-quinate and shikimate. The activity of this enzyme is greatly increased by in vivo irradiation of the cells with blue/uv light. We conclude that the biosynthesis of the predominant caffeic acid conjugates in carrot cells occurs via the corresponding 4-coumaric acid esters. Thus, in this system, 5-O-(4-coumaroyl)-D-quinate can be seen as the final intermediate in the chlorogenic acid pathway.

  11. Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering.

    PubMed

    Zhao, Shujuan; Jones, J Andrew; Lachance, Daniel M; Bhan, Namita; Khalidi, Omar; Venkataraman, Sylesh; Wang, Zhengtao; Koffas, Mattheos A G

    2015-03-01

    Reconstruction of highly efficient biosynthesis pathways is essential for the production of valuable plant secondary metabolites in recombinant microorganisms. In order to improve the titer of green tea catechins in Escherichia coli, combinatorial strategies were employed using the ePathBrick vectors to express the committed catechin pathway: flavanone 3β-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Three F3H, three DFR, and two LAR genes originating from different plant species were selected and synthesized, to create 18 pathway variants to be screened in E. coli. Constructs containing F3H(syn) originally from Camellia sinensis, DFR(syn) from Anthurium andraeanum, C. sinensis, or Fragaria ananass, and LAR(syn) from Desmodium uncinatum (p148, p158 and p168) demonstrated high conversion efficiency with either eriodictyol or naringenin as substrate. A highly efficient construct was created by assembling additional copies of DFR(syn) and LAR(syn) enabling a titer of 374.6 ± 43.6 mg/L of (+)-catechin. Improving the NADPH availability via the ΔpgiΔppc mutation, BLΔpgiΔppc-p148 produced the highest titer of catechin at 760.9 ± 84.3 mg/L. After utilizing a library of scaffolding proteins, the strain BLΔpgiΔppc-p168-759 reached the highest titer of (+)-catechin of 910.9 ± 61.3 mg/L from 1.0 g/L of eriodictyol in batch culture with M9 minimal media. The impact of oxygen availability on the biosynthesis of catechin was also investigated. PMID:25527438

  12. Flavonoids and their qualitative variation in Calystegia soldanella and related species (Convolvulaceae).

    PubMed

    Murai, Yoshinori; Setoguchi, Hiroaki; Ono, Eiichiro; Iwashina, Tsukasa

    2015-03-01

    Coastal species are exposed to severe environmental stresses, e.g. salt and UV-B. The plants adapt themselves to such harsh environment by controlling morphological features and chemical defense systems. Flavonoids are known as efficient anti-stress polyphenols produced by plants. Most flavonoids show antioxidant activity, and their properties are important for plants to survive under high-stress conditions such as those in a coastal area. Among the compounds, ortho-dihydroxylated flavonoids act as strong antioxidants. In this survey, we elucidated the flavonoid composition of a seashore species Calystegia soldanella, which is distributed not only on the seashore, but also by the inland freshwater lake, Lake Biwa. Seven flavonol glycosides, i.e. quercetin 3-0- rutinoside, 3-O-glucoside, 3-O-rhamnoside and 3-O-apiosyl-(1-->2)-[rhamnosyl-(1-->6)-glucoside], and kaempferol 3-O-rutinoside, 3-O-glucoside and 3-0- rhamnoside were isolated from the leaves of C. soldanella. In addition, it was shown that the quercetin (Qu) to kaempferol (Km) ratio of coastal populations was higher than that of lakeshore populations. In general, these differences of Qu/Km ratio depend on flavonoid 3'-hydroxylase (F3'H) transcription. RT-PCR analysis suggested that F3'H of C. soldanella is regulated translationally or post-translationally, but not transcriptionally. Furthermore, quantitative and qualitative differences in flavonoid composition occurred among three Calystegia species, C. soldanella, C. japonica and C. hederacea. PMID:25924521

  13. The Gene Encoding Dihydroflavonol 4-Reductase Is a Candidate for the anthocyaninless Locus of Rapid Cycling Brassica rapa (Fast Plants Type)

    PubMed Central

    Wendell, Douglas L.; Vaziri, Anoumid; Shergill, Gurbaksh

    2016-01-01

    Rapid cycling Brassica rapa, also known as Wisconsin Fast Plants, are a widely used organism in both K-12 and college science education. They are an excellent system for genetics laboratory instruction because it is very easy to conduct genetic crosses with this organism, there are numerous seed stocks with variation in both Mendelian and quantitative traits, they have a short generation time, and there is a wealth of educational materials for instructors using them. Their main deficiency for genetics education is that none of the genetic variation in RCBr has yet been characterized at the molecular level. Here we present the first molecular characterization of a gene responsible for a trait in Fast Plants. The trait under study is purple/nonpurple variation due to the anthocyaninless locus, which is one of the Mendelian traits most frequently used for genetics education with this organism. We present evidence that the DFR gene, which encodes dihyroflavonol 4-reductase, is the candidate gene for the anthocyaninless (ANL) locus in RCBr. DFR shows complete linkage with ANL in genetic crosses with a total of 948 informative chromosomes, and strains with the recessive nonpurple phenotype have a transposon-related insertion in the DFR which is predicted to disrupt gene function. PMID:27548675

  14. The Gene Encoding Dihydroflavonol 4-Reductase Is a Candidate for the anthocyaninless Locus of Rapid Cycling Brassica rapa (Fast Plants Type).

    PubMed

    Wendell, Douglas L; Vaziri, Anoumid; Shergill, Gurbaksh

    2016-01-01

    Rapid cycling Brassica rapa, also known as Wisconsin Fast Plants, are a widely used organism in both K-12 and college science education. They are an excellent system for genetics laboratory instruction because it is very easy to conduct genetic crosses with this organism, there are numerous seed stocks with variation in both Mendelian and quantitative traits, they have a short generation time, and there is a wealth of educational materials for instructors using them. Their main deficiency for genetics education is that none of the genetic variation in RCBr has yet been characterized at the molecular level. Here we present the first molecular characterization of a gene responsible for a trait in Fast Plants. The trait under study is purple/nonpurple variation due to the anthocyaninless locus, which is one of the Mendelian traits most frequently used for genetics education with this organism. We present evidence that the DFR gene, which encodes dihyroflavonol 4-reductase, is the candidate gene for the anthocyaninless (ANL) locus in RCBr. DFR shows complete linkage with ANL in genetic crosses with a total of 948 informative chromosomes, and strains with the recessive nonpurple phenotype have a transposon-related insertion in the DFR which is predicted to disrupt gene function. PMID:27548675

  15. Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines

    PubMed Central

    Castellarin, Simone D; Di Gaspero, Gabriele

    2007-01-01

    Background Fruit coloration of red-skinned grapevines is mainly due to anthocyanin pigments. We analysed a panel of nine cultivars that included extreme phenotypes for berry colour, ranging from green (absence of anthocyanins) to red, purple, violet and blue. Expression of six genes of the anthocyanin pathway coding for flavanone-hydroxylase (F3H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), UDP-glucose:flavonoid-3-O-glucosyltransferase (UFGT), glutathione-S-transferase (GST), O-methyltransferase (OMT) and four transcription factors (MybA, MybB, MybC, MybD) was analysed by quantitative RT-PCR at four developmental stages from before the onset of ripening until full maturity and compared to anthocyanin metabolites. Results Total anthocyanin content at full maturity correlated well with the cumulative expression of F3H, UFGT and GST throughout ripening. Transcripts of the last two genes were absent in the green-skinned cultivar 'Sauvignonasse', also known as 'Tocai friulano', and were at least 10-fold less abundant in pale red cultivars, such as 'Pinot gris' and 'Gewürztraminer', compared to fully coloured cultivars. Predominance of tri-hydroxylated anthocyanins (delphinidin, petunidin and malvidin) in cultivars bearing dark berries with violet and blue hue was associated with higher ratios of F3'5'H/F3'H transcription, compared to red-skinned cultivars. Higher levels of OMT transcripts were observed in berries of cultivars that accumulated methoxylated forms of anthocyanins more abundantly than non-methoxylated forms. Conclusion Colour variation of the grape berry conforms to a peculiar pattern of genotype-specific expression of the whole set of anthocyanin genes in a direct transcript-metabolite-phenotype relationship. Cumulative mRNA levels of the structural genes and their relative abundance throughout ripening explained per se the final phenotype for anthocyanin content, anthocyanin composition, colour intensity and colour hue of

  16. Generation and analysis of expressed sequence tags from a normalized cDNA library of young leaf from Ma bamboo (Dendrocalamus latiflorus Munro).

    PubMed

    Gao, Z M; Li, C L; Peng, Z H

    2011-11-01

    Ma bamboo (Dendrocalamus latiflorus Munro) belongs to Dendrocalamus genus, Bambusease tribe, Bambusoideae subfamily, Poaceae family. It is a representative species of clumping bamboo, and a principal commercial species for various construction purposes using mature culms and for human consumption using young shoots. A normalized cDNA library was constructed from young leaves of Ma bamboo and 9,574 high-quality ESTs were generated, from which 5,317 unigenes including 1,502 contigs and 3,815 singletons were assembled. The unigenes were assigned into different gene ontology (GO) categories and summarized into 13 broad biologically functional groups according to similar functional characteristics or cellular roles by BLAST search against public databases. Eight hundred and ninety-one unigenes were assigned by KO identifiers and mapped to six KEGG biochemical pathways. The transcripts involved in biosynthesis of secondary metabolites such as cytochrome 450, flavonol synthase/flavanone 3-hydroxylase, and dihydroflavonol-4-reductase were well represented by 14 unigenes in the unigene set. The candidate genes involved in phytohormone metabolism, signal transduction and encoding cell wall-associated receptor kinases were also identified. Sixty-seven unigenes related to plant resistance (R) genes, including RPP genes, RGAs and RDL/RF genes, were discovered. These results will provide genome-wide knowledge about the molecular physiology of Ma bamboo young leaves and tools for advanced studies of molecular mechanism underlying leaf growth and development. PMID:21713530

  17. Red Clover Coumarate 3'-Hydroxylase (CYP98A44) is Capable of Hydroxylating P-Coumaroyl-Shikimate but not P-Coumaroyl-Malate: Implications for the Biosynthesis of Phaselic Acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Red clover (Trifolium pratense) leaves accumulate several µmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicag...

  18. Violet/blue chrysanthemums--metabolic engineering of the anthocyanin biosynthetic pathway results in novel petal colors.

    PubMed

    Brugliera, Filippa; Tao, Guo-Qing; Tems, Ursula; Kalc, Gianna; Mouradova, Ekaterina; Price, Kym; Stevenson, Kim; Nakamura, Noriko; Stacey, Iolanda; Katsumoto, Yukihisa; Tanaka, Yoshikazu; Mason, John G

    2013-10-01

    Chrysanthemums (Chrysanthemum×morifolium Ramat.) are an important cut-flower and potted plant crop in the horticultural industry world wide. Chrysanthemums express the flavonoid 3'-hydroxylase (F3'H) gene and thus accumulate anthocyanins derived from cyanidin in their inflorescences which appear pink/red. Delphinidin-based anthocyanins are lacking due to the deficiency of a flavonoid 3', 5'-hydroxylase (F3'5'H), and so violet/blue chrysanthemum flower colors are not found. In this study, together with optimization of transgene expression and selection of the host cultivars and gene source, F3'5'H genes have been successfully utilized to produce transgenic bluish chrysanthemums that accumulate delphinidin-based anthocyanins. HPLC analysis and feeding experiments with a delphinidin precursor identified 16 cultivars of chrysanthemums out of 75 that were predicted to turn bluish upon delphinidin accumulation. A selection of eight cultivars were successfully transformed with F3'5'H genes under the control of different promoters. A pansy F3'5'H gene under the control of a chalcone synthase promoter fragment from rose resulted in the effective diversion of the anthocyanin pathway to produce delphinidin in transgenic chrysanthemum flower petals. The resultant petal color was bluish, with 40% of total anthocyanidins attributed to delphinidin. Increased delphinidin levels (up to 80%) were further achieved by hairpin RNA interference-mediated silencing of the endogenous F3'H gene. The resulting petal colors were novel bluish hues, not possible by hybridization breeding. This is the first report of the production of anthocyanins derived from delphinidin in chrysanthemum petals leading to novel flower color. PMID:23926066

  19. Direct comparison between genomic constitution and flavonoid contents in Allium multiple alien addition lines reveals chromosomal locations of genes related to biosynthesis from dihydrokaempferol to quercetin glucosides in scaly leaf of shallot (Allium cepa L.).

    PubMed

    Masuzaki, S; Shigyo, M; Yamauchi, N

    2006-02-01

    The extrachromosome 5A of shallot (Allium cepa L., genomes AA) has an important role in flavonoid biosynthesis in the scaly leaf of Allium fistulosum-shallot monosomic addition lines (FF+nA). This study deals with the production and biochemical characterisation of A. fistulosum-shallot multiple alien addition lines carrying at least 5A to determine the chromosomal locations of genes for quercetin formation. The multiple alien additions were selected from the crossing between allotriploid FFA (female symbol) and A. fistulosum (male symbol). The 113 plants obtained from this cross were analysed by a chromosome 5A-specific PGI isozyme marker of shallot. Thirty plants were preliminarily selected for an alien addition carrying 5A. The chromosome numbers of the 30 plants varied from 18 to 23. The other extrachromosomes in 19 plants were completely identified by using seven other chromosome markers of shallot. High-performance liquid chromatography analyses of the 19 multiple additions were conducted to identify the flavonoid compounds produced in the scaly leaves. Direct comparisons between the chromosomal constitution and the flavonoid contents of the multiple alien additions revealed that a flavonoid 3'-hydroxylase (F3'H) gene for the synthesis of quercetin from kaempferol was located on 7A and that an anonymous gene involved in the glucosidation of quercetin was on 3A or 4A. As a result of supplemental SCAR analyses by using genomic DNAs from two complete sets of A. fistulosum-shallot monosomic additions, we have assigned F3'H to 7A and flavonol synthase to 4A. PMID:16411131

  20. The Reaumuria trigyna leucoanthocyanidin dioxygenase (RtLDOX) gene complements anthocyanidin synthesis and increases the salt tolerance potential of a transgenic Arabidopsis LDOX mutant.

    PubMed

    Zhang, Huirong; Du, Chao; Wang, Yan; Wang, Jia; Zheng, Linlin; Wang, Yingchun

    2016-09-01

    Reaumuria trigyna is a typical, native desert halophyte that grows under extreme conditions in Inner Mongolia. In a previous transcriptomic profiling analysis, flavonoid pathway-related genes in R. trigyna showed significant differences in transcript abundance under salt stress. Leucoanthocyanidin dioxygenase (LDOX, EC 1.14.11.19) is one of three dioxygenases in the flavonoid pathway that catalyzes the formation of anthocyanidins from leucoanthocyanidins. In this study, we cloned the full-length cDNA of R. trigyna LDOX (RtLDOX), and found RtLDOX recombinant protein was able to replace flavanone-3-hydroxylase (F3H, EC 1.14.11.9), another dioxygenase in the flavonoid pathway, to convert naringenin to dihydrokaempferol in vitro. R. trigyna LDOX can complement the Arabidopsis LDOX mutant transparent testa11 (tt11-11), which has reduced proanthocyanin (PA) and anthocyanin levels in seeds, to accumulate these two compounds. Thus, RtLDOX acts as a multifunctional dioxygenase to effect the synthesis of PA and anthocyanins and can perform F3H dioxygenase activities in the flavonoid biosynthesis pathway. The RtLDOX promoter harbored many cis-acting elements that might be recognized and bound by transcription factors related to stress response. RtLDOX expression was strongly increased under salt stress, and RtLDOX transgenic Arabidopsis mutant under NaCl stress accumulated the content of flavonoids leading to an increased antioxidant activities and plant biomass. These results suggest that RtLDOX as a multifunctional dioxygenase in flavonoid biosynthesis involves in enhancing plant response to NaCl stress. PMID:27219053

  1. Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry (Fragaria × ananassa) fruits.

    PubMed

    Pillet, Jeremy; Yu, Hao-Wei; Chambers, Alan H; Whitaker, Vance M; Folta, Kevin M

    2015-08-01

    New modulators of the strawberry flavonoid pathway were identified through correlation network analysis. The transcriptomes of red, ripe fruit from two parental lines and 14 of their progeny were compared, and uncharacterized transcripts matching the expression patterns of known flavonoid-pathway genes were identified. Fifteen transcripts corresponded with putative transcription factors, and several of these were examined experimentally using transient expression in developing fruits. The results suggest that two of the newly-identified regulators likely contribute to discrete nodes of the flavonoid pathway. One increases only LEUCOANTHOCYANIDIN REDUCTASE (LAR) and FLAVONOL 3'-HYDROXYLASE (F3'H) transcript accumulation upon overexpression. Another affects LAR and FLAVONOL SYNTHASE (FLS) after overexpression. The third putative transcription factor appears to be a universal regulator of flavonoid-pathway genes, as many pathway transcripts decrease in abundance when this gene is silenced. This report demonstrates that such systems-level approaches may be especially powerful when connected to an effective transient expression system, helping to provide rapid and strong evidence of gene function in key fruit-ripening processes. PMID:25979996

  2. Impact of cluster thinning on transcriptional regulation of anthocyanin biosynthesis-related genes in 'Summer Black' grapes.

    PubMed

    Xi, Xiaojun; Zha, Qian; Jiang, Aili; Tian, Yihua

    2016-07-01

    Cluster thinning is an agronomic practice that strongly affects anthocyanin biosynthesis in the skin of grape berries. However, the impact of cluster thinning on anthocyanin biosynthesis has not been fully elucidated at the molecular level. Here, we investigated its effects on the berry quality, the biosynthesis of anthocyanins, and the expression levels of related genes from the onset of véraison to harvest in 'Summer Black' grapes. It was observed that the total soluble solid and anthocyanin content in berry skin significantly increased under cluster thinning, whereas the berry weight and titratable acidity showed no differences from the beginning of véraison to harvest. The expression level of most anthocyanin biosynthesis-related genes was significantly up-regulated by cluster thinning from the beginning of véraison and was higher at its end compared to the control. Up-regulation of flavonoid 3',5'-hydroxylase (F3'5'H) and O-methyltransferase (OMT) expression, and down-regulation of flavonoid 3'-hydroxylase (F3'H) expression were observed, which might be the cause of shift in the anthocyanin profile. These findings provide insights into the molecular basis of the relationship between cluster thinning and anthocyanin biosynthesis in the grape berry skin. PMID:27035257

  3. Characterization of the regulatory network of BoMYB2 in controlling anthocyanin biosynthesis in purple cauliflower.

    PubMed

    Chiu, Li-Wei; Li, Li

    2012-10-01

    Purple cauliflower (Brassica oleracea L. var. botrytis) Graffiti represents a unique mutant in conferring ectopic anthocyanin biosynthesis, which is caused by the tissue-specific activation of BoMYB2, an ortholog of Arabidopsis PAP2 or MYB113. To gain a better understanding of the regulatory network of anthocyanin biosynthesis, we investigated the interaction among cauliflower MYB-bHLH-WD40 network proteins and examined the interplay of BoMYB2 with various bHLH transcription factors in planta. Yeast two-hybrid studies revealed that cauliflower BoMYBs along with the other regulators formed the MYB-bHLH-WD40 complexes and BobHLH1 acted as a bridge between BoMYB and BoWD40-1 proteins. Different BoMYBs exhibited different binding activity to BobHLH1. Examination of the BoMYB2 transgenic lines in Arabidopsis bHLH mutant backgrounds demonstrated that TT8, EGL3, and GL3 were all involved in the BoMYB2-mediated anthocyanin biosynthesis. Expression of BoMYB2 in Arabidopsis caused up-regulation of AtTT8 and AtEGL3 as well as a subset of anthocyanin structural genes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and leucoanthocyanidin dioxygenase. Taken together, our results show that MYB-bHLH-WD40 network transcription factors regulated the bHLH gene expression, which may represent a critical feature in the control of anthocyanin biosynthesis. BoMYB2 together with various BobHLHs specifically regulated the late anthocyanin biosynthetic pathway genes for anthocyanin biosynthesis. Our findings provide additional information for the complicated regulatory network of anthocyanin biosynthesis and the transcriptional regulation of transcription factors in vegetable crops. PMID:22644767

  4. Three R2R3-MYB transcription factors regulate distinct floral pigmentation patterning in Phalaenopsis spp.

    PubMed

    Hsu, Chia-Chi; Chen, You-Yi; Tsai, Wen-Chieh; Chen, Wen-Huei; Chen, Hong-Hwa

    2015-05-01

    Orchidaceae are well known for their fascinating floral morphologic features, specialized pollination, and distinctive ecological strategies. With their long-lasting flowers of various colors and pigmentation patterning, Phalaenopsis spp. have become important ornamental plants worldwide. In this study, we identified three R2R3-MYB transcription factors PeMYB2, PeMYB11, and PeMYB12. Their expression profiles were concomitant with red color formation in Phalaenopsis spp. flowers. Transient assay of overexpression of three PeMYBs verified that PeMYB2 resulted in anthocyanin accumulation, and these PeMYBs could activate the expression of three downstream structural genes Phalaenopsis spp. Flavanone 3-hydroxylase5, Phalaenopsis spp. Dihydroflavonol 4-reductase1, and Phalaenopsis spp. Anthocyanidin synthase3. In addition, these three PeMYBs participated in the distinct pigmentation patterning in a single flower, which was revealed by virus-induced gene silencing. In the sepals/petals, silencing of PeMYB2, PeMYB11, and PeMYB12 resulted in the loss of the full-red pigmentation, red spots, and venation patterns, respectively. Moreover, different pigmentation patterning was regulated by PeMYBs in the sepals/petals and lip. PeMYB11 was responsive to the red spots in the callus of the lip, and PeMYB12 participated in the full pigmentation in the central lobe of the lip. The differential pigmentation patterning was validated by RNA in situ hybridization. Additional assessment was performed in six Phalaenopsis spp. cultivars with different color patterns. The combined expression of these three PeMYBs in different ratios leads to a wealth of complicated floral pigmentation patterning in Phalaenopsis spp. PMID:25739699

  5. Expression of Genes Involved in Anthocyanin Biosynthesis in Relation to Anthocyanin, Proanthocyanidin, and Flavonol Levels during Bilberry Fruit Development1

    PubMed Central

    Jaakola, Laura; Määttä, Kaisu; Pirttilä, Anna Maria; Törrönen, Riitta; Kärenlampi, Sirpa; Hohtola, Anja

    2002-01-01

    The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented. PMID:12376640

  6. Three R2R3-MYB Transcription Factors Regulate Distinct Floral Pigmentation Patterning in Phalaenopsis spp.1[OPEN

    PubMed Central

    Hsu, Chia-Chi; Chen, You-Yi; Tsai, Wen-Chieh; Chen, Wen-Huei; Chen, Hong-Hwa

    2015-01-01

    Orchidaceae are well known for their fascinating floral morphologic features, specialized pollination, and distinctive ecological strategies. With their long-lasting flowers of various colors and pigmentation patterning, Phalaenopsis spp. have become important ornamental plants worldwide. In this study, we identified three R2R3-MYB transcription factors PeMYB2, PeMYB11, and PeMYB12. Their expression profiles were concomitant with red color formation in Phalaenopsis spp. flowers. Transient assay of overexpression of three PeMYBs verified that PeMYB2 resulted in anthocyanin accumulation, and these PeMYBs could activate the expression of three downstream structural genes Phalaenopsis spp. Flavanone 3-hydroxylase5, Phalaenopsis spp. Dihydroflavonol 4-reductase1, and Phalaenopsis spp. Anthocyanidin synthase3. In addition, these three PeMYBs participated in the distinct pigmentation patterning in a single flower, which was revealed by virus-induced gene silencing. In the sepals/petals, silencing of PeMYB2, PeMYB11, and PeMYB12 resulted in the loss of the full-red pigmentation, red spots, and venation patterns, respectively. Moreover, different pigmentation patterning was regulated by PeMYBs in the sepals/petals and lip. PeMYB11 was responsive to the red spots in the callus of the lip, and PeMYB12 participated in the full pigmentation in the central lobe of the lip. The differential pigmentation patterning was validated by RNA in situ hybridization. Additional assessment was performed in six Phalaenopsis spp. cultivars with different color patterns. The combined expression of these three PeMYBs in different ratios leads to a wealth of complicated floral pigmentation patterning in Phalaenopsis spp. PMID:25739699

  7. AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco

    PubMed Central

    Bharti, Poonam; Mahajan, Monika; Vishwakarma, Ajay K.; Bhardwaj, Jyoti; Yadav, Sudesh Kumar

    2015-01-01

    In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants. PMID:26116024

  8. AtROS1 overexpression provides evidence for epigenetic regulation of genes encoding enzymes of flavonoid biosynthesis and antioxidant pathways during salt stress in transgenic tobacco.

    PubMed

    Bharti, Poonam; Mahajan, Monika; Vishwakarma, Ajay K; Bhardwaj, Jyoti; Yadav, Sudesh Kumar

    2015-09-01

    In plants, epigenetic changes have been identified as regulators of developmental events during normal growth as well as environmental stress exposures. Flavonoid biosynthetic and antioxidant pathways play a significant role in plant defence during their exposure to environmental cues. The aim of this study was to unravel whether genes encoding enzymes of flavonoid biosynthetic and antioxidant pathways are under epigenetic regulation, particularly DNA methylation, during salt stress. For this, a repressor of silencing from Arabidopsis, AtROS1, was overexpressed in transgenic tobacco. Generated transgenics were evaluated to examine the influence of AtROS1 on methylation status of promoters as well as on coding regions of genes encoding enzymes of flavonoids biosynthesis and antioxidant pathways. Overexpression of AtROS1 increases the demethylation levels of both promoters as well as coding regions of genes encoding chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, and anthocyanidin synthase of the flavonoid biosynthetic pathway, and glutathione S-transferase, ascorbate peroxidase, glutathione peroxidase, and glutathione reductase of the antioxidant pathway during control conditions. The level of demethylation was further increased at promoters as well as coding regions of these genes during salt-stress conditions. Transgenic tobacco overexpressing AtROS1 showed tolerance to salt stress that could have been due to the higher expression levels of the genes encoding enzymes of the flavonoid biosynthetic and antioxidant pathways. This is the first comprehensive study documenting the epigenetic regulation of flavonoid biosynthetic and antioxidant pathways during salt-stress exposure of plants. PMID:26116024

  9. Rain-Shelter Cultivation Modifies Carbon Allocation in the Polyphenolic and Volatile Metabolism of Vitis vinifera L. Chardonnay Grapes.

    PubMed

    Gao, Yuan; Li, Xiao-Xi; Han, Mei-Mei; Yang, Xiao-Fan; Li, Zheng; Wang, Jun; Pan, Qiu-Hong

    2016-01-01

    This study investigated the effect of rain-shelter cultivation on the biosynthesis of flavonoids and volatiles in grapes, with an aim of determining whether rain-shelter application could help to improve the sensory attributes and quality of grapes. Vitis vinifera L. Chardonnay grapes, grown in the Huaizhuo basin region of northern China, were selected within two consecutive years. A rain-shelter roof was constructed using a colorless polyethylene (PE) film with a light transmittance of 80%. Results showed that rain-shelter treatment did not affect the accumulation of soluble solids during grape maturation. However, the allocation of assimilated carbon in phenolic and volatile biosynthetic pathways varied significantly, leading to alterations in polyphenolic and volatile profiles. The rain-shelter cultivation enhanced the concentration of flavan-3-ols via the flavonoid-3'5'-hydroxylase (F3'5'H) pathway, but reduced the level of flavonols and flavan-3-ols via the flavonoid-3'-hydroxylase (F3'H) pathway. In addition, the rain-shelter cultivation significantly enhanced the synthesis of fatty acid-derived volatiles, isoprene-derived terpenoids and amino acid-derived branched-chain aliphatics, but led to a decrease in the accumulation of isoprene-derived norisoprenoids and amino acid-derived benzenoids. Principal component analysis revealed some key compounds that differentiated the grapes cultivated under open-field and rain-shelter conditions. Moreover, the effect of the rain-shelter application on the accumulation of these compounds appeared to be vintage dependent. The alteration of their profiles caused by the rain-shelter treatment was significant in the vintage that received higher rainfall, which usually took place in the first rapid growth and veraison phases. PMID:27218245

  10. The Flavonoid Pathway Regulates the Petal Colors of Cotton Flower

    PubMed Central

    Tan, Jiafu; Wang, Maojun; Tu, Lili; Nie, Yichun; Lin, Yongjun; Zhang, Xianlong

    2013-01-01

    Although biochemists and geneticists have studied the cotton flower for more than one century, little is known about the molecular mechanisms underlying the dramatic color change that occurs during its short developmental life following blooming. Through the analysis of world cotton germplasms, we found that all of the flowers underwent color changes post-anthesis, but there is a diverse array of petal colors among cotton species, with cream, yellow and red colors dominating the color scheme. Genetic and biochemical analyses indicated that both the original cream and red colors and the color changes post-anthesis were related to flavonoid content. The anthocyanin content and the expression of biosynthesis genes were both increased from blooming to one day post-anthesis (DPA) when the flower was withering and undergoing abscission. Our results indicated that the color changes and flavonoid biosynthesis of cotton flowers were precisely controlled and genetically regulated. In addition, flavonol synthase (FLS) genes involved in flavonol biosynthesis showed specific expression at 11 am when the flowers were fully opened. The anthocyanidin reductase (ANR) genes, which are responsible for proanthocyanidins biosynthesis, showed the highest expression at 6 pm on 0 DPA, when the flowers were withered. Light showed primary, moderate and little effects on flavonol, anthocyanin and proanthocyanidin biosynthesis, respectively. Flavonol biosynthesis was in response to light exposure, while anthocyanin biosynthesis was involved in flower color changes. Further expression analysis of flavonoid genes in flowers of wild type and a flavanone 3-hydroxylase (F3H) silenced line showed that the development of cotton flower color was controlled by a complex interaction between genes and light. These results present novel information regarding flavonoids metabolism and flower development. PMID:23951318

  11. Suppression subtractive hybridization identifies genes induced in response to UV-B irradiation in apple skin: isolation of a putative UDP-glucose 4-epimerase.

    PubMed

    Ban, Yusuke; Honda, Chikako; Bessho, Hideo; Pang, Xiao-Ming; Moriguchi, Takaya

    2007-01-01

    Suppression subtractive hybridization (SSH) successfully identified 11 cDNAs in apple skin with highly induced expression as a result of ultraviolet (UV)-B irradiation. Apart from three putative flavonoid biosynthetic genes, chalcone synthase (CHS; A5C), flavanone-3-hydroxylase (F3H; B5F), and flavonol synthase (FLS; D1F), five clones (A1H, A10E, B11G, D5F, and D11H) were induced by low temperature (17 degrees C) as well, which is also known to induce anthocyanin accumulation in apple skin. Moreover, four clones (A1H, A10E, B11G, and D11H), showing higher expression levels in the skin, accumulated higher anthocyanin concentrations than their counterparts. Of the four clones, only A10E, a putative UDP-glucose 4-epimerase (UGE), was deemed to play an important role in anthocyanin accumulation in apple skin based on the facts that: (i) its transcription level was higher in the deep red cultivar, 'Jonathan', than in the pale red cultivar, 'Tsugaru'; and (ii) it could reversibly catalyse UDP-glucose to UDP-galactose, and the latter molecule is a major sugar donor for cyanidin-glycoside in apple. Therefore, the full-length cDNA of A10E was isolated by rapid amplification of cDNA ends (RACE) and designated as MdUGE1. Further analysis demonstrated that UGE enzymatic activity was positively correlated with anthocyanin accumulation in apple skin. Thus, MdUGE1 isolated by SSH could play an important role in anthocyanin biosynthesis in apple skin in concert with other flavonoid biosynthetic genes. PMID:17404384

  12. Exogenous application of pectin-derived oligosaccharides to grape berries modifies anthocyanin accumulation, composition and gene expression.

    PubMed

    Villegas, Daniel; Handford, Michael; Alcalde, José Antonio; Perez-Donoso, Alonso

    2016-07-01

    -glucose flavonoid 3-O-glucosyltransferase (UFGT) expression, probably mediated by a rise in MYBA1 was also observed. Regarding the anthocyanin profile, despite the increase observed in MYB5A expression in PDO and Ethrel treatments, no changes in flavonoid 3'-hydroxylase (F-3'-H); flavonoid 3'5'-hydroxylase (F-3'5'-H) or O-methyltransferase (OMT) could be related with the profile modifications described. Overall, this study highlights that application of PDO is a novel means of altering specific grape berry anthocyanins, and could be a means of positively influencing wine quality without the addition of agrochemicals. PMID:27031424

  13. Transposon tagging of a male-sterility, female-sterility gene, St8, revealed that the meiotic MER3 DNA helicase activity is essential for fertility in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion fr...

  14. DIBENZODIOXOCIN STRUCTURES INVOLVING P-HYDROXYPHENYL UNITS IN C3H DOWN-REGULATED LIGNINS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We previously reported that downregulation of the gene encoding 4-coumarate 3-hydroxylase (C3H) in alfalfa markedly increased the proportion of p-hydroxyphenyl (H) units relative to the normally dominant guaiacyl (G) and syringyl (S) units, as determined by 2D 13C-1H correlative NMR methods, thioaci...

  15. TOOLS FOR LIGNIN STRUCTURAL ANALYSIS; APPLICATIONS TO C3H-DOWNREGULATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects on lignification of downregulating most of the genes for enzymes on the monolignol biosynthetic pathway have been reasonably well studied. The exception to this is the crucial hydroxylase, cinnamate 3-hydroxylase (C3H), taking p-coumarate to caffeate. The Noble Foundation group has been ...

  16. Identification of the Pr1 gene product completes the anthocyanin biosynthesis pathway of maize

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In maize, mutations in the pr1 locus lead to the accumulation of pelargonidin (red) rather than cyanidin (purple) pigments in aleurone cells where the anthocyanin biosynthetic pathway is active. We characterized pr1 mutation and isolated a putative F3'H encoding gene (Zmf3'h1), and showed by segrega...

  17. Flavonoid Properties in Plant Families Synthesizing Betalain Pigments (Review).

    PubMed

    Iwashina, Tsukasa

    2015-06-01

    The anthocyanin pigments are contained in the flowers, fruits, leaves and roots of almost plant species. On the other hand, distribution of the betacyanins are limited in eight families of the order Caryophyllales, i.e. Aizoaceae, Amaranthaceae, Basellaceae, Cactaceae, Didiereaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae. However, other flavonoids, i.e. flavones, C-glycosylflavones, flavonols, flavanones, dihydroflavonols, chalcones, aurones, and flavan and proanthocyanidins, are synthesized in betalain-containing families. In this review, distribution and properties of the flavonoids in eight betalain-containing families are described. PMID:26197560

  18. Xanthine oxidase inhibitors from Vietnamese Blumea balsamifera L.

    PubMed

    Nguyen, Mai Thanh Thi; Nguyen, Nhan Trung

    2012-08-01

    From the MeOH extract of the aerial part of Blumea balsamifera L., a new dihydroflavonol, (2R,3S)-(-)-4'-O-methyldihydroquercetin (1), together with seven known compounds has been isolated. Their structures were elucidated on the basis of spectroscopic data. Compounds 1-4 and 6-8 displayed significant xanthine oxidase inhibitory activity in a concentration-dependent manner, and compounds 1, 6 and 8 showed more potent inhibitory activity, with IC₅₀ values ranging from 0.23 to 1.91 µM, than that of a positive control allopurinol (IC₅₀ 2.50 µM). PMID:22821854

  19. Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia).

    PubMed

    Regos, Ionela; Treutter, Dieter

    2010-10-01

    A pentafluorophenylpropyl (PFP) stationary phase was tested for the simultaneous determination of several classes of phenolic compounds. The chromatographic results were compared with those obtained by using a bifunctional phase constituted of octadecyl and phenylpropyl bonded silica and three conventional C18 columns. The elution gradient was optimized with 5% formic acid and sodium acetate in combination with acetic acid as additives and methanol as solvents. For these evaluations, a complex phenolic extract of Onobrychis viciifolia (sainfoin) and test mixtures containing 54 standard substances including 2 simple phenolic compounds, 1 amino acid, 4 hydroxybenzoic acids (HBA), 6 hydroxycinnamic acids (HCA), 3 flavan-3-ols, 9 anthocyanins, 2 dihydroflavonols, 1 chalcone, 4 flavones, 1 isoflavone and 21 flavonols have been assayed. The perfluorinated column showed good resolution for the studied phenolic compounds which have the following elution order: HBA, HCA, flavan-3-ols, anthocyanins, dihydroflavonols, flavones, flavonols and isoflavones. Compared with other columns, it provides longer elution ranges for HBA, HCA and flavan-3-ols and increased retention times for all compound classes except anthocyanins which were similarly retained on a C18 column. Its selectivity is different from C18 and bifunctional phases. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) and post-column derivatization with p-dimethyl-aminocinnamic aldehyde (DMACA) has been validated for the analysis of individual phenolic compounds from a sainfoin plant extract (O. viciifolia). PMID:20817190

  20. Glycosyltransferase efficiently controls phenylpropanoid pathway

    PubMed Central

    Aksamit-Stachurska, Anna; Korobczak-Sosna, Alina; Kulma, Anna; Szopa, Jan

    2008-01-01

    Background In a previous study, anthocyanin levels in potato plants were increased by manipulating genes connected with the flavonoid biosynthesis pathway. However, starch content and tuber yield were dramatically reduced in the transgenic plants, which over-expressed dihydroflavonol reductase (DFR). Results Transgenic plants over-expressing dihydroflavonol reductase (DFR) were subsequently transformed with the cDNA coding for the glycosyltransferase (UGT) of Solanum sogarandinum in order to obtain plants with a high anthocyanin content without reducing tuber yield and quality. Based on enzyme studies, the recombinant UGT is a 7-O-glycosyltransferase whose natural substrates include both anthocyanidins and flavonols such as kaempferol and quercetin. In the super-transformed plants, tuber production was much higher than in the original transgenic plants bearing only the transgene coding for DFR, and was almost the same as in the control plants. The anthocyanin level was lower than in the initial plants, but still higher than in the control plants. Unexpectedly, the super-transformed plants also produced large amounts of kaempferol, chlorogenic acid, isochlorogenic acid, sinapic acid and proanthocyanins. Conclusion In plants over-expressing both the transgene for DFR and the transgene for UGT, the synthesis of phenolic acids was diverted away from the anthocyanin branch. This represents a novel approach to manipulating phenolic acids synthesis in plants. PMID:18321380

  1. Negative Regulation of Anthocynanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor

    SciTech Connect

    Gou, J.Y.; Liu, C.; Felippes, F. F.; Weigel, D.; Wang, J.-W.

    2011-04-01

    Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.

  2. De Novo Transcriptome Analysis to Identify Anthocyanin Biosynthesis Genes Responsible for Tissue-Specific Pigmentation in Zoysiagrass (Zoysia japonica Steud.)

    PubMed Central

    Ahn, Jong Hwa; Kim, June-Sik; Kim, Seungill; Soh, Hye Yeon; Shin, Hosub; Jang, Hosung; Ryu, Ju Hyun; Kim, Ahyeong; Yun, Kil-Young; Kim, Shinje; Kim, Ki Sun; Choi, Doil; Huh, Jin Hoe

    2015-01-01

    Zoysiagrass (Zoysia japonica Steud.) is commonly found in temperate climate regions and widely used for lawns, in part, owing to its uniform green color. However, some zoysiagrass cultivars accumulate red to purple pigments in their spike and stolon tissues, thereby decreasing the aesthetic value. Here we analyzed the anthocyanin contents of two zoysiagrass cultivars ‘Anyang-jungji’ (AJ) and ‘Greenzoa’ (GZ) that produce spikes and stolons with purple and green colors, respectively, and revealed that cyanidin and petunidin were primarily accumulated in the pigmented tissues. In parallel, we performed a de novo transcriptome assembly and identified differentially expressed genes between the two cultivars. We found that two anthocyanin biosynthesis genes encoding anthocyanidin synthase (ANS) and dihydroflavonol 4-reductase (DFR) were preferentially upregulated in the purple AJ spike upon pigmentation. Both ANS and DFR genes were also highly expressed in other zoysiagrass cultivars with purple spikes and stolons, but their expression levels were significantly low in the cultivars with green tissues. We observed that recombinant ZjDFR1 and ZjANS1 proteins successfully catalyze the conversions of dihydroflavonols into leucoanthocyanidins and leucoanthocyanidins into anthocyanidins, respectively. These findings strongly suggest that upregulation of ANS and DFR is responsible for tissue-specific anthocyanin biosynthesis and differential pigmentation in zoysiagrass. The present study also demonstrates the feasibility of a de novo transcriptome analysis to identify the key genes associated with specific traits, even in the absence of reference genome information. PMID:25905914

  3. Osteogenesis imperfecta due to compound heterozygosity for the LEPRE1 gene.

    PubMed

    Moul, Adrienne; Alladin, Amanda; Navarrete, Cristina; Abdenour, George; Rodriguez, Maria M

    2013-10-01

    Osteogenesis imperfecta is a rare connective tissue disorder characterized by bone fragility and low bone density. Most cases are caused by an autosomal dominant mutation in either COL1A1 or COL1A2 gene encoding type I collagen. However, autosomal recessive forms have been identified. We present a patient with severe respiratory distress due to osteogenesis imperfecta simulating type II, born to a non-consanguineous couple with mixed African-American and African-Hispanic ethnicity. Cultured skin fibroblasts demonstrated compound heterozygosity for mutations in the LEPRE1 gene encoding prolyl 3-hydroxylase 1 confirming the diagnosis of autosomal recessive osteogenesis imperfecta type VIII, perinatal lethal type. PMID:23301918

  4. Molecular mechanism for cadmium-induced anthocyanin accumulation in Azolla imbricata.

    PubMed

    Dai, Ling-Peng; Dong, Xin-Jiao; Ma, Hai-Hu

    2012-04-01

    Anthocyanins inducibly synthesized by Cd treatment showed high antioxidant activity and might be involved in internal detoxification mechanisms of Azolla imbricata against Cd toxicity. In order to understand anthocyanin biosynthesis mechanism during Cd stress, the cDNAs encoding chalcone synthase (CHS) and dihydroflavonol reductase (DFR), two key enzymes in the anthocyanin synthesis pathway, were isolated from A. imbricata. Deduced amino acid sequences of the cDNAs showed high homology to the sequences from other plants. Expression of AiDFR, and to a lesser extent AiCHS, was significantly induced in Cd treatment plant in comparison with the control. CHS and DFR enzymatic activities showed similar pattern changes with these genes expression during Cd stress. These results strongly indicate that Cd induced anthocyanin accumulation is probably mediated by up-regulation of structural genes including CHS and DFR, which might further increase the activities of enzymes encoded by these structural genes that control the anthocyanin biosynthetic steps. PMID:22225708

  5. Studies on search for bioactive natural products targeting TRAIL signaling leading to tumor cell apoptosis.

    PubMed

    Ishibashi, Masami; Ohtsuki, Takashi

    2008-09-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in many transformed cells but not in normal cells and, hence, has been expected as a new anticancer strategy. During our studies on search for bioactive natural products from various natural resources such as plants and microorganisms, we recently identified several natural products which exhibited activities related to TRAIL signaling. Dimeric sesquiterpenoids isolated from Zingiberaceous plant, Curcuma parviflora, showed enhancement activity of gene expression of TRAIL-receptor and TRAIL-receptor protein level. Several new isoflavone natural products, named brandisianins, were isolated from Leguminosaeous plant, Millettia brandisiana, by our screening study targeting TRAIL-receptor expression enhancement activity. A dihydroflavonol (BB1) that was extracted from Compositaeous plant, Blumea balsamifera, and fuligocandin B, a new anthranilylproline-indole alkaloid isolated from myxomycete were found to exhibit reversal effect of TRAIL resistance activity. PMID:18273883

  6. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression.

    PubMed Central

    van der Krol, A R; Mur, L A; Beld, M; Mol, J N; Stuitje, A R

    1990-01-01

    To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits. PMID:2152117

  7. Polyphenol metabolism provides a screening tool for beneficial effects of Onobrychis viciifolia (sainfoin).

    PubMed

    Thill, Jana; Regos, Ionela; Farag, Mohamed A; Ahmad, Asma F; Kusek, Justyna; Castro, Ana; Schlangen, Karin; Carbonero, Christine Hayot; Gadjev, Ilya Z; Smith, Lydia M J; Halbwirth, Heidi; Treutter, Dieter; Stich, Karl

    2012-10-01

    Onobrychis viciifolia (sainfoin) is a traditional fodder legume showing multiple benefits for the environment, animal health and productivity but weaker agronomic performance in comparison to other legumes. Benefits can be mainly ascribed to the presence of polyphenols. The polyphenol metabolism in O. viciifolia was studied at the level of gene expression, enzyme activity, polyphenol accumulation and antioxidant activity. A screening of 37 accessions regarding each of these characters showed a huge variability between individual samples. Principal component analysis revealed that flavonols and flavan 3-ols are the most relevant variables for discrimination of the accessions. The determination of the activities of dihydroflavonol 4-reductase and flavonol synthase provides a suitable screening tool for the estimation of the ratio of flavonols to flavan 3-ols and can be used for the selection of samples from those varieties that have a specific optimal ratio of these compounds for further breeding. PMID:22818525

  8. Ericoside, a new antibacterial biflavonoid from Erica mannii (Ericaceae).

    PubMed

    Bitchagno, Gabin Thierry M; Tankeo, Simplice B; Tsopmo, Apollinaire; Simo Mpetga, James D; Tchinda, Alembert T; Fobofou, Serge Alain T; Nkuete, Antoine Honoré L; Wessjohann, Ludger A; Kuete, Victor; Tane, Pierre

    2016-03-01

    A new dihydroflavonol-flavonol biflavonoid derivative, named ericoside was isolated from the ethanol extract of the whole plant of Erica mannii along with the known flavonoid, taxifolin 3-O-α-L-rhamnopyranoside; and two readily available sterols (sitosterol, sitosterol 3-O-β-D-glucopyranoside). The isolation was performed using chromatographic methods and the structure of purified molecules were elucidated using spectroscopic techniques (e.g. MS, NMR) and by comparison with literature data. The crude ethanol extract, ericoside, and taxifolin 3-O-α-L-rhamnopyranoside were tested against ten Gram-negative bacteria including multidrug resistant clinical isolates using a broth microdilution method. The crude ethanol extract showed no noteworthy activity. Of the purified compounds, ericoside displayed moderate activity against the resistant Escherichia coli AG100 with a MIC of 64 μg/mL. PMID:26802608

  9. Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae)

    PubMed Central

    Casimiro-Soriguer, Inés; Narbona, Eduardo; Buide, M. L.; del Valle, José C.; Whittall, Justen B.

    2016-01-01

    Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a. PMID:26973662

  10. Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae).

    PubMed

    Casimiro-Soriguer, Inés; Narbona, Eduardo; Buide, M L; Del Valle, José C; Whittall, Justen B

    2016-01-01

    Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a. PMID:26973662

  11. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn).

    PubMed

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum 'Hokkai T10' cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3(,) H1, FtF3'H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat. PMID:27014239

  12. Can main group systems act as superior catalysts for dihydrogen generation reactions? A computational investigation.

    PubMed

    Kuriakose, Nishamol; Vanka, Kumar

    2016-03-30

    The protolytic cleavage of the O-H bond in water and alcohols is a very important reaction, and an important method for producing dihydrogen. Full quantum chemical studies with density functional theory (DFT) reveal that germanium based complexes, such as HC{CMeArB}2GeH (Ar = 2,6-(i)Pr2C6H3), with the assistance of silicon based compounds such as SiF3H, can perform significantly better than the existing state-of-the-art post-transition metal based systems for catalyzing dihydrogen generation from water and alcohols through the protolysis reaction. PMID:26084994

  13. Expression of flavonoid biosynthesis genes and accumulation of flavonoid in wheat leaves in response to drought stress.

    PubMed

    Ma, Dongyun; Sun, Dexiang; Wang, Chenyang; Li, Yaoguang; Guo, Tiancai

    2014-07-01

    Flavonoids are the low molecular weight polyphenolic secondary metabolic compounds, and have various functions in growth, development, reproduction, and stress defense. However, little is known about the roles of the key enzymes in the flavonoids biosynthesis pathway in response to drought stress in winter wheat. Here, we investigated the expression pattern of flavonoids biosynthesis genes and accumulation of flavonoids in wheat leaves under drought stress. Quantitative real-time PCR analysis showed that there were a rapid increase in expression levels of TaCHS, TaCHI, TaF3H, TaFNS, TaFLS, TaDFR, and TaANS under drought stress in two wheat cultivars Aikang 58 (AK) and Chinese Spring (CS). The cultivar CS exhibited higher genes expression levels of TaCHS, TaCHI, TaF3H, TaFLS, TaDFR, and TaANS, and the cultivar AK showed a higher expression level of TaFNS gene during drought treatment. The increase rates of genes expression were superior in AK compared to CS. Total phenolics content, total flavonoids content, anthocyanin content, and schaftoside content in wheat leaves were enhanced during drought treatment and cultivar CS had a relative higher accumulation. These results suggest that the flavonoids pathway genes expression and accumulation of flavonoids compounds may be closely related to drought tolerant in wheat. Further, flavonoids response mechanism may be different between wheat cultivars. PMID:24727789

  14. Phytochrome-interacting factors PIF4 and PIF5 negatively regulate anthocyanin biosynthesis under red light in Arabidopsis seedlings.

    PubMed

    Liu, Zhongjuan; Zhang, Yongqiang; Wang, Jianfeng; Li, Ping; Zhao, Chengzhou; Chen, Yadi; Bi, Yurong

    2015-09-01

    Light is an important environmental factor inducing anthocyanin accumulation in plants. Phytochrome-interacting factors (PIFs) have been shown to be a family of bHLH transcription factors involved in light signaling in Arabidopsis. Red light effectively increased anthocyanin accumulation in wild-type Col-0, whereas the effects were enhanced in pif4 and pif5 mutants but impaired in overexpression lines PIF4OX and PIF5OX, indicating that PIF4 and PIF5 are both negative regulators for red light-induced anthocyanin accumulation. Consistently, transcript levels of several genes involved in anthocyanin biosynthesis and regulatory pathway, including CHS, F3'H, DFR, LDOX, PAP1 and TT8, were significantly enhanced in mutants pif4 and pif5 but decreased in PIF4OX and PIF5OX compared to in Col-0, indicating that PIF4 and PIF5 are transcriptional repressor of these gene. Transient expression assays revealed that PIF4 and PIF5 could repress red light-induced promoter activities of F3'H and DFR in Arabidopsis protoplasts. Furthermore, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) test and electrophoretic mobility shift assay (EMSA) showed that PIF5 could directly bind to G-box motifs present in the promoter of DFR. Taken together, these results suggest that PIF4 and PIF5 negatively regulate red light-induced anthocyanin accumulation through transcriptional repression of the anthocyanin biosynthetic genes in Arabidopsis. PMID:26259175

  15. Complete assignment of structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of the shallot (Allium cepa L.).

    PubMed

    Masuzaki, Shin-ichi; Shigyo, Masayoshi; Yamauchi, Naoki

    2006-08-01

    We analyzed Japanese bunching onion (Allium fistulosum L.) - shallot (Allium cepa L. Aggregatum group) alien chromosome addition lines in order to assign the genes involved in the flavonoid biosynthesis pathway to chromosomes of the shallot. Two complete sets of alien monosomic additions (2n = 2x + 1 = 17) were used for determining the chromosomal locations of several partial sequences of candidate genes, CHS, CHI, F3H, DFR, and ANS via analyses of PCR-based markers. The results of DNA marker analyses showed that the CHS-A, CHS-B, CHI, F3H, DFR, and ANS genes should be assigned to chromosomes 2A, 4A, 3A, 3A, 7A, and 4A, respectively. HPLC analyses of 14 A. fistulosum - shallot multiple alien additions (2n = 2x + 2 - 2x + 7 = 18 - 23) were conducted to identify the anthocyanin compounds produced in the scaly leaves. A direct comparison between the genomic constitution and the anthocyanin compositions of the multiple additions revealed that a 3GT gene for glucosylation of anthocyanidin was located on 4A. Thus, we were able to assign all structural genes involved in flavonoid biosynthesis influencing bulb color to individual chromosomes of A. cepa. PMID:17038797

  16. Cloning and expression analyses of the anthocyanin biosynthetic genes in mulberry plants.

    PubMed

    Qi, Xiwu; Shuai, Qin; Chen, Hu; Fan, Li; Zeng, Qiwei; He, Ningjia

    2014-10-01

    Anthocyanins are natural food colorants produced by plants that play important roles in their growth and development. Mulberry fruits are rich in anthocyanins, which are the most important active components of mulberry and have many potentially beneficial effects on human health. The study of anthocyanin biosynthesis will bring benefits for quality improvement and industrial exploration of mulberry fruits. In the present study, nine putative genes involved in anthocyanin biosynthesis in mulberry plants were identified and cloned. Sequence analysis revealed that the mulberry anthocyanin biosynthetic genes were conserved and had counterparts in other plants. Spatial transcriptional analysis showed detectable expression of eight of these genes in different tissues. The results of expression and UPLC analyses in two mulberry cultivars with differently colored fruit indicated that anthocyanin concentrations correlated with the expression levels of genes associated with anthocyanin biosynthesis including CHS1, CHI, F3H1, F3'H1, and ANS during the fruit ripening process. The present studies provide insight into anthocyanin biosynthesis in mulberry plants and may facilitate genetic engineering for improvement of the anthocyanin content in mulberry fruit. PMID:24748075

  17. Anthocyanins and flavonols are responsible for purple color of Lablab purpureus (L.) sweet pods.

    PubMed

    Cui, Baolu; Hu, Zongli; Zhang, Yanjie; Hu, Jingtao; Yin, Wencheng; Feng, Ye; Xie, Qiaoli; Chen, Guoping

    2016-06-01

    Lablab pods, as dietary vegetable, have high nutritional values similar to most of edible legumes. Moreover, our studies confirmed that purple lablab pods contain the natural pigments of anthocyanins and flavonols. Compared to green pods, five kinds of anthocyanins (malvidin, delphinidin and petunidin derivatives) were found in purple pods by HPLC-ESI-MS/MS and the major contents were delphinidin derivatives. Besides, nine kinds of polyphenol derivatives (quercetin, myricetin, kaempferol and apigenin derivatives) were detected by UPLC-ESI-MS/MS and the major components were quercetin and myricetin derivatives. In order to discover their molecular mechanism, expression patterns of biosynthesis and regulatory gens of anthocyanins and flavonols were investigated. Experimental results showed that LpPAL, LpF3H, LpF3'H, LpDFR, LpANS and LpPAP1 expressions were significantly induced in purple pods compared to green ones. Meanwhile, transcripts of LpFLS were more abundant in purple pods than green or yellow ones, suggestind that co-pigments of anthocyanins and flavonols are accumulated in purple pods. Under continuously dark condition, no anthocyanin accumulation was detected in purple pods and transcripts of LpCHS, LpANS, LpFLS and LpPAP1 were remarkably repressed, indicating that anthocyanins and flavonols biosynthesis in purple pods was regulated in light-dependent manner. These results indicate that co-pigments of anthocyanins and flavonols contribute to purple pigmentations of pods. PMID:26995313

  18. Capturing the spin state diversity of iron(III)-aryl porphyrins: OLYP is better than TPSSh.

    PubMed

    Conradie, Marrigje M; Conradie, Jeanet; Ghosh, Abhik

    2011-01-01

    DFT calculations with a variety of exchange-correlation functionals, including PW91, OLYP, TPSSh, B3LYP and B3LYP*, have been carried out on the low-energy spin states of chloroiron(III) porphyrin and four aryliron(III) porphyrins, viz. Fe(III)(P)Ph (S=1/2), Fe(III)(P)C(6)F(5) (S=5/2), Fe(III)(P)(3,4,5-C(6)F(3)H(2)) (S=1/2), Fe(III)(P)(2,4,6-C(6)F(3)H(2)) (S=5/2), where the expected spin states have been indicated within parentheses. Qualitatively, OLYP reproduces all the expected ground spin states. B3LYP appears to have some difficulty yielding the observed sextet ground states. B3LYP*, TPSSh and PW91 all fail to reproduce the sextet ground states, the latter two by rather large margins of energy. As far as this study is concerned, the overall performance of the functionals appears to be OLYP/OPBE>B3LYP>B3LYP*>TPSSh>PW91/BLYP/BP86/TPSS. PMID:21134606

  19. Seed coat phenolics and the developing silique transcriptome of Brassica carinata.

    PubMed

    Li, Xiang; Westcott, Neil; Links, Matthew; Gruber, Margaret Y

    2010-10-27

    Structures for nine compounds were elucidated in seed coats of two genetically related Brassica carinata lines. The yellow-seeded line accumulated monomeric kaempferols, phenylpropanoids, and lignans, while extractable and unextractable proanthocyanidins and a high-performance liquid chromatography peak containing polymeric-like quercetin/lignan structures were strongly reduced. The brown-seeded line accumulated large amounts of both types of proanthocyanidins (extractable and unextractable), as well as phenylpropanoids and lignans equivalent to the amounts in the yellow-seeded seed coats, but the brown-seeded seed coats lacked kaempferols. A Brassica napus 15K oligoarray experiment indicated that yellow-seeded siliques had more extreme gene expression changes and a 2.4-fold higher number of upregulated genes than brown-seeded siliques, including a host of transcription factors and genes with unknown function. Transcripts for six flavonoid genes (CHS, F3H, FOMT, DFR, GST, and TTG1) were lower and two (F3'H and FLS) were higher in yellow-seeded siliques, but expression of CHI, PAP1, and phenylpropanoid genes was unchanged. PMID:20925379

  20. Accumulation of phenylpropanoids and correlated gene expression in hairy roots of tartary buckwheat under light and dark conditions.

    PubMed

    Thwe, Aye Aye; Kim, YeJi; Li, Xiaohua; Kim, Yeon Bok; Park, Nam-Il; Kim, Haeng Hoon; Kim, Sun-Ju; Park, Sang Un

    2014-12-01

    Differential expression patterns of flavonoid biosynthetic pathway genes in the hairy roots of tartary buckwheat cultivars "Hokkai T8" and "Hokkai T10" were studied over a time course of the light-dark cycle. The Agrobacterium rhizogenes-mediated transformation system was applied for inducing hairy roots. Further, a total of six phenolic compounds and two anthocyanins were analyzed in the hairy roots which were exposed to both light and dark conditions, and their amounts were estimated by HPLC. The gene expression levels peaked on day 5 of culture during the time course of both dark and light conditions. Notably, FtPAL, Ft4CL, FtC4H, FtCHI, FtF3H, FtF3'H-1, and FtFLS-1 were more highly expressed in Hokkai T10 than in Hokkai T8 under dark conditions, among which FtPAL and FtCHI were found to be significantly upregulated, except on day 20 of culture. Significantly higher levels of phenolic compound, rutin, along with two anthocyanins were detected in the hairy roots of Hokkai T10 under both conditions. Furthermore, among all the phenolic compounds detected, the amount of rutin in Hokkai T10 hairy roots was found to be ∼5-fold (59,01 mg/g dry weight) higher than that in the control (12.45 mg/g dry weight) at the respective time periods under light and dark conditions. PMID:25194705

  1. Transcriptional control of monolignol biosynthesis in Pinus taeda: factors affecting monolignol ratios and carbon allocation in phenylpropanoid metabolism

    NASA Technical Reports Server (NTRS)

    Anterola, Aldwin M.; Jeon, Jae-Heung; Davin, Laurence B.; Lewis, Norman G.

    2002-01-01

    Transcriptional profiling of the phenylpropanoid pathway in Pinus taeda cell suspension cultures was carried out using quantitative real time PCR analyses of all known genes involved in the biosynthesis of the two monolignols, p-coumaryl and coniferyl alcohols (lignin/lignan precursors). When the cells were transferred to a medium containing 8% sucrose and 20 mm potassium iodide, the monolignol/phenylpropanoid pathway was induced, and transcript levels for phenylalanine ammonia lyase, cinnamate 4-hydroxylase, p-coumarate 3-hydroxylase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase, and cinnamyl alcohol dehydrogenase were coordinately up-regulated. Provision of increasing levels of exogenously supplied Phe to saturating levels (40 mm) to the induction medium resulted in further up-regulation of their transcript levels in the P. taeda cell cultures; this in turn was accompanied by considerable increases in both p-coumaryl and coniferyl alcohol formation and excretion. By contrast, transcript levels for both cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase were only slightly up-regulated. These data, when considered together with metabolic profiling results and genetic manipulation of various plant species, reveal that carbon allocation to the pathway and its differential distribution into the two monolignols is controlled by Phe supply and differential modulation of cinnamate 4-hydroxylase and p-coumarate 3-hydroxylase activities, respectively. The coordinated up-regulation of phenylalanine ammonia lyase, 4-coumarate:CoA ligase, caffeoyl-CoA O-methyltransferase, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase in the presence of increasing concentrations of Phe also indicates that these steps are not truly rate-limiting, because they are modulated according to metabolic demand. Finally, the transcript profile of a putative acid/ester O-methyltransferase, proposed as an alternative catalyst for O-methylation leading

  2. Mixed-function oxidase activity in seabirds and its relationship to oil pollution.

    PubMed

    Peakall, D B; Jeffrey, D A; Boersma, D

    1987-01-01

    1. The hepatic activity of epoxide hydrolase, aldrin epoxidase, aminopyrine N-demethylase, 7-ethoxyresorufin O-deethylase, benzo(a)pyrene 3-hydroxylase and UDP glucuronyl transferase was determined in adult herring gulls (Larus argentatus) at various stages of the breeding season. 2. MFO activity was measured for adult Leach's storm-petrels (Oceanodroma leucorhoa), guillemot (Uria aalge) and Atlantic puffins (Fratercula arctica). For most assays the values were highest for the puffin. 3. MFO activity in both nestling and adult Atlantic puffins was determined. The degree of induction caused by a single internal dose of Prudhoe Bay crude oil in adult puffins and that caused by multiple internal doses in nestling puffins was measured. PMID:2890477

  3. Ommochrome genesis in an albino strain of a terrestrial isopod.

    PubMed

    Hasegawa, Y; Negishi, S; Naito, J; Ikeda, R; Hasegawa, H; Nagamura, Y; Ishiguro, I

    1999-01-01

    The contents of tryptophan (Trp) metabolites and the activities of the enzymes involved in ommochrome biosynthesis were measured in an albino strain of a terrestrial isopod Armadillidium vulgare. There was little difference between the Trp content in the albino mutant and that in the wild type, although the contents of 3-hydroxykynurenine (3-OH-Kyn), 3-hydroxyanthranilic acid (3-OH-AA) and xanthommatin in the albino were significantly lower than those in the wild type. Tryptophan 2,3-dioxygenase (TDO) activity in the albino was extremely low, while the activities of Kyn-3-hydroxylase and kynureninase did not differ significantly between the two phenotypes. The extremely low activity of TDO is probably one of main reasons why almost no ommochrome pigment is produced in the albino mutant. PMID:10721113

  4. Sc65-Null Mice Provide Evidence for a Novel Endoplasmic Reticulum Complex Regulating Collagen Lysyl Hydroxylation

    PubMed Central

    Weis, MaryAnn; Rai, Jyoti; Hudson, David M.; Dimori, Milena; Zimmerman, Sarah M.; Hogue, William R.; Swain, Frances L.; Burdine, Marie S.; Mackintosh, Samuel G.; Tackett, Alan J.; Suva, Larry J.; Eyre, David R.

    2016-01-01

    Collagen is a major component of the extracellular matrix and its integrity is essential for connective tissue and organ function. The importance of proteins involved in intracellular collagen post-translational modification, folding and transport was recently highlighted from studies on recessive forms of osteogenesis imperfecta (OI). Here we describe the critical role of SC65 (Synaptonemal Complex 65, P3H4), a leprecan-family member, as part of an endoplasmic reticulum (ER) complex with prolyl 3-hydroxylase 3. This complex affects the activity of lysyl-hydroxylase 1 potentially through interactions with the enzyme and/or cyclophilin B. Loss of Sc65 in the mouse results in instability of this complex, altered collagen lysine hydroxylation and cross-linking leading to connective tissue defects that include low bone mass and skin fragility. This is the first indication of a prolyl-hydroxylase complex in the ER controlling lysyl-hydroxylase activity during collagen synthesis. PMID:27119146

  5. How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population

    PubMed Central

    2013-01-01

    Background Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. Results An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H

  6. [Flavonoids of Artemisia campestris, ssp. glutinosa].

    PubMed

    Hurabielle, M; Eberle, J; Paris, M

    1982-10-01

    Four flavanones (pinostrobin, pinocembrin, sakuranetin and naringenin), one dihydroflavonol (7-methyl aromadendrin) and one flavone (hispidulin) have been isolated from Artemisia campestris L. ssp. glutinosa Gay and identified by spectroscopic methods. Artemisia campestris L. sous-espèce glutinosa Gay est une Composée Anthémidée largement répandue sur les sables du littoral méditerranéean et abondante dans certaines régions d'Espagne et d'Italie. Dans le cadre d'une étude chimiotaxonomique du genre Artemisia Tourn., nous nous sommes intéressés à l'analyse des flavonoïdes, composés jamais décrits, à notre connaissance, dans cette espèce d' Artemisia. Les sommités fleuries d' Artemisia campestris sous-espèce glutinosa, séchées et pulvérisées, sont dégraissées à l'ether de pétrole et épuisées par le chloroforme. Le fractionnement de l'extrait chloroformique, par chromatographie sur colonne de silice, et la purification de certaines fractions conduisent à l'isolement de six génines flavoniques, à l'etat pur. L' étude des spectres UV, des spectres de masse et des spectres de RMN [1,2] et la comparaison avec des échantillons authentiques permettent de proposer, pour ces flavonoïdes, les structures de la pinostrobine [3], de la pinocembrine [4], de la sakuranétine, de la naringénine [5] (flavanones), de la méthyl-7-aromadendrine, [6, 7] (dihydroflavonol) et de l'hispiduline [8, 9] (flavone); quatre de ces génines sont méthylées. Parmi ces flavonoïdes, la pinostrobine n'a jamais été décrite, à notre connaissance, dans la famille des Composées; la pinocembrine, la sakuranétine et la naringénine ont déjà été signalées chez quelques Astéracées et Eupatoriées [10], et l'hispiduline dans la tribu des Anthémidées ( Santolina chamaecyparissus L.) [8]. Seule, la méthyl-7-aromadendrine semble décrite, à ce jour, dans le genre Artemisia Tourn. [7]. PMID:17396957

  7. Completion of Tricin Biosynthesis Pathway in Rice: Cytochrome P450 75B4 Is a Unique Chrysoeriol 5'-Hydroxylase.

    PubMed

    Lam, Pui Ying; Liu, Hongjia; Lo, Clive

    2015-08-01

    Flavones are ubiquitously accumulated in land plants, but their biosynthesis in monocots remained largely elusive until recent years. Recently, we demonstrated that the rice (Oryza sativa) cytochrome P450 enzymes CYP93G1 and CYP93G2 channel flavanones en route to flavone O-linked conjugates and C-glycosides, respectively. In tricin, the 3',5'-dimethoxyflavone nucleus is formed before O-linked conjugations. Previously, flavonoid 3',5'-hydroxylases belonging to the CYP75A subfamily were believed to generate tricetin from apigenin for 3',5'-O-methylation to form tricin. However, we report here that CYP75B4 a unique flavonoid B-ring hydroxylase indispensable for tricin formation in rice. A CYP75B4 knockout mutant is tricin deficient, with unusual accumulation of chrysoeriol (a 3'-methoxylated flavone). CYP75B4 functions as a bona fide flavonoid 3'-hydroxylase by restoring the accumulation of 3'-hydroxylated flavonoids in Arabidopsis (Arabidopsis thaliana) transparent testa7 mutants and catalyzing in vitro 3'-hydroxylation of different flavonoids. In addition, overexpression of both CYP75B4 and CYP93G1 (a flavone synthase II) in Arabidopsis resulted in tricin accumulation. Specific 5'-hydroxylation of chrysoeriol to selgin by CYP75B4 was further demonstrated in vitro. The reaction steps leading to tricin biosynthesis are then reconstructed as naringenin → apigenin → luteolin → chrysoeriol → selgin → tricin. Hence, chrysoeriol, instead of tricetin, is an intermediate in tricin biosynthesis. CYP75B4 homologous sequences are highly conserved in Poaceae, and they are phylogenetically distinct from the canonical CYP75B flavonoid 3'-hydroxylase sequences. Recruitment of chrysoeriol-specific 5'-hydroxylase activity by an ancestral CYP75B sequence may represent a key event leading to the prevalence of tricin-derived metabolites in grasses and other monocots today. PMID:26082402

  8. Gallium fluoroarsenates.

    PubMed

    Marshall, Kayleigh L; Armstrong, Jennifer A; Weller, Mark T

    2015-07-28

    Six new phases in the gallium-fluoride-arsenate system have been synthesised hydrofluorothermally using a fluoride-rich medium and "HAsF6" (HF : AsF5) as a reactant. RbGaF3(H2AsO4), KGaF(H2AsO4) and [piperazine-H2]2[Ga2F8(HAsO4)]·H2O have one dimensional structures, [DABCO-H2]2[Ga4F7O2H(AsO4)2]·4H2O consists of two dimensionally connected polyhedral layers, while GaF(AsO3[OH,F])2 and (NH4)3Ga4F9(AsO4)2 both have three-dimensionally connected polyhedral frameworks. PMID:26095086

  9. Laser induced fluorescence spectra of fluorophenol cations in a Ne matrix

    USGS Publications Warehouse

    Bondybey, V.E.; English, J.H.; Miller, T.A.; Shiley, R.H.

    1983-01-01

    Laser induced fluorescence and/or absorption spectra of the cations of 2,3,5,6‐tetrafluorophenol, 2,3,5,6‐tetrafluorothiophenol, and 3,5‐difluorophenol have been obtained in a Ne matrix. The spectra of C6HF4OH+ are much better resolved than in the gas phase. The gas phase congestion is likely caused by the near degeneracy of the and electronic states whose separation is now measured at 207 cm−1. The spectrum of C6H3F2OH+ represents a deperturbed example of the Jahn–Teller distorted sym‐C6F3H3+ ion. C6H3F2SH+ shows only a broad featureless absorption.

  10. Synthesis, antifungal activities and molecular docking studies of novel 2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl dithiocarbamates.

    PubMed

    Zou, Yan; Yu, Shichong; Li, Renwu; Zhao, Qingjie; Li, Xiang; Wu, Maocheng; Huang, Ting; Chai, Xiaoxun; Hu, Honggang; Wu, Qiuye

    2014-03-01

    A series of 2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl dithiocarbamates as new analogs of fluconazole were synthesized and their antifungal activities were evaluated. Among these compounds, 2a-f and 3a-q exhibited higher activities than fluconazole against nearly all fungi tested except Aspergillus fumigatus. Noticeably, the in vitro biological activities of 2b, 3a, 3c, 3h-k, and 3o-q against Candida species were much better than those of fluconazole and ketoconazole. Also, 2a-d, 3a-d, 3e-f, 3h-k, 3p and 3q showed higher activities against A. fumi than fluconazole. Computational docking experiments indicated that the inhibition of CYP51 involved a coordination bond with iron of the heme group, the hydrophilic H-bonding region, the hydrophobic region, and the narrow hydrophobic cleft. PMID:24487187

  11. Genetic and environmental effects influencing fruit colour and QTL analysis in raspberry.

    PubMed

    McCallum, Susan; Woodhead, Mary; Hackett, Christine A; Kassim, Angzzas; Paterson, Alistair; Graham, Julie

    2010-08-01

    Raspberry (Rubus idaeus) fruit colour was assessed in the Latham x Glen Moy mapping population using a colour meter and visual scores over three seasons and three environments. The colour measurements were found to be significantly associated with pigment content, have high heritability, and stable QTL were identified across environments and seasons. Anthocyanin content has previously been shown to be the major contributor to fruit colour in red raspberry. Major structural genes (F3'H, FLS, DFR, IFR, OMT and GST) and transcription factors (bZIP, bHLH and MYB) influencing flavonoid biosynthesis have been identified, mapped and shown to underlie QTL for quantitative and qualitative anthocyanin composition. Favourable alleles for the selected traits were identified for the aspects of fruit colour and partitioning of individual pigments. PMID:20419285

  12. [Expression of the genes involved in anthocyanin biosynthesis of 'Tsuda' turnip].

    PubMed

    Xu, Zhi-Ru; Li, Yu-Hua

    2006-10-01

    'Tsuda' turnip (Brassica campestris L. ssp. rapa), in which roots anthocyanin pigmentation is light-sensitive, was used as the material. 'Tsuda' plants were held in darkness or irradiated with sun light and constant light for different time. Anthocyanins in root peel of 'Tsuda' turnip exposed to constant light were identified and quantified with a UV-visual spectrophotometer. The results demonstrated that the anthocyanins accumulation in 'Tsuda' was related with light-exposure time (Fig.1 and Table 1). Fragments of genes selected from the subtraction library of 'Tsuda' turnip involved in anthocyanin biosynthesis were used as probes. The Northern blotting results showed that the expression of PAL, CHS, F3H, DFR and ANS could be induced by irradiation with light and the expression of these genes was related with light exposure time. The expression of MYB was basically the same whether in darkness or in light (Figs.2,3). PMID:17075183

  13. Ultraviolet-B radiation and water deficit interact to alter flavonol and anthocyanin profiles in grapevine berries through transcriptomic regulation.

    PubMed

    Martínez-Lüscher, Johann; Sánchez-Díaz, Manuel; Delrot, Serge; Aguirreolea, Jone; Pascual, Inmaculada; Gomès, Eric

    2014-11-01

    UV-B radiation and water deficit may trigger flavonol and anthocyanin biosynthesis in plant tissues. In addition, previous research has showed strong qualitative effects on grape berry skin flavonol and anthocyanin profiles in response to UV-B and water deficit. The aim of this study is to identify the mechanisms leading to quantitative and qualitative changes in flavonol and anthocyanin profiles, in response to separate and combined UV-B and water deficit. Grapevines (Vitis vinifera L. cv. Tempranillo) were exposed to three levels of UV-B radiation (0, 5.98 and 9.66 kJ m(-2) day(-1)) and subjected to two water regimes. A strong effect of UV-B on flavonol and anthocyanin biosynthesis was found, resulting in an increased anthocyanin concentration and a change in their profile. Concomitantly, two key biosynthetic genes (FLS1 and UFGT) were up-regulated by UV-B, leading to increased flavonol and anthocyanin skin concentration. Changes in flavonol and anthocyanin composition were explained to a large extend by transcript levels of F3'H, F3'5'H and OMT2. A significant interaction between UV-B and water deficit was found in the relative abundance of 3'4' and 3'4'5' substituted flavonols, but not in their anthocyanin homologues. The ratio between 3'4'5' and 3'4' substituted flavonols was linearly related to the ratios of F3'5'H and FLS1 transcription, two steps up-regulated independently by water deficit and UV-B radiation, respectively. Our results indicate that changes in flavonol profiles in response to environmental conditions are not only a consequence of changes in the expression of flavonoid hydroxylases; but also the result of the competition of FLS, F3'5'H and F3'H enzymes for the same flavonol substrates. PMID:25231967

  14. Comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves in chrysanthemum.

    PubMed

    Hong, Yan; Yang, Li-Wen; Li, Meng-Ling; Dai, Si-Lan

    2016-06-01

    Light is one of the key environmental factors that affect anthocyanin biosynthesis. However, the underlying molecular mechanism remains unclear, and many problems regarding phenotypic change and corresponding gene regulation have not been solved. In the present study, comparative analyses of light-induced anthocyanin accumulation and gene expression between the ray florets and leaves were performed in Chrysanthemum × morifolium 'Purple Reagan'. After contrasting the variations in the flower color phenotype and relative pigment content, as well as expression patterns of structural and regulator genes responsible for anthocyanin biosynthesis and photoreceptor between different plant organs under light and dark conditions, we concluded that (1) both the capitulum and foliage are key organs responding to light for chrysanthemum coloration; (2) compared with flavones, shading makes a greater decrease on the anthocyanins accumulation; (3) most of the structural and regulatory genes in the light-induced anthocyanin pathway specifically express in the ray florets; and (4) CmCHS, CmF3H, CmF3'H, CmANS, CmDFR, Cm3GT, CmMYB5-1, CmMYB6, CmMYB7-1, CmbHLH24, CmCOP1 and CmHY5 are key genes for light-induced anthocyanin biosynthesis in chrysanthemum ray florets, while on the transcriptional level, the expressions of CmPHYA, CmPHYB, CmCRY1a, CmCRY1b and CmCRY2 are insignificantly changed. Moreover, the inferred comprehensive effect of multiple signals on the accumulation of anthocyanins and transmission channel of light signal that exist between the leaves and ray florets were further discussed. These results further our understanding of the relationship between the gene expression and light-induced anthocyanin biosynthesis, and lay foundations for the promotion of the molecular breeding of novel flower colors in chrysanthemums. PMID:26990403

  15. Effect of Different Agrobacterium rhizogenes Strains on Hairy Root Induction and Phenylpropanoid Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum Gaertn)

    PubMed Central

    Thwe, Aye; Valan Arasu, Mariadhas; Li, Xiaohua; Park, Chang Ha; Kim, Sun Ju; Al-Dhabi, Naif Abdullah; Park, Sang Un

    2016-01-01

    The development of an efficient protocol for successful hairy root induction by Agrobacterium rhizogenes is the key step toward an in vitro culturing method for the mass production of secondary metabolites. The selection of an effective Agrobacterium strain for the production of hairy roots is highly plant species dependent and must be determined empirically. Therefore, our goal was to investigate the transformation efficiency of different A. rhizogenes strains for the induction of transgenic hairy roots in Fagopyrum tataricum ‘Hokkai T10’ cultivar; to determine the expression levels of the polypropanoid biosynthetic pathway genes, such as ftpAL, FtC4H, Ft4CL, FrCHS, FrCH1, FrF3H, FtFLS1, FtFLS2, FtF3, H1, FtF3′H2, FtANS, and FtDFR; and to quantify the in vitro synthesis of phenolic compounds and anthocyanins. Among different strains, R1000 was the most promising candidate for hairy root stimulation because it induced the highest growth rate, root number, root length, transformation efficiency, and total anthocyanin and rutin content. The R1000, 15834, and A4 strains provided higher transcript levels for most metabolic pathway genes for the synthesis of rutin (22.31, 15.48, and 13.04 μg/mg DW, respectively), cyanidin 3-O-glucoside (800, 750, and 650 μg/g DW, respectively), and cyanidin 3-O-rutinoside (2410, 1530, and 1170 μg/g DW, respectively). A suitable A. rhizogenes strain could play a vital role in the fast growth of the bulk amount of hairy roots and secondary metabolites. Overall, R1000 was the most promising strain for hairy root induction in buckwheat. PMID:27014239

  16. Transcriptional regulation of anthocyanin biosynthesis in red cabbage.

    PubMed

    Yuan, Youxi; Chiu, Li-Wei; Li, Li

    2009-11-01

    The color of red cabbage (Brassica oleracea var. capitata) is due to anthocyanin accumulation. To investigate the regulatory control of anthocyanin production in red cabbage, the expression of anthocyanin biosynthetic and regulatory genes from eight commercial cultivars was examined. While the four green varieties had negligible amount of anthocyanins under normal growth condition, the four red cultivars contained up to 1.60 mg g(-1) fresh weight. HPLC analysis of the four red cultivars revealed that they produced similar composition of various forms of cyanidin glucosides but at different concentrations. Molecular analysis indicated that all the red cabbage shared common mechanism of regulatory control for anthocyanin biosynthesis. Except CHI which showed similar expression levels between green and red cultivars, the other structural genes, CHS, F3H, F3'H, DFR, LDOX, and GST, were constitutively up-regulated during all stages of vegetative growth in red varieties. The expression of these structural genes was also dramatically increased in green and red cabbage under nutrient stresses. The increased expression of the structural genes coincided with a coordinated increase in transcript levels of a bHLH gene, BoTT8, and a MYB transcription factor, BoMYB2. These results indicate that activation of these two regulatory factors by unknown mechanisms constitutively up-regulates nearly the entire pathway genes for the onset of anthocyanin biosynthesis in red cabbage. Moreover, the amount of total anthocyanins in red cabbage was found to be positively correlated with total antioxidant power, implicating the potential health benefit of red cabbage to human health. PMID:19756724

  17. Transcriptome Analysis of Differentially Expressed Genes Relevant to Variegation in Peach Flowers

    PubMed Central

    Yu, Faxin; Li, Shuxian; Yin, Tongming

    2014-01-01

    Background Variegation in flower color is commonly observed in many plant species and also occurs on ornamental peaches (Prunus persica f. versicolor [Sieb.] Voss). Variegated plants are highly valuable in the floricultural market. To gain a global perspective on genes differentially expressed in variegated peach flowers, we performed large-scale transcriptome sequencing of white and red petals separately collected from a variegated peach tree. Results A total of 1,556,597 high-quality reads were obtained, with an average read length of 445 bp. The ESTs were assembled into 16,530 contigs and 42,050 singletons. The resulting unigenes covered about 60% of total predicted genes in the peach genome. These unigenes were further subjected to functional annotation and biochemical pathway analysis. Digital expression analysis identified a total of 514 genes differentially expressed between red and white flower petals. Since peach flower coloration is determined by the expression and regulation of structural genes relevant to flavonoid biosynthesis, a detailed examination detected four key structural genes, including C4H, CHS, CHI and F3H, expressed at a significantly higher level in red than in white petal. Except for the structural genes, we also detected 11 differentially expressed regulatory genes relating to flavonoid biosynthesis. Using the differentially expressed structural genes as the test objects, we validated the digital expression results by using quantitative real-time PCR, and the differential expression of C4H, CHS and F3H were confirmed. Conclusion In this study, we generated a large EST collection from flower petals of a variegated peach. By digital expression analysis, we identified an informative list of candidate genes associated with variegation in peach flowers, which offered a unique opportunity to uncover the genetic mechanisms underlying flower color variegation. PMID:24603808

  18. Functional conservation analysis and expression modes of grape anthocyanin synthesis genes responsive to low temperature stress.

    PubMed

    Zhang, Cheng; Jia, Haifeng; Wu, Weimin; Wang, Xicheng; Fang, Jinggui; Wang, Chen

    2015-12-10

    In grape cultivation, low temperature generally increases the expression of genes involved in synthesis of anthocyanin. In this study, multi-type structural analysis of the proteins encoded by five anthocyanin biosynthesis genes VvF3H, VvPAL, VvCHS3, VvCHS2 and VvLDOX, in addition to nine of their homologous genes revealed that proteins in grapevine shared a high similarity with that in kiwi, red orange and some other species in which the biosynthesis of anthocyanin significantly influenced by low temperature as proved by previous studies. Low temperature regulatory elements were also found in the promoter region of the grapevine genes VvCHS2, VvPAL and VvF3H. These findings indicate that the functions of anthocyanin biosynthesis genes in grapevine are conservative and might be sensitive to low temperature. In order to identify the specific expression patterns of the five anthocyanin biosynthesis genes and the changes of polyphenols, anthocyanins and flavonoids under low temperature stress. The transcription analysis of the five genes and the content of polyphenols, anthocyanins and flavonoids in grape skins were examined, by using Vitis vinifera L. cv. 'Yongyou 1' and 'Juxing' berries as experimental material and treated at 4°C and 25°C for 24h, 48 h, 72 h and 96 h. The results showed that low temperature greatly enhanced the expression of the five anthocyanin biosynthesis genes. Low temperature greatly slowed down the decomposition of polyphenol, anthocyanin, and flavonoid in grape skins. Our study also found that cv. 'Juxing' responded more sensitively to low temperature than cv. 'Yongyou 1'. All the findings would provide a basis for further study on the mechanism of anthocyanin biosynthesis under environmental stress. PMID:26253159

  19. Transposon Tagging of a Male-Sterility, Female-Sterility Gene, St8, Revealed that the Meiotic MER3 DNA Helicase Activity Is Essential for Fertility in Soybean

    PubMed Central

    Baumbach, Jordan; Pudake, Ramesh N.; Johnson, Callie; Kleinhans, Kaylin; Ollhoff, Alexandrea; Palmer, Reid G.; Bhattacharyya, Madan K.; Sandhu, Devinder

    2016-01-01

    The W4 locus in soybean encodes a dihydroflavonol-4-reductase (DFR2) that regulates pigmentation patterns in flowers and hypocotyl. The mutable w4-m allele that governs variegated flowers has arisen through insertion of a CACTA-type transposable element, Tgm9, in DFR2. In the w4-m line, reversion from variegated to purple flower indicates excision of Tgm9, and its insertion at a new locus. Previously, we have identified a male-sterile, female-sterile mutant among the selfed progenies of a revertant plant carrying only purple flowers. Co-segregation between Tgm9 and the sterility phenotype suggested that the mutant was generated by insertion of Tgm9 at the St8 locus. The transposon was localized to exon 10 of Glyma.16G072300 that shows high identity to the MER3 DNA helicase involved in crossing over. Molecular analysis of fertile branches from two independent revertant plants confirmed precise excision of Tgm9 from the st8 allele, which restored fertility. In soybean, the gene is expressed in flower-buds, trifoliate leaves and stem. Phylogenetic analysis placed St8 in a clade with the Arabidopsis and rice MER3 suggesting that St8 is most likely the orthologous MER3 soybean gene. This study established the utility of Tgm9 in gene identification as well as in forward and reverse genetics studies. PMID:26930200

  20. Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth

    PubMed Central

    Lou, Qian; Liu, Yali; Qi, Yinyan; Jiao, Shuzhen; Tian, Feifei; Jiang, Ling; Wang, Yuejin

    2014-01-01

    Grape hyacinth (Muscari) is an important ornamental bulbous plant with an extraordinary blue colour. Muscari armeniacum, whose flowers can be naturally white, provides an opportunity to unravel the complex metabolic networks underlying certain biochemical traits, especially colour. A blue flower cDNA library of M. armeniacum and a white flower library of M. armeniacum f. album were used for transcriptome sequencing. A total of 89 926 uni-transcripts were isolated, 143 of which could be identified as putative homologues of colour-related genes in other species. Based on a comprehensive analysis relating colour compounds to gene expression profiles, the mechanism of colour biosynthesis was studied in M. armeniacum. Furthermore, a new hypothesis explaining the lack of colour phenotype of the grape hyacinth flower is proposed. Alteration of the substrate competition between flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) may lead to elimination of blue pigmentation while the multishunt from the limited flux in the cyanidin (Cy) synthesis pathway seems to be the most likely reason for the colour change in the white flowers of M. armeniacum. Moreover, mass sequence data obtained by the deep sequencing of M. armeniacum and its white variant provided a platform for future function and molecular biological research on M. armeniacum. PMID:24790110

  1. Chemical and functional characterization of Italian propolis obtained by different harvesting methods.

    PubMed

    Papotti, Giulia; Bertelli, Davide; Bortolotti, Laura; Plessi, Maria

    2012-03-21

    The composition and antioxidant activity of Italian poplar propolis obtained using three harvesting methods and extracted with different solvents were evaluated. Waxes, balsams, and resins contents were determined. Flavones and flavonols, flavanones and dihydroflavonols, and total phenolics were also analyzed. To characterize the phenolic composition, the presence of 15 compounds was verified through HPLC-MS/MS. The antioxidant activity was evaluated through 1,1-diphenyl-2-picrylhydrazyl radical and reducing power assays. The ability of propolis to inhibit lipid oxidation was monitored by analyzing hydroperoxide and TBARS formation in lipids incorporated into an oil-in-water (O/W) emulsion. Acetone shows the highest extraction capacity. Wedge propolis has the highest concentration of active phenolic compounds (TP = 359.1 ± 16.3 GAEs/g; TFF = 5.83 ± 0.42%; TFD = 7.34 ± 1.8%) and seems to be the most promising for obtaining high-value propolis more suitable to prepare high-quality dietary supplements (TBARS = 0.012 ± 0.009 mmol std/g; RP = 0.77 ± 0.07 TEs/g). PMID:22360702

  2. The use of relative quantitative RT-PCR for expression analysis in azalea flower color sports.

    PubMed

    De Keyser, E; De Riek, J; Van Bockstaele, E

    2003-01-01

    The fastest way to create new azalea (Rhododendron simsii hybrids) cultivars is by making use of flower colour sports, which appear spontaneously on azalea plants. Unfortunately, there is still very little known on how bud sport induction occurs. Therefore, genes coding for two key enzymes of the azalea flavonoid biosynthesis pathway, chalcon synthase (chs) and dihydroflavonol 4-reductase (dfr) that were reported before to be apt for modification by the action of bud sporting, were isolated and characterized. The expression of these two flower colour genes in the petals of azalea flowers will be compared between all 'Hellmut Vogel' flower colour sports. To measure the expression levels of both genes, relative quantitative RT-PCR analysis will be worked out on a real-time PCR machine. The expression of housekeeping genes, which is expected to be the same for all sports, will be used to calculate the relative expression level of the two genes of interest. The optimisation of this technique will be discussed. PMID:24757769

  3. Association of Polyphenols from Oranges and Apples with Specific Intestinal Microorganisms in Systemic Lupus Erythematosus Patients

    PubMed Central

    Cuervo, Adriana; Hevia, Arancha; López, Patricia; Suárez, Ana; Sánchez, Borja; Margolles, Abelardo; González, Sonia

    2015-01-01

    Our group has recently shown the existence of a gut microbial dysbiosis in systemic lupus erythematosus (SLE), supporting previous evidence involving intestinal bacteria in the initiation and amplification of autoimmune diseases. While several studies have addressed the use of dietary fibres to modify intestinal microbiota, information about other correlated components, such as polyphenols, is scarce. The aim of this work was to identify dietary components able to influence this altered microbiota in 20 SLE women and 20 age-matched controls. Food intake was recorded by means of a food frequency questionnaire. The intake of fibres was calculated from Marlett tables, and Phenol-Explorer was used for polyphenol consumption. Results showed positive associations between flavone intake and Blautia, flavanones and Lactobacillus, and dihydrochalcones and Bifidobacterium in the SLE group. Regarding the controls, dihydroflavonols were directly associated with Faecalibacterium, whereas flavonol intake was inversely associated with Bifidobacterium. From the food sources of these polyphenols related to microbiota, orange intake was directly associated with Lactobacillus and apple with Bifidobacterium in SLE, whilst red wine was the best contributor to Faecalibacterium variation. The association between common foods and particular microbial genera, reported to be decreased in SLE, could be of great importance for these patients. PMID:25690419

  4. [Effect of bagging with different colors on the fruit coloration of 'yunhongli No.2' pear].

    PubMed

    Ma, Ce; Xiao, Chang-Cheng; Hu, Hong-Ju; Huang, Xiao-San; Zhang, Shao-Ling; Wu, Jun

    2014-03-01

    The present study was conducted to reveal the effect of bags with different colors on the fruit coloration of 'Yunhongli No. 2'. The differences in fruit skin color, chlorophyll, flavonoids, total phenol, anthocyanin contents and the activities of related enzymes involved in anthocyanin synthesis among different bagging treatments were evaluated. The results showed that dark treatment at the fruit development stage was beneficial to skin coloration after bag removing. After removing bags, the anthocyanin content in the treatment of natural light was highest and the red coloration of the fruit skin were best, followed by the treatment of white bags. The different bagging treatments significantly affected the contents of chlorophyll, flavonoids, total phenol, anthocyanin in the fruit skin, thereby affected the skin coloration. The activities of related enzymes for anthocyanin synthesis showed significant differences among the different bagging treatments. The correlation analysis suggested that the anthocyanin content was significantly positively related with the activities of dihydroflavonol 4-reductase (DFR) and UDP-glucose flavonoid-3-O-glucosyltransf-erase (UFGT), however, it had no significant correlation with the activity of phenylalanin ammo-nialyase (PAL). PMID:24984501

  5. Effect of BTH on anthocyanin content and activities of related enzymes in Strawberry after harvest.

    PubMed

    Cao, Shifeng; Hu, Zhichao; Zheng, Yonghua; Lu, Binhong

    2010-05-12

    The effect of benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) at 0.2 g L(-1) on anthocyanin content and the enzymes involved in its metabolism such as glucose-6-phosphate dehydrogenase (G6PDH), shikimate dehydrogenase (SKDH), tyrosine ammonia lyase (TAL), phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate/coenzyme A ligase (4-CL), and dihydroflavonol 4-reductase (DFR) in strawberry (Fragaria x ananassa Duch.) fruit was investigated in this study. The result showed that BTH treatment gave higher levels of anthocyanin in strawberries during 10 days of storage at 1 degrees C. Meanwhile, the treatment also increased the activities of G6PDH, SKDH, TAL, PAL, C4H, and DFR. These results indicated that the increase in anthocyanin content by BTH might result from the activation of its related enzymes. These data are the first evidence that BTH induces enzyme activities related to anthocyanin metabolism in strawberry fruit after harvest. PMID:20377227

  6. Induction of Anthocyanin Accumulation by Cytokinins in Arabidopsis thaliana.

    PubMed Central

    Deikman, J.; Hammer, P. E.

    1995-01-01

    Arabidopsis thaliana plants treated with exogenous cytokinins accumulate anthocyanin pigments. We have characterized this response because it is potentially useful as a genetic marker for cytokinin responsiveness. Levels of mRNAs for four genes of the anthocyanin biosynthesis pathway, phenylalanine ammonia lyase 1 (PAL1), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) were shown to increase coordinately in response to benzyladenine (BA). However, nuclear run-on transcription experiments suggested that although CHS and DFR are controlled by BA at the transcriptional level, PAL1 and CHI are controlled by BA posttranscriptionally. CHS mRNA levels increased within 2 h of BA spray application, and peaked by 3 h. Levels of PAL1 mRNA did not increase within 6 h of BA spray. We also showed that PAL1, CHS, CHI, and DFR mRNA levels fluctuate during a 24-h period and appear to be controlled by a circadian clock. The relation between cytokinin regulation and light regulation of CHS gene transcription is discussed. PMID:12228453

  7. Redirection of anthocyanin synthesis in Osteospermum hybrida by a two-enzyme manipulation strategy.

    PubMed

    Seitz, Christian; Vitten, Matthias; Steinbach, Peter; Hartl, Sabrina; Hirsche, Jörg; Rathje, Wiebke; Treutter, Dieter; Forkmann, Gert

    2007-03-01

    Modern biotechnology has developed powerful tools for genetic engineering and flower colours are an excellent object to study possibilities and limitations of engineering strategies. Osteospermum hybrida became a popular ornamental plant within the last 20 years. Many cultivars display rose to lilac flower colours mainly based on delphinidin-derived anthocyanins. The predominant synthesis of delphinidin derivatives is referred to a strong endogenous flavonoid 3',5'-hydroxylase (F3'5'H) activity. Furthermore, since dihydroflavonol 4-reductase (DFR) of Osteospermum does not convert dihydrokaempferol (DHK) to leucopelargonidin, synthesis of pelargonidin-based anthocyanins is naturally not realised. In order to redirect anthocyanin biosynthesis in Osteospermum towards pelargonidin derivatives, we introduced cDNAs coding for DFRs which efficiently convert DHK to LPg. But neither the expression of Gerbera hybrida DFR nor of Fragaria x ananassa DFR - the latter is characterised by an unusual high substrate preference for DHK - altered anthocyanin composition in flowers of transgenic plants. However, chemical inhibition of F3'5'H activity in ray florets of dfr transgenic plants resulted in the accumulation of pelargonidin derivatives. Accordingly, retransformation of a transgenic plant expressing Gerbera DFR with a construct for RNAi-mediated suppression of F3'5'H activity resulted in double transgenic plants accumulating predominantly pelargonidin derivatives in flowers. PMID:17286993

  8. Prenylated flavonoids from the stems and leaves of Desmodium caudatum and evaluation of their inhibitory activity against the film-forming growth of Zygosaccharomyces rouxii F51.

    PubMed

    Sasaki, Hisako; Shibata, Hirofumi; Imabayashi, Kiyoshi; Takaishi, Yoshihisa; Kashiwada, Yoshiki

    2014-07-01

    In order to provide scientific evidence for the relationship between the traditional usage, stems and leaves of Desmodium caudatum being used for protecting miso from spoilage, and its Japanese name (miso-naoshi), phytochemical study on the stems and leaves of this plant was carried out. Seven new prenylated flavonoids (1-3, 15-18), together with 19 known compounds (4-14, 19-26), were isolated, and the structures of new compounds were elucidated by extensive spectroscopic analyses. The minimum inhibitory concentrations (MICs) of 28 flavonoids, including 17 compounds (1, 2, 4, 5, 7-14, 20-22, 24, 25) isolated in this study and 11 flavonoids (27-37) previously isolated from the roots of this plant, against the film-forming yeast of Zygosaccharomyces rouxii F51 were determined. Fifteen compounds (2, 4, 5, 11, 12, 14, 21, 22, 25, 27, 28, 32-35) inhibited the film-forming growth of Z. rouxii F51 (MIC values, 7.8-62.5 μg/mL), among which 2",2"-dimethylpyran-(5",6":7,8)-5,2'-dihydroxy-4'-methoxy-(2R,3R)-dihydroflavonol (11) demonstrated potent inhibitory activity with an MIC value of 7.8 μg/mL. PMID:24956381

  9. Growth inhibition in Chinese cabbage (Brassica rapa var. chinensis) growth exposed to di-n-butyl phthalate.

    PubMed

    Liao, Chien-Sen; Yen, Jui-Hung; Wang, Yei-Shung

    2009-04-30

    The toxicity and effects of di-n-butyl phthalate (DBP), an endocrine disruptor, on the growth of Chinese cabbage (Brassica rapa var. chinensis) were studied. Etiolation occurred on leaves of Chinese cabbage plant treated with 50mg/L of DBP for 42 d. DBP even below 1mg/L had a significant effect on the concentration of chlorophyll in Chinese cabbage and the biomass showed a severe decrease under treatment with more than 30 mg/L of DBP. At a concentration below 1mg/L of DBP, no significant difference in accumulation was found, but treatments with concentration exceeding 10, 30, 50 and 100mg/L all resulted in significant accumulation of DBP. Six protein spots extracted from leaf tissue of DBP-treated Chinese cabbage displaying a differential expression are shown in 2-DE maps. According to proteome level studies, three protein spots were found to increase and were identified, respectively, as acyl-[acyl-carrier-protein] desaturase (acyl-ACP desaturase), root phototropism protein 3 (RPT3) and ferredoxin-nitrite reductase (Fd-NiR). The other three protein spots were found to decrease and were identified respectively as dihydroflavonol-4-reductase (DFR), aminoacyl-tRNA synthetase (aaRS) and ATP synthase subunit beta. The key finding is that the other closely related plant, Bok choy (Brassica rapa subsp. chinensis), the subspecies of Chinese cabbage, respond differently to the same chemicals. PMID:18678443

  10. Studies of intestinal permeability of 36 flavonoids using Caco-2 cell monolayer model.

    PubMed

    Tian, Xiao-Juan; Yang, Xiu-Wei; Yang, Xiaoda; Wang, Kui

    2009-02-01

    To investigate the structure-permeability relationship of dietary/nutriceutic flavonoids, the transepithelial transport and cellular uptake of 36 flavonoids (including flavones, flavonols, dihydroflavones, dihydroflavonols, isoflavones, chalcones, flavanes, flavanols, methylated and glycosidic derivatives) were investigated using the Caco-2 cell monolayer. The apparent permeability coefficients (P(app)) of the flavonoids were calculated from bilateral transport assays in the Transwell system with flavonoid determination using a high performance liquid chromatography (HPLC) coupled with a UV detector. The most flavonoids exhibited concentration-independent P(app) values and a ratio of 0.5-1.8 for P(app AB to BL)/P(app BL to AB), suggesting passive diffusion pathways. However, certain flavonoids e.g. morin and some flavonoid glycosides may involve the efflux mechanisms. For isoflavones, flavones, and dihydroflavones, the oil/water partition coefficients (additionally modified by the number and position of the three hydroxyl groups) was the key determinant for Caco-2 cell permeation. However, the permeability of flavonols is more complex with their structure possibly related to their high rate of cell accumulation. Overall, the parental skeleton structure, the number and position of free hydroxyl groups, accumulation and efflux in Caco-2 cell play the key roles in the transport of flavonoids across Caco-2 cell monolayer. PMID:18848870

  11. Short-term UV-B exposure induces metabolic and anatomical changes in peel of harvested lemons contributing in fruit protection against green mold.

    PubMed

    Ruiz, V E; Interdonato, R; Cerioni, L; Albornoz, P; Ramallo, J; Prado, F E; Hilal, M; Rapisarda, V A

    2016-06-01

    UV-B radiation (UVBR) is a small fraction of the solar spectrum from 280 to 315nm. UVBR produces photomorphogenic acclimation responses in plants, modulating their cellular structure and physiology. Here, changes in the peel of harvested lemons after short time exposure to UVBR were analyzed and its potential effects against fungal infection were studied. In the flavedo, UVBR treatment induced variations in the respiratory profiles and increased the phenolic compound contents. Final products of the flavonoid pathway (flavones, flavonols and anthocyanins) increased more markedly than their precursors (flavanones and dihydroflavonols). The increased accumulation of soluble phenolics in the flavedo of treated lemons is associated with the high antioxidant activity found in the flavedo of these samples. Supporting the biochemical determinations, anatomical observations showed abundant intravacuolar deposits of phenolic compounds and an increase in the cell wall thickness in UVBR-treated samples. Metabolic and anatomical modifications associated to UVBR improved natural defenses against Penicillium digitatum, the causal agent of green mold disease. Our results suggest that mature postharvest lemons exposed to the artificial radiation showed phenotypic plasticity, allowing an acclimation response to UVBR which confers fruit resistance to pathogens. Thus, combination of UVBR with other treatments could represent an important improvement to control postharvest diseases on citrus. PMID:27017432

  12. Early Steps in Proanthocyanidin Biosynthesis in the Model Legume Medicago truncatula1[W][OA

    PubMed Central

    Pang, Yongzhen; Peel, Gregory J.; Wright, Elane; Wang, Zengyu; Dixon, Richard A.

    2007-01-01

    Oligomeric proanthocyanidins (PAs) composed primarily of epicatechin units accumulate in the seed coats of the model legume Medicago truncatula, reaching maximal levels at around 20 d after pollination. Genes encoding the single Medicago anthocyanidin synthase (ANS; EC 1.14.11.19) and leucoanthocyanidin reductase (LAR; EC 1.17.1.3) were cloned and the corresponding enzymes functionally identified. Recombinant MtANS converted leucocyanidin to cyanidin, and, more efficiently, dihydroquercetin to the flavonol quercetin. Levels of transcripts encoding dihydroflavonol reductase, ANS, and anthocyanidin reductase (ANR), the enzyme responsible for conversion of anthocyanidin to (−)-epicatechin, paralleled the accumulation of PAs in developing seeds, whereas LAR transcripts appeared to be more transiently expressed. LAR, ANS, and ANR proteins were localized to the cytosol in transfected tobacco (Nicotiana tabacum) leaves. Antisense down-regulation of ANS in M. truncatula resulted in reduced anthocyanin and PA levels, but had no impact on flavonol levels. Transgenic tobacco plants constitutively overexpressing MtLAR showed reduced anthocyanin content, but no catechin or increased levels of PAs were detected either in leaves or in flowers. Our results confirm previously ascribed in vivo functions for ANS and ANR. However, the apparent lack of catechin in M. truncatula PAs, the poor correlation between LAR expression and PA accumulation, and the lack of production of catechin monomers or oligomers in transgenic plants overexpressing MtLAR question the role of MtLAR in PA biosynthesis in Medicago. PMID:17885080

  13. Secondary metabolites and phenylpropanoid pathway enzymes as influenced under supplemental ultraviolet-B radiation in Withania somnifera Dunal, an indigenous medicinal plant.

    PubMed

    Takshak, Swabha; Agrawal, S B

    2014-11-01

    The present study aims to investigate the effects of supplemental ultraviolet B (3.6 kJ m(-2)day(-1) above ambient) radiation on secondary metabolites and phenylpropanoid pathway enzymes of Withania somnifera under field conditions at 40, 70, and 100 days after transplantation. Secondary metabolites' (alkaloids, anthocyanins, carotenoids, flavonoids, lignin, phytosterols, saponins, and tannins) concentrations were analysed at the end of the treatments. Activities of phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone-flavanone isomerase (CHI), and dihydroflavonol reductase (DFR) were also determined. In treated plants, secondary metabolite-concentrations generally increased (higher concentrations being recorded in roots compared to leaves). Anomalies were recorded for lycopene in roots and phytosterols in leaves (all sampling ages); β-carotene declined in leaves at third sampling age. s-UV-B-treated plants depicted decrease in withanolide A content with concomitant increase in withaferin A (two major alkaloids analysed by HPLC) compared to their respective controls. Phenylpropanoid pathway enzyme-activities increased in leaves and roots under s-UV-B treatment, the latter showing greater increase. The study concludes that s-UV-B is a potent factor in increasing the concentrations of secondary metabolites and their biosynthetic pathway enzymes in W. somnifera. PMID:25226342

  14. Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: Augmentation of secondary metabolites and antioxidants.

    PubMed

    Takshak, Swabha; Agrawal, S B

    2015-12-01

    Supplementary ultraviolet-B (ambient+3.6  kJ m(-2) day(-1)) induced changes on morphological, physiological, and biochemical characteristics (specifically the defence strategies: UV-B protective compounds and antioxidants) of Coleus forskohlii were investigated under field conditions at 30, 60, and 90 days after transplantation. Levels of secondary metabolites increased under s-UV-B stress; flavonoids and phenolics (primary UV-B screening agents) were recorded to be higher in leaves which are directly exposed to s-UV-B. This was also verified by enhanced activities of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase (PAL), cinnamyl alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone-flavanone isomerase (CHI), and dihydroflavonol reductase (DFR). Antioxidants, both enzymatic (ascorbate peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide dismutase) and non-enzymatic (ascorbic acid and α-tocopherol) also increased in the treated organs of the test plant, higher contents being recorded in roots except for ascorbic acid. On the contrary, protein and chlorophyll content (directly implicated in regulating plant growth and development) declined under s-UV-B. These alterations in plant biochemistry led the plant to compromise on its photosynthate allocation towards growth and biomass production as evidenced by a reduction in its height and biomass. The study concludes that s-UV-B is a potent stimulating factor in increasing the concentrations of defense compounds and antioxidants in C. forskohlii to optimize its performance under stress. PMID:26461242

  15. Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

    PubMed Central

    Kubasek, WL; Shirley, BW; McKillop, A; Goodman, HM; Briggs, W; Ausubel, FM

    1992-01-01

    Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor. PMID:12297632

  16. Arabidopsis CAPRICE (MYB) and GLABRA3 (bHLH) Control Tomato (Solanum lycopersicum) Anthocyanin Biosynthesis

    PubMed Central

    Wada, Takuji; Kunihiro, Asuka; Tominaga-Wada, Rumi

    2014-01-01

    In Arabidopsis thaliana the MYB transcription factor CAPRICE (CPC) and the bHLH transcription factor GLABRA3 (GL3) are central regulators of root-hair differentiation and trichome initiation. By transforming the orthologous tomato genes SlTRY (CPC) and SlGL3 (GL3) into Arabidopsis, we demonstrated that these genes influence epidermal cell differentiation in Arabidopsis, suggesting that tomato and Arabidopsis partially use similar transcription factors for epidermal cell differentiation. CPC and GL3 are also known to be involved in anthocyanin biosynthesis. After transformation into tomato, 35S::CPC inhibited anthocyanin accumulation, whereas GL3::GL3 enhanced anthocyanin accumulation. Real-time reverse transcription PCR analyses showed that the expression of anthocyanin biosynthetic genes including Phe-ammonia lyase (PAL), the flavonoid pathway genes chalcone synthase (CHS), dihydroflavonol reductase (DFR), and anthocyanidin synthase (ANS) were repressed in 35S::CPC tomato. In contrast, the expression levels of PAL, CHS, DFR, and ANS were significantly higher in GL3::GL3 tomato compared with control plants. These results suggest that CPC and GL3 also influence anthocyanin pigment synthesis in tomato. PMID:25268379

  17. High-Throughput Sequencing and De Novo Assembly of Red and Green Forms of the Perilla frutescens var. crispa Transcriptome

    PubMed Central

    Fukushima, Atsushi; Nakamura, Michimi; Suzuki, Hideyuki; Saito, Kazuki; Yamazaki, Mami

    2015-01-01

    Perilla frutescens var. crispa (Labiatae) has two chemo-varietal forms, i.e. red and green forms of perilla, that differ in the production of anthocyanins. To facilitate molecular biological and biochemical studies in perilla-specialized metabolism we used Illumina RNA-sequencing technology in our comprehensive comparison of the transcriptome map of the leaves of red and green forms of perilla. Sequencing generated over 1.2 billion short reads with an average length of 101 nt. De novo transcriptome assembly yielded 47,788 and 47,840 unigenes in the red and green forms of perilla plants, respectively. Comparison of the assembled unigenes and existing perilla cDNA sequences showed highly reliable alignment. All unigenes were annotated with gene ontology (GO) and Enzyme Commission numbers and entered into the Kyoto Encyclopedia of Genes and Genomes. We identified 68 differentially expressed genes (DEGs) in red and green forms of perilla. GO enrichment analysis of the DEGs showed that genes involved in the anthocyanin metabolic process were enriched. Differential expression analysis revealed that the transcript level of anthocyanin biosynthetic unigenes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase was significantly higher in red perilla, while the transcript level of unigenes encoding limonene synthase was significantly higher in green perilla. Our data serve as a basis for future research on perilla bio-engineering and provide a shortcut for the characterization of new functional genes in P. frutescens. PMID:26070213

  18. Characterization of two tartary buckwheat R2R3-MYB transcription factors and their regulation of proanthocyanidin biosynthesis.

    PubMed

    Bai, Yue-Chen; Li, Cheng-Lei; Zhang, Jin-Wen; Li, Shuang-Jiang; Luo, Xiao-Peng; Yao, Hui-Peng; Chen, Hui; Zhao, Hai-Xia; Park, Sang-Un; Wu, Qi

    2014-11-01

    Tartary buckwheat (Fagopyrum tataricum Gaertn.) contains high concentrations of flavonoids. The flavonoids are mainly represented by rutin, anthocyanins and proanthocyanins in tartary buckwheat. R2R3-type MYB transcription factors (TFs) play key roles in the transcriptional regulation of the flavonoid biosynthetic pathway. In this study, two TF genes, FtMYB1 and FtMYB2, were isolated from F. tataricum and characterized. The results of bioinformatic analysis indicated that the putative FtMYB1 and FtMYB2 proteins belonged to the R2R3-MYB family and displayed a high degree of similarity with TaMYB14 and AtMYB123/TT2. In vitro and in vivo evidence both showed the two proteins were located in the nucleus and exhibited transcriptional activation activities. During florescence, both FtMYB1 and FtMYB2 were more highly expressed in the flowers than any other organ. The overexpression of FtMYB1 and FtMYB2 significantly enhanced the accumulation of proanthocyanidins (PAs) and showed a strong effect on the target genes' expression in Nicotiana tabacum. The expression of dihydroflavonol-4-reductase (DFR) was upregulated to 5.6-fold higher than that of control, and the expression level was lower for flavonol synthase (FLS). To our knowledge, this is the first functional characterization of two MYB TFs from F. tataricum that control the PA pathway. PMID:24730512

  19. Association of polyphenols from oranges and apples with specific intestinal microorganisms in systemic lupus erythematosus patients.

    PubMed

    Cuervo, Adriana; Hevia, Arancha; López, Patricia; Suárez, Ana; Sánchez, Borja; Margolles, Abelardo; González, Sonia

    2015-01-01

    Our group has recently shown the existence of a gut microbial dysbiosis in systemic lupus erythematosus (SLE), supporting previous evidence involving intestinal bacteria in the initiation and amplification of autoimmune diseases. While several studies have addressed the use of dietary fibres to modify intestinal microbiota, information about other correlated components, such as polyphenols, is scarce. The aim of this work was to identify dietary components able to influence this altered microbiota in 20 SLE women and 20 age-matched controls. Food intake was recorded by means of a food frequency questionnaire. The intake of fibres was calculated from Marlett tables, and Phenol-Explorer was used for polyphenol consumption. Results showed positive associations between flavone intake and Blautia, flavanones and Lactobacillus, and dihydrochalcones and Bifidobacterium in the SLE group. Regarding the controls, dihydroflavonols were directly associated with Faecalibacterium, whereas flavonol intake was inversely associated with Bifidobacterium. From the food sources of these polyphenols related to microbiota, orange intake was directly associated with Lactobacillus and apple with Bifidobacterium in SLE, whilst red wine was the best contributor to Faecalibacterium variation. The association between common foods and particular microbial genera, reported to be decreased in SLE, could be of great importance for these patients. PMID:25690419

  20. The Birth of a Black Rice Gene and Its Local Spread by Introgression

    PubMed Central

    Oikawa, Tetsuo; Maeda, Hiroaki; Oguchi, Taichi; Yamaguchi, Takuya; Tanabe, Noriko; Ebana, Kaworu; Yano, Masahiro; Izawa, Takeshi

    2015-01-01

    The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice. PMID:26362607

  1. Discovery of Highly Sweet Compounds from Natural Sources

    NASA Astrophysics Data System (ADS)

    Kinghorn, A. Douglas; Kennelly, Edward J.

    1995-08-01

    Sucrose, the most widely used sweetener globally, is of plant origin. In addition, a number of other plant constituents are employed as dietary sucrose substitutes in one or more countries, including the diterpenoid, stevioside, the triterpenoid, glycyrrhizin, and the protein, thaumatin. Accordingly, there has been much interest in discovering further examples of potently sweet compounds of natural origin, for potential use in foods, beverages, and medicines. Approximately 75 plant-derived compounds are presently known, mainly representative of the flavonoid, proanthocyandin, protein, steroidal saponin, and terpenoid chemotypes. In our program directed towards the elucidation of further highly sweet molecules from plants, candidate sweet-tasting plants for laboratory investigation are obtained from ethnobotanical observations in the field or in the existing literature. Examples of novel sweet-tasting compounds obtained so far are the sesquiterpenoids, hernandulcin and 4beta-hydroxyhemandulcin; the triterpenoids, abrusosides A-D; a semi-synthetic dihydroflavonol based on the naturally occurring substance, dihydroquercetin 3-acetate; and the proanthocyanidin, selligueain A. Natural product sweeteners may be of potential commercial use per se, and can be used for synthetic modification to produce improved sweeteners, and can also be of value scientifically to aid in the better understanding of structure-sweetness relationships.

  2. The transcriptome of Populus in elevated CO2 reveals increased anthocyanin biosynthesis during delayed autumnal senescence

    SciTech Connect

    Tallis, M.J.; Rogers, A.; Lin, Y.; Zhang, J.; Street, N. R.; Miglietta, F.; Karnosky, D. F.; Angelis, P. D.; Calfapietra, C.; Taylor, G.

    2010-03-01

    The delay in autumnal senescence that has occurred in recent decades has been linked to rising temperatures. Here, we suggest that increasing atmospheric CO{sub 2} may partly account for delayed autumnal senescence and for the first time, through transcriptome analysis, identify gene expression changes associated with this delay. Using a plantation of Populus x euramericana grown in elevated [CO{sub 2}] (e[CO{sub 2}]) with free-air CO{sub 2} enrichment (FACE) technology, we investigated the molecular and biochemical basis of this response. A Populus cDNA microarray was used to identify genes representing multiple biochemical pathways influenced by e[CO{sub 2}] during senescence. Gene expression changes were confirmed through real-time quantitative PCR, and leaf biochemical assays. Pathways for secondary metabolism and glycolysis were significantly up-regulated by e[CO{sub 2}] during senescence, in particular, those related to anthocyanin biosynthesis. Expressed sequence tags (ESTs) representing the two most significantly up-regulated transcripts in e[CO{sub 2}], LDOX (leucoanthocyanidin dioxgenase) and DFR (dihydroflavonol reductase), gave (e[CO{sub 2}]/ambient CO{sub 2} (a[CO{sub 2}])) expression ratios of 39.6 and 19.3, respectively. We showed that in e[CO{sub 2}] there was increased autumnal leaf sugar accumulation and up-regulation of genes determining anthocyanin biosynthesis which, we propose, prolongs leaf longevity during natural autumnal senescence.

  3. Expression Profiling of the Maize Flavonoid Pathway Genes Controlled by Estradiol-Inducible Transcription Factors CRC and P

    PubMed Central

    Bruce, Wesley; Folkerts, Otto; Garnaat, Carl; Crasta, Oswald; Roth, Brad; Bowen, Ben

    2000-01-01

    To determine the scope of gene expression controlled by the maize transcription factors C1/R and P, which are responsible for activating flavonoid synthesis, we used GeneCalling, an open-ended, gel-based, mRNA-profiling technology, to analyze cell suspension lines of the maize inbred Black Mexican Sweet (BMS) that harbored estradiol-inducible versions of these factors. BMS cells were transformed with a continually expressed estrogen receptor/maize C1 activator domain fusion gene (ER–C1) and either a fusion of C1 and R (CRC), P, or luciferase genes regulated by a promoter containing four repeats of an estrogen receptor binding site. Increasing amounts of luciferase activity, anthocyanins, and flavan-4-ols were detected in the respective cell lines after the addition of estradiol. The expression of both known and novel genes was detected simultaneously in these BMS lines by profiling the mRNA isolated from replicate samples at 0, 6, and 24 hr after estradiol treatment. Numerous cDNA fragments were identified that showed a twofold or greater difference in abundance at 6 and 24 hr than at 0 hr. The cDNA fragments from the known flavonoid genes, except chalcone isomerase (chi1), were induced in the CRC-expressing line after hormone induction, whereas only the chalcone synthase (c2) and flavanone/dihydroflavonol reductase (a1) genes were induced in the P-expressing line, as was expected. Many novel cDNA fragments were also induced or repressed by lines expressing CRC alone, P alone, or both transcription factors in unique temporal patterns. The temporal differences and the evidence of repression indicate a more diverse set of regulatory controls by CRC or P than originally expected. GeneCalling analysis was successful in detecting members of complex metabolic pathways and uncovering novel genes that were either coincidentally regulated or directly involved in such pathways. PMID:10634908

  4. Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin

    PubMed Central

    Han, Yuepeng; Vimolmangkang, Sornkanok; Soria-Guerra, Ruth Elena; Korban, Schuyler S.

    2012-01-01

    Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated. PMID:22238451

  5. Novel drought-inducible genes in the highly drought-tolerant cowpea: cloning of cDNAs and analysis of the expression of the corresponding genes.

    PubMed

    Iuchi, S; Yamaguchi-Shinozaki, K; Urao, T; Terao, T; Shinozaki, K

    1996-12-01

    Ten cDNAs of genes that were induced by dehydration stress were cloned by differential screening from the highly drought-tolerant legume, cowpea (Vigna unguiculata), a major crop in West Africa. The clones were collectively named CPRD (cowpea clones responsive to dehydration). Northern blot analysis revealed that nine of the CPRD genes were induced by dehydration stress, but the timing of induction of mRNA synthesis varied among the CPRD genes. We analyzed the effects of other environmental stresses on the expression of the CPRD8, CPRD14 and CPRD22 genes, and we found that these genes were strongly induced by high-salinity stress but not by cold or heat stress. Drought-stressed cowpea plants accumulated abscisic acid (ABA) to a level that was 160 times higher than that in unstressed plants. The CPRD8 and CPRD22 genes were induced to a significant extent by the application of exogenous ABA but the CPRD14 gene was not. These results indicate the existence of at least two signal-transduction pathways between the detection of water stress and the expression of CPRD genes in cowpea. Sequence analysis of CPRD8 and CPRD22 cDNAs revealed that they encoded putative proteins that were related to old yellow enzyme and group 2 LEA proteins, respectively. The protein encoded by CPRD14 exhibited sequence homology to dihydroflavonol-4-reductase (DFR) and vestitone reductase (VR). Old yellow enzyme, DFR and VR have not been identified as drought-inducible proteins in other plants, whereas LEA genes have been well characterized as drought-inducible genes. The various gene products might function to protect cells from environmental stress. PMID:9032963

  6. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species.

    PubMed

    Yuan, Yao-Wu; Rebocho, Alexandra B; Sagawa, Janelle M; Stanley, Lauren E; Bradshaw, Harvey D

    2016-03-01

    Flower color patterns have long served as a model for developmental genetics because pigment phenotypes are visually striking, yet generally not required for plant viability, facilitating the genetic analysis of color and pattern mutants. The evolution of novel flower colors and patterns has played a key role in the adaptive radiation of flowering plants via their specialized interactions with different pollinator guilds (e.g., bees, butterflies, birds), motivating the search for allelic differences affecting flower color pattern in closely related plant species with different pollinators. We have identified LIGHT AREAS1 (LAR1), encoding an R2R3-MYB transcription factor, as the causal gene underlying the spatial pattern variation of floral anthocyanin pigmentation between two sister species of monkeyflower: the bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated Mimulus cardinalis. We demonstrated that LAR1 positively regulates FLAVONOL SYNTHASE (FLS), essentially eliminating anthocyanin biosynthesis in the white region (i.e., light areas) around the corolla throat of M. lewisii flowers by diverting dihydroflavonol into flavonol biosynthesis from the anthocyanin pigment pathway. FLS is preferentially expressed in the light areas of the M. lewisii flower, thus prepatterning the corolla. LAR1 expression in M. cardinalis flowers is much lower than in M. lewisii, explaining the unpatterned phenotype and recessive inheritance of the M. cardinalis allele. Furthermore, our gene-expression analysis and genetic mapping results suggest that cis-regulatory change at the LAR1 gene played a critical role in the evolution of different pigmentation patterns between the two species. PMID:26884205

  7. Differential expression of MYB gene (OgMYB1) determines color patterning in floral tissue of Oncidium Gower Ramsey.

    PubMed

    Chiou, Chung-Yi; Yeh, Kai-Wun

    2008-03-01

    The yellow coloration pattern in Oncidium floral lip associated with red sepal and petal tissues is an ideal model to study coordinate regulation of anthocyanin synthesis. In this study, chromatography analysis revealed that the red coloration in floral tissues was composed of malvidin-3-O-galactoside, peonidin-3-O-glucoside, delphinidin-3-O-glucoside and cyanidin-3-O-glucoside compounds. By contrary, these pigments were not detected in yellow lip tissue. Four key genes involved in anthocyanin biosynthetic pathway, i.e. chalcone synthase (OgCHS), chalcone isomerase (OgCHI), dihydroflavonol 4-reductase (OgDFR) and anthocyanidin synthase (OgANS) were isolated and their expression patterns were characterized. Northern blot analysis confirmed that although they are active during floral development, OgCHI and OgDFR genes are specifically down-regulated in yellow lip tissue. Bombardment with OgCHI and OgDFR genes into lip tissue driven by a flower-specific promoter, Pchrc (chromoplast-specific carotenoid-associated gene), demonstrated that transient expression of these two genes resulted in anthocyanin production in yellow lip. Further analysis of a R2R3 MYB transcription factor, OgMYB1, revealed that although it is actively expressed during floral development, it is not expressed in yellow lip tissue. Transient expression of OgMYB1 in lip tissues by bombardment can also induce formation of red pigments through the activation of OgCHI and OgDFR transcription. These results demonstrate that differential expression of OgMYB1 is critical to determine the color pattern of floral organ in Oncidium Gower Ramsey. PMID:18161007

  8. New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.

    PubMed

    Pérez-Díaz, J Ricardo; Pérez-Díaz, Jorge; Madrid-Espinoza, José; González-Villanueva, Enrique; Moreno, Yerko; Ruiz-Lara, Simón

    2016-01-01

    In grapevine, anthocyanins and proanthocyanidins are the main flavonoids in berries, which are associated to organoleptic properties in red wine such as color and astringency. Flavonoid pathway is specifically regulated at transcriptional level and several R2R3-MYB proteins have shown to act as positive regulators. However, some members of this family have shown to repress the flavonoid biosynthesis. In this work, we present the characterization of VvMYB4-like gene, which encodes a putative transcriptional factor highly expressed in the skin of berries at the pre veraison stage in grapevine. Its over-expression in tobacco resulted in the loss of pigmentation in flowers due a decrease in anthocyanin accumulation. Severity in anthocyanin suppression observed in petals could be associated with the expression level of the VvMYB4-like transgene. Expression analysis of flavonoid structural genes revealed the strong down-regulation of the flavonoid-related genes anthocyanidin synthase (ANS) and dihydroflavonol reductase (DFR) genes and also the reduction of the anthocyanin-related gene UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), which was dependent of the transgene expression. In addition, expression of VvMYB4-like in the model plant Arabidopsis showed similar results, with the higher down-regulation observed in the AtDFR and AtLDOX genes. These results suggest that VvMYB4-like may play an important role in regulation of anthocyanin biosynthesis in grapevine acting as a transcriptional repressor of flavonoid structural genes. PMID:26497001

  9. A large-scale genetic screen in Arabidopsis to identify genes involved in pollen exine production.

    PubMed

    Dobritsa, Anna A; Geanconteri, Aliza; Shrestha, Jay; Carlson, Ann; Kooyers, Nicholas; Coerper, Daniel; Urbanczyk-Wochniak, Ewa; Bench, Bennie J; Sumner, Lloyd W; Swanson, Robert; Preuss, Daphne

    2011-10-01

    Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production. PMID:21849515

  10. Identification and characterisation of CYP75A31, a new flavonoid 3'5'-hydroxylase, isolated from Solanum lycopersicum

    PubMed Central

    2010-01-01

    Background Understanding the regulation of the flavonoid pathway is important for maximising the nutritional value of crop plants and possibly enhancing their resistance towards pathogens. The flavonoid 3'5'-hydroxylase (F3'5'H) enzyme functions at an important branch point between flavonol and anthocyanin synthesis, as is evident from studies in petunia (Petunia hybrida), and potato (Solanum tuberosum). The present work involves the identification and characterisation of a F3'5'H gene from tomato (Solanum lycopersicum), and the examination of its putative role in flavonoid metabolism. Results The cloned and sequenced tomato F3'5'H gene was named CYP75A31. The gene was inserted into the pYeDP60 expression vector and the corresponding protein produced in yeast for functional characterisation. Several putative substrates for F3'5'H were tested in vitro using enzyme assays on microsome preparations. The results showed that two hydroxylation steps occurred. Expression of the CYP75A31 gene was also tested in vivo, in various parts of the vegetative tomato plant, along with other key genes of the flavonoid pathway using real-time PCR. A clear response to nitrogen depletion was shown for CYP75A31 and all other genes tested. The content of rutin and kaempferol-3-rutinoside was found to increase as a response to nitrogen depletion in most parts of the plant, however the growth conditions used in this study did not lead to accumulation of anthocyanins. Conclusions CYP75A31 (NCBI accession number GQ904194), encodes a flavonoid 3'5'-hydroxylase, which accepts flavones, flavanones, dihydroflavonols and flavonols as substrates. The expression of the CYP75A31 gene was found to increase in response to nitrogen deprivation, in accordance with other genes in the phenylpropanoid pathway, as expected for a gene involved in flavonoid metabolism. PMID:20128892

  11. Elucidation of Molecular Identity of the W3 Locus and Its Implication in Determination of Flower Colors in Soybean

    PubMed Central

    Park, Gyu Tae; Sundaramoorthy, Jagadeesh; Lee, Jeong-Dong; Kim, Jeong Hoe; Seo, Hak Soo; Song, Jong Tae

    2015-01-01

    The wide range of flower colors in soybean is controlled by six independent loci (W1, W2, W3, W4, Wm, and Wp). Among these loci, mutations in the W3 locus under the w4 allelic background (i.e., w3w4) produce near-white flowers, while the W3w4 genotype produces purple throat flowers. Although a gene encoding dihydroflavonol 4-reductase, DFR1, has been known to be closely associated with the W3 locus, its molecular identity has not yet been characterized. In the present study, we aimed to determine whether DFR1 is responsible for allelic variations in the W3 locus. On the basis of the sequence of a DFR probe, Glyma.14G072700 was identified as a candidate gene for DFR1, and nucleotide sequences of Glyma.14G072700 from cultivars with previously validated genotypes for the W3 locus were determined. As a result, a number of nucleotide polymorphisms, mainly single-base substitutions, between both coding and 5′-upstream region sequences of the W3 and w3 alleles were identified. Among them, an indel of 311-bp in the 5′-upstream region was noteworthy, since the Glyma.14G072700 in all the w3 alleles examined contained the indel, whereas that in all the W3 alleles did not; the former was barely expressed, but the latter was well expressed. These results suggest that Glyma.14G072700 is likely to correspond to DFR1 for the W3 locus and that its expression patterns may lead to allelic color phenotypes of W3 and w3 alleles under the w4 allelic background. PMID:26555888

  12. Aqueous Extracts from Tunisian Diplotaxis: Phenol Content, Antioxidant and Anti-Acetylcholinesterase Activities, and Impact of Exposure to Simulated Gastrointestinal Fluids.

    PubMed

    Bahloul, Nada; Bellili, Sana; Aazza, Smail; Chérif, Ameur; Faleiro, Maria Leonor; Antunes, Maria Dulce; Miguel, Maria Graça; Mnif, Wissem

    2016-01-01

    Antioxidants have been considered essential for preventing cell damage by scavenging deleterious free radicals. The consumption of antioxidant-rich plants is associated with a reduced risk of some chronic diseases. This study evaluates the antioxidant and acetylcholinesterase inhibition activities of aqueous extracts obtained from different parts of Diplotaxis simplex and Diplotaxis harra from Tunisia. The study also aimed to investigate the action of simulated gastrointestinal juice on antioxidant activities of both extracts. The total phenolic, flavone and flavonol, and flavanone and dihydroflavonol contents were determined by Folin-Ciocalteau, aluminum chloride and 2,4-dinitrophenylhydrazine colorimetric methods, respectively. The metal ion chelating activity, acetylcholinesterase inhibition capacity, and free radical scavenging potential of the extracts towards ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), hydroxyl, superoxide and nitric oxide were also evaluated. The action of simulated gastro-intestinal fluids on the flavone and flavonol content and total antioxidant activity of the flower extracts was surveyed. Extracts from the seeds and flowers of D. simplex and D. harra displayed the highest amounts of phenols (2691.7 and 2694.5 mg Caffeic Acid Equivalent (CAE)/100 mg; 3433.4 and 2647.2 mg CAE/100 mg, respectively) and flavonols/flavones (2144.4 and 2061.1 mg Rutin Equivalent (RE)/100 g; 1922.6 and 1461.1 mg RE/100 g, respectively). The flower and seed extracts exhibited the highest rates of antioxidant and acetylcholinesterase inhibition activities. A decrease in the flavonoid content and antioxidant activity was observed after extract exposure to simulated saliva. Antioxidant and acetylcholinesterase inhibition activities were noted to depend on plant species and plant parts. In vitro gastrointestinal digestion is useful in assessing the bio-accessibility of compounds with biological activities from

  13. In silico study for diversing the molecular pathway of pigment formation: an alternative to manual coloring in cotton fibers

    PubMed Central

    Ahad, Ammara; Ahmad, Aftab; Din, Salah ud; Rao, Abdul Q.; Shahid, Ahmad A.; Husnain, Tayyab

    2015-01-01

    Diversity of colors in flowers and fruits is largely due to anthocyanin pigments. The flavonoid/anthocyanin pathway has been most extensively studied. Dihydroflavonol 4-reductase (DFR) is a vital enzyme of the flavonoid pathway which displays major impact on the formation of anthocyanins, flavan 3-ols and flavonols. The substrate specificity of the DFR was found to play a crucial role in determination of type of anthocyanidins. Altering the flavonoid/anthocyanin pathway through genetic engineering to develop color of our own choice is an exciting subject of future research. In the present study, comparison among four DFR genes (Gossypium hirsutum, Iris × hollandica, Ang. DFRI and DFRII), sequence alignment for homology as well as protein modeling and docking is demonstrated. Estimation of catalytic sites, prediction of substrate preference and protein docking were the key features of this article. For specific substrate uptake, a proline rich region and positions 12 plus 26 along with other positions emphasizing the 26-amino acid residue region (132–157) was tested. Results showed that proline rich region position 12, 26, and 132–157 plays an important role in selective attachment of DFRs with respective substrates. Further, “Expasy ProtParam tool” results showed that Iris × hollandica DFR amino acids (Asn 9: Asp 23) are favorable for reducing DHQ and DHM thus accumulating delphinidin, while Gossypium hirsutum DFR has (Asn 13: Asp 21) hypothesized to consume DHK. Protein docking data showed that amino acid residues in above mentioned positions were just involved in attachment of DFR with substrate and had no role in specific substrate uptake. Advanced bioinformatics analysis has revealed that all above mentioned positions have role in substrate attachment. For substrate specificity, other residues region is involved. It will help in color manipulations in different plant species. PMID:26442064

  14. A Large-Scale Genetic Screen in Arabidopsis to Identify Genes Involved in Pollen Exine Production1[C][W][OA

    PubMed Central

    Dobritsa, Anna A.; Geanconteri, Aliza; Shrestha, Jay; Carlson, Ann; Kooyers, Nicholas; Coerper, Daniel; Urbanczyk-Wochniak, Ewa; Bench, Bennie J.; Sumner, Lloyd W.; Swanson, Robert; Preuss, Daphne

    2011-01-01

    Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production. PMID:21849515

  15. Molecular characterization of a nonautonomous transposable element (dTph1) of petunia.

    PubMed Central

    Gerats, A G; Huits, H; Vrijlandt, E; Maraña, C; Souer, E; Beld, M

    1990-01-01

    An insertion sequence of 283 base pairs has been isolated from the DFR-C gene (dihydroflavonol-4-reductase) of petunia. This insert was found only in a line unstable for the An1 locus (anthocyanin 1, located on chromosome VI) and not in fully pigmented progenitor and revertant lines or in stable white derivative lines. This implies that the An1 locus encodes the DFR-C gene. The unstable An1 system in the line W138 is known to be a two-element system, the autonomous element being located on chromosome I. In the presence of the autonomous element, W138 flowers exhibit a characteristic pattern of red revertant spots and sectors on a white background. In the absence of the autonomous element, the W138 allele gives rise to a stable recessive (white) phenotype. Sequence analysis of progenitor, unstable, and revertant alleles revealed dTph1 to contain perfect terminal inverted repeats of 12 base pairs. In DFR-C, it is flanked by an 8-base pair target site duplication. Sequences homologous to dTph1 are present in at least 50 copies in the line W138. Sequence analysis of An1 revertant alleles indicated that excision, including removal of the target site duplication, is required for reversion to the wild-type phenotype. Derivative stable recessive alleles showed excision of dTph1 and a rearrangement of the target site duplication. dTph1 is the smallest transposable element described to date that is still capable of transposition. The use of dTph1 in tagging experiments and subsequent gene isolation is discussed. PMID:1967052

  16. Site-selected insertional mutagenesis of tomato with maize Ac and Ds elements.

    PubMed

    Cooley, M B; Goldsbrough, A P; Still, D W; Yoder, J I

    1996-08-27

    Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maize Ac or Ds transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-range Ds insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multiple Ac (HiAc) or Ds (HiDs) elements allowed detection of somatic insertions at two single-copy genes, PG (polygalacturonase) and DFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion into PG than others. Insertions in PG and DFR were found throughout the gene regions monitored and, with the exception of one insertion in PG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions in PG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinal PG insertions in tomato. PMID:8804392

  17. Comparative Leaves Transcriptome Analysis Emphasizing on Accumulation of Anthocyanins in Brassica: Molecular Regulation and Potential Interaction with Photosynthesis

    PubMed Central

    Mushtaq, Muhammad A.; Pan, Qi; Chen, Daozong; Zhang, Qinghua; Ge, Xianhong; Li, Zaiyun

    2016-01-01

    The purple leaf pigmentation mainly associated with anthocyanins accumulation is common in Brassica but the mechanisms of its production and its potential physiological functions are poorly understood. Here, we performed the phenotypic, cytological, physiological, and comparative leaves transcriptome analyses of 11 different varieties belonging to five Brassica species with purple or green leaves. We observed that the anthocyanin was accumulated in most of vegetative tissues in all species and also in reproduction organs of B. carinata. Anthocyanin accumulated in different part of purple leaves including adaxial and abaxial epidermal cells as well as palisade and spongy mesophyll cells. Leave transcriptome analysis showed that almost all late biosynthetic genes (LBGs) of anthocyanin, especially Dihydroflavonol 4-Reductase (DFR), Anthocyanidin Synthase (ANS) and Transparent Testa 19 (TT19), were highly up-regulated in all purple leaves. However, only one of transcript factors in anthocyanin biosynthesis pathway, Transparent Testa 8 (TT8), was up regulated along with those genes in all purple leaves, indicating its pivotal role for anthocyanin production in Brassica. Interestingly, with the up-regulation of genes for anthocyanin synthesis, Cytosolic 6-phosphogluconolactonase (PLG5) which involved in the oxidative pentose-phosphate pathway was up-regulated in all purple leaves and three genes FTSH PROTEASE 8 (FTS8), GLYCOLATE OXIDASE 1 (GOX1), and GLUTAMINE SYNTHETASE 1;4 (GLN1;4) related to degradation of photo-damaged proteins in photosystem II and light respiration were down-regulated. These results highlighted the potential physiological functions of anthocyanin accumulation related to photosynthesis which might be of great worth in future. PMID:27047501

  18. Metabolism and pharmacokinetics of 8-hydroxypiperidinylmethyl-baicalein (BA-j) as a novel selective CDK1 inhibitor in monkey.

    PubMed

    Guo, Hong-Min; Sun, Yu-Ming; Zhang, Shi-Xuan; Ju, Xiu-Lan; Xie, Ai-Yun; Li, Jing; Zou, Liang; Sun, Xiao-Dan; Li, Hai-Liang; Zheng, Yang

    2015-12-01

    Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in the cell proliferation process and a new target in the research and development of anti-cancer drugs. 8-Hydroxypiperidinemethyl-baicalein (BA-j) is a Mannich base derivative of baicalein (BA) isolated from Scutellaria baicalensis, as a novel selective CDK1 inhibitor. 12 metabolites of BA-j in the monkey urine were identified by LC-MS-MS and (1)H NMR. The major metabolic pathways of BA-j, by capturing oxygen free radicals ((.)O2(-)) and releasing peroxides (H2O2), are degraded into active intermediate metabolite dihydroflavonol, then into main metabolite M179 by Shiff reaction, second metabolite M264 by sulfation, trace amount of metabolite M559 by glucuronidation UGT1A9, and without metabolism by CYP3A4. The metabolic process of BA-j by regulating intracellular reactive oxygen species (ROS) was related with BA-j selectively inducing apoptosis in cancer cells. Pharmacokinetics of 10mg/kg oral BA-j in monkey by HPLC-UV was best fitted to a two-compartment open model, with t1/2(β) of 4.2h, Cmax 25.4μM at 2h, and Vd 12.6L, meaning the drug distributing widely in body fluids with no special selectivity to certain tissues, and being able to permeate through the blood-brain barrier. The protein binding rate of BA-j was 91.8%. BA-j has excellent druggability for oral administration or injection, and it may be developed into a novel anti-cancer drug as a selective CDK1 inhibitor. PMID:26474673

  19. Antibacterial Activity, Antioxidant Effect and Chemical Composition of Propolis from the Región del Maule, Central Chile.

    PubMed

    Nina, Nélida; Quispe, Cristina; Jiménez-Aspee, Felipe; Theoduloz, Cristina; Feresín, Gabriela Egly; Lima, Beatriz; Leiva, Elba; Schmeda-Hirschmann, Guillermo

    2015-01-01

    Propolis is commercialized in Chile as an antimicrobial agent. It is obtained mainly from central and southern Chile, but is used for the same purposes regardless of its origin. To compare the antimicrobial effect, the total phenolic (TP), the total flavonoid (TF) content and the phenolic composition, 19 samples were collected in the main production centers in the Región del Maule, Chile. Samples were extracted with MeOH and assessed for antimicrobial activity against Gram (+) and Gram (-) bacteria. TP and TF content, antioxidant activity by the DPPH, FRAP and TEAC methods were also determined. Sample composition was assessed by HPLD-DAD-ESI-MS/MS. Differential compounds in the samples were isolated and characterized. The antimicrobial effect of the samples showed MICs ranging from 31.5 to > 1000 µg/mL. Propolis from the central valley was more effective as antibacterial than those from the coastal area or Andean slopes. The samples considered of interest (MIC ≤ 62.5 µg/mL) showed effect on Escherichia coli, Pseudomonas sp., Yersinia enterocolitica and Salmonella enteritidis. Two new diarylheptanoids, a diterpene, the flavonoids pinocembrin and chrysin were isolated and elucidated by spectroscopic and spectrometric means. Some 29 compounds were dereplicated by HPLC-MS and tentatively identified, including nine flavones/flavonol derivatives, one flavanone, eight dihydroflavonols and nine phenyl-propanoids. Propolis from the Región del Maule showed large variation in antimicrobial effect, antioxidant activity and composition. So far the presence of diarylheptanoids in samples from the coastal area of central Chile can be considered as a marker of a new type of propolis. PMID:26457694

  20. Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the Pink allele of the ANS gene in onion (Allium cepa L.).

    PubMed

    Kim, Sunggil; Park, Jee Young; Yang, Tae-Jin

    2015-06-01

    Intact retrotransposon and DNA transposons inserted in a single gene were characterized in onions (Allium cepa) and their transcription and copy numbers were estimated in this study. While analyzing diverse onion germplasm, large insertions in the DFR-A gene encoding dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway were found in two accessions. A 5,070-bp long terminal repeat (LTR) retrotransposon inserted in the active DFR-A (R4) allele was identified from one of the large insertions and designated AcCOPIA1. An intact ORF encoded typical domains of copia-like LTR retrotransposons. However, AcCOPIA1 contained atypical 'TG' and 'TA' dinucleotides at the ends of the LTRs. A 4,615-bp DNA transposon was identified in the other large insertion. This DNA transposon, designated AcCACTA1, contained an ORF coding for a transposase showing homology with the CACTA superfamily transposable elements (TEs). Another 5,073-bp DNA transposon was identified from the DFR-A (TRN) allele. This DNA transposon, designated AchAT1, belonged to the hAT superfamily with short 4-bp terminal inverted repeats (TIRs). Finally, a 6,258-bp non-autonomous DNA transposon, designated AcPINK, was identified in the ANS-p allele encoding anthocyanidin synthase, the next downstream enzyme to DFR in the anthocyanin biosynthesis pathway. AcPINK also possessed very short 3-bp TIRs. Active transcription of AcCOPIA1, AcCACTA1, and AchAT1 was observed through RNA-Seq analysis and RT-PCR. The copy numbers of AcPINK estimated by mapping the genomic DNA reads produced by NextSeq 500 were predominantly high compared with the other TEs. A series of evidence indicated that these TEs might have transposed in these onion genes very recently, providing a stepping stone for elucidation of enormously large-sized onion genome structure. PMID:25515665

  1. Competition between anthocyanin and flavonol biosynthesis produces spatial pattern variation of floral pigments between Mimulus species

    PubMed Central

    Yuan, Yao-Wu; Rebocho, Alexandra B.; Sagawa, Janelle M.; Stanley, Lauren E.; Bradshaw, Harvey D.

    2016-01-01

    Flower color patterns have long served as a model for developmental genetics because pigment phenotypes are visually striking, yet generally not required for plant viability, facilitating the genetic analysis of color and pattern mutants. The evolution of novel flower colors and patterns has played a key role in the adaptive radiation of flowering plants via their specialized interactions with different pollinator guilds (e.g., bees, butterflies, birds), motivating the search for allelic differences affecting flower color pattern in closely related plant species with different pollinators. We have identified LIGHT AREAS1 (LAR1), encoding an R2R3-MYB transcription factor, as the causal gene underlying the spatial pattern variation of floral anthocyanin pigmentation between two sister species of monkeyflower: the bumblebee-pollinated Mimulus lewisii and the hummingbird-pollinated Mimulus cardinalis. We demonstrated that LAR1 positively regulates FLAVONOL SYNTHASE (FLS), essentially eliminating anthocyanin biosynthesis in the white region (i.e., light areas) around the corolla throat of M. lewisii flowers by diverting dihydroflavonol into flavonol biosynthesis from the anthocyanin pigment pathway. FLS is preferentially expressed in the light areas of the M. lewisii flower, thus prepatterning the corolla. LAR1 expression in M. cardinalis flowers is much lower than in M. lewisii, explaining the unpatterned phenotype and recessive inheritance of the M. cardinalis allele. Furthermore, our gene-expression analysis and genetic mapping results suggest that cis-regulatory change at the LAR1 gene played a critical role in the evolution of different pigmentation patterns between the two species. PMID:26884205

  2. Aqueous Extracts from Tunisian Diplotaxis: Phenol Content, Antioxidant and Anti-Acetylcholinesterase Activities, and Impact of Exposure to Simulated Gastrointestinal Fluids

    PubMed Central

    Bahloul, Nada; Bellili, Sana; Aazza, Smail; Chérif, Ameur; Faleiro, Maria Leonor; Antunes, Maria Dulce; Miguel, Maria Graça; Mnif, Wissem

    2016-01-01

    Antioxidants have been considered essential for preventing cell damage by scavenging deleterious free radicals. The consumption of antioxidant-rich plants is associated with a reduced risk of some chronic diseases. This study evaluates the antioxidant and acetylcholinesterase inhibition activities of aqueous extracts obtained from different parts of Diplotaxis simplex and Diplotaxis harra from Tunisia. The study also aimed to investigate the action of simulated gastrointestinal juice on antioxidant activities of both extracts. The total phenolic, flavone and flavonol, and flavanone and dihydroflavonol contents were determined by Folin–Ciocalteau, aluminum chloride and 2,4-dinitrophenylhydrazine colorimetric methods, respectively. The metal ion chelating activity, acetylcholinesterase inhibition capacity, and free radical scavenging potential of the extracts towards ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), hydroxyl, superoxide and nitric oxide were also evaluated. The action of simulated gastro-intestinal fluids on the flavone and flavonol content and total antioxidant activity of the flower extracts was surveyed. Extracts from the seeds and flowers of D. simplex and D. harra displayed the highest amounts of phenols (2691.7 and 2694.5 mg Caffeic Acid Equivalent (CAE)/100 mg; 3433.4 and 2647.2 mg CAE/100 mg, respectively) and flavonols/flavones (2144.4 and 2061.1 mg Rutin Equivalent (RE)/100 g; 1922.6 and 1461.1 mg RE/100 g, respectively). The flower and seed extracts exhibited the highest rates of antioxidant and acetylcholinesterase inhibition activities. A decrease in the flavonoid content and antioxidant activity was observed after extract exposure to simulated saliva. Antioxidant and acetylcholinesterase inhibition activities were noted to depend on plant species and plant parts. In vitro gastrointestinal digestion is useful in assessing the bio-accessibility of compounds with biological activities

  3. Eriosema (Fabaceae) Species Represent a Rich Source of Flavonoids with Interesting Pharmacological Activities.

    PubMed

    Awouafack, Maurice Ducret; Tane, Pierre; Spiteller, Michael; Eloff, Jacobus Nicolaas

    2015-07-01

    Many flavonoids have so far been isolated as main secondary metabolites in plant species of the genus Eriosema (Fabaceae), which contains approximately 160 species. A total of 52 flavonoids including isoflavones, dihydroflavonols, flavonols, flavanones, dihydrochalcones, isoflavanone and their pyrano or glucoside derivatives were isolated and characterized from the five species of this genus investigated to date. Total synthesis and semi-synthesis (acetylation, methylation, hydrogenation, and cyclization) of some isolated flavonoids were reported. Due to several significant pharmacological properties (antimicrobial, cytotoxicity, anti-mycobacterial, antioxidant, antiviral, erectile-dysfunction, vasodilatory and hypoglycemic) of the isolated flavonoids and derivatives, more scientists should be interested in investigating Eriosema species. The present review is the first to document all flavonoids that have been reported from the genus Eriosema to date together with their synthetic and semi-synthetic derivatives, and their pharmacological properties. Dihydrochalcones, which are precursors of other classes of flavonoids, are very rare in natural sources and their isolation from Eriosema species may explain the large number of flavonoids found in this genus. It appears that isoflavone could be a marker for species in this genus. The 83 flavonoids (1-83) documented include 52 isolates, 31 semi-synthetic and 3 totally synthetic derivatives. Data were obtained from Google scholar, Pubmed, Scifinder, Sciencedirect, and Scopus. With 52 different flavonoids isolated from only 5 of the approximately 160 species it shows the remarkable chemical diversity of this genus. This compilation of the biological activities and chemical composition may renew the interest of pharmacologists and phytochemists in this genus. PMID:26411043

  4. Comparative Leaves Transcriptome Analysis Emphasizing on Accumulation of Anthocyanins in Brassica: Molecular Regulation and Potential Interaction with Photosynthesis.

    PubMed

    Mushtaq, Muhammad A; Pan, Qi; Chen, Daozong; Zhang, Qinghua; Ge, Xianhong; Li, Zaiyun

    2016-01-01

    The purple leaf pigmentation mainly associated with anthocyanins accumulation is common in Brassica but the mechanisms of its production and its potential physiological functions are poorly understood. Here, we performed the phenotypic, cytological, physiological, and comparative leaves transcriptome analyses of 11 different varieties belonging to five Brassica species with purple or green leaves. We observed that the anthocyanin was accumulated in most of vegetative tissues in all species and also in reproduction organs of B. carinata. Anthocyanin accumulated in different part of purple leaves including adaxial and abaxial epidermal cells as well as palisade and spongy mesophyll cells. Leave transcriptome analysis showed that almost all late biosynthetic genes (LBGs) of anthocyanin, especially Dihydroflavonol 4-Reductase (DFR), Anthocyanidin Synthase (ANS) and Transparent Testa 19 (TT19), were highly up-regulated in all purple leaves. However, only one of transcript factors in anthocyanin biosynthesis pathway, Transparent Testa 8 (TT8), was up regulated along with those genes in all purple leaves, indicating its pivotal role for anthocyanin production in Brassica. Interestingly, with the up-regulation of genes for anthocyanin synthesis, Cytosolic 6-phosphogluconolactonase (PLG5) which involved in the oxidative pentose-phosphate pathway was up-regulated in all purple leaves and three genes FTSH PROTEASE 8 (FTS8), GLYCOLATE OXIDASE 1 (GOX1), and GLUTAMINE SYNTHETASE 1;4 (GLN1;4) related to degradation of photo-damaged proteins in photosystem II and light respiration were down-regulated. These results highlighted the potential physiological functions of anthocyanin accumulation related to photosynthesis which might be of great worth in future. PMID:27047501

  5. Identification and Molecular Analysis of Four New Alleles at the W1 Locus Associated with Flower Color in Soybean

    PubMed Central

    Sundaramoorthy, Jagadeesh; Park, Gyu Tae; Chang, Jeong Ho; Lee, Jeong-Dong; Kim, Jeong Hoe; Seo, Hak Soo; Chung, Gyuhwa; Song, Jong Tae

    2016-01-01

    In soybean, flavonoid 3′5′-hydroxylase (F3′5′H) and dihydroflavonol-4-reductase (DFR) play a crucial role in the production of anthocyanin pigments. Loss-of-function of the W1 locus, which encodes the former, or W3 and W4, which encode the latter, always produces white flowers. In this study, we searched for new genetic components responsible for the production of white flowers in soybean and isolated four white-flowered mutant lines, i.e., two Glycine soja accessions (CW12700 and CW13381) and two EMS-induced mutants of Glycine max (PE1837 and PE636). F3′5′H expression in CW12700, PE1837, and PE636 was normal, whereas that in CW13381 was aberrant and missing the third exon. Sequence analysis of F3′5′H of CW13381 revealed the presence of an indel (~90-bp AT-repeat) in the second intron. In addition, the F3′5′H of CW12700, PE1837, and PE636 harbored unique single-nucleotide substitutions. The single nucleotide polymorphisms resulted in substitutions of amino acid residues located in or near the SRS4 domain of F3′5′H, which is essential for substrate recognition. 3D structure modeling of F3′5′H indicated that the substitutions could interfere with an interaction between the substrate and heme group and compromise the conformation of the active site of F3′5′H. Recombination analysis revealed a tight correlation between all of the mutant alleles at the W1 locus and white flower color. On the basis of the characterization of the new mutant alleles, we discussed the biological implications of F3′5′H and DFR in the determination of flower colors in soybean. PMID:27442124

  6. Cloning, Overexpression, and Mutagenesis of the Sporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

    PubMed Central

    Kita, Keiko; Fukura, Takanobu; Nakase, Koh-Ichi; Okamoto, Kenji; Yanase, Hideshi; Kataoka, Michihiko; Shimizu, Sakayu

    1999-01-01

    We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced in Escherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G19-X-X-G22-X-X-A25, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G19→A and G22→A mutant enzymes by 4-COBE did not occur. The A25→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH. PMID:10583966

  7. Deficiency of CRTAP in non-lethal recessive osteogenesis imperfecta reduces collagen deposition into matrix

    PubMed Central

    Valli, M; Barnes, AM; Gallanti, A; Cabral, WA; Viglio, S; Weis, MA; Makareeva, E; Eyre, D; Leikin, S; Antoniazzi, F; Marini, JC; Mottes, M

    2013-01-01

    Deficiency of any component of the ER-resident collagen prolyl 3-hydroxylation complex causes recessive osteogenesis imperfecta (OI). The complex modifies the α1(I)Pro986 residue and contains cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 (P3H1) and cyclophilin B (CyPB). Fibroblasts normally secrete about 10% of CRTAP. Most CRTAP mutations cause a null allele and lethal type VII OI. We identified a 7-year-old Egyptian boy with non-lethal type VII OI and investigated the effects of his null CRTAP mutation on collagen biochemistry, the prolyl 3-hydroxylation complex, and collagen in extracellular matrix. The proband is homozygous for an insertion/deletion in CRTAP (c.118_133del16insTACCC). His dermal fibroblasts synthesize fully overmodified type I collagen, and 3-hydroxylate only 5% of α1(I)Pro986. CRTAP transcripts are 10% of control. CRTAP protein is absent from proband cells, with residual P3H1 and normal CyPB levels. Dermal collagen fibril diameters are significantly increased. By immunofluorescence of long-term cultures, we identified a severe deficiency (10–15% of control) of collagen deposited in extracellular matrix, with disorganization of the minimal fibrillar network. Quantitative pulse-chase experiments corroborate deficiency of matrix deposition, rather than increased matrix turnover. We conclude that defects of extracellular matrix, as well as intracellular defects in collagen modification, contribute to the pathology of type VII OI. PMID:21955071

  8. Increase in 4-coumaryl alcohol units during lignification in alfalfa (Medicago sativa) alters the extractability and molecular weight of lignin.

    PubMed

    Ziebell, Angela; Gracom, Kristen; Katahira, Rui; Chen, Fang; Pu, Yunqiao; Ragauskas, Art; Dixon, Richard A; Davis, Mark

    2010-12-10

    The lignin content of biomass can impact the ease and cost of biomass processing. Lignin reduction through breeding and genetic modification therefore has potential to reduce costs in biomass-processing industries (e.g. pulp and paper, forage, and lignocellulosic ethanol). We investigated compositional changes in two low-lignin alfalfa (Medicago sativa) lines with antisense down-regulation of p-coumarate 3-hydroxylase (C3H) or hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase (HCT). We investigated whether the difference in reactivity during lignification of 4-coumaryl alcohol (H) monomers versus the naturally dominant sinapyl alcohol and coniferyl alcohol lignin monomers alters the lignin structure. Sequential base extraction readily reduced the H monomer content of the transgenic lines, leaving a residual lignin greatly enriched in H subunits; the extraction profile highlighted the difference between the control and transgenic lines. Gel permeation chromatography of isolated ball-milled lignin indicated significant changes in the weight average molecular weight distribution of the control versus transgenic lines (CTR1a, 6000; C3H4a, 5500; C3H9a, 4000; and HCT30a, 4000). PMID:20921228

  9. Kdo hydroxylase is an inner core assembly enzyme in the Ko-containing lipopolysaccharide biosynthesis.

    PubMed

    Chung, Hak Suk; Yang, Eun Gyeong; Hwang, Dohyeon; Lee, Ji Eun; Guan, Ziqiang; Raetz, Christian R H

    2014-09-26

    The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d-glycero-d-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe(2+)/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVAin vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis. PMID:25204504

  10. Overview on the biotechnological production of L-DOPA.

    PubMed

    Min, Kyoungseon; Park, Kyungmoon; Park, Don-Hee; Yoo, Young Je

    2015-01-01

    L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process. PMID:25432672

  11. Collagen prolyl3-hydroxylation: a major role for a minor post-translational modification?

    PubMed Central

    Hudson, David M.; Eyre, David R.

    2014-01-01

    Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap−/− mouse was missing 3-hydroxyproline in type I collagen α-chains. This finding led to the discovery mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzymes have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent. PMID:23772978

  12. Biochemical Characterization of a Spearmint Mutant That Resembles Peppermint in Monoterpene Content 1

    PubMed Central

    Croteau, Rodney; Karp, Frank; Wagschal, Kurt C.; Satterwhite, D. Michael; Hyatt, David C.; Skotland, Calvin B.

    1991-01-01

    A radiation-induced mutant of Scotch spearmint (Mentha × gracilis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the C6-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and C6-oxygenated families of monoterpenes from the common precursor (−)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (−)-limonene hydroxylase; the C6-hydroxylase producing (−)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (−)-trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the α,β-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3- and C6-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha. PMID:16668250

  13. Proteomic and Epigenetic Analyses of Lotus (Nelumbo nucifera) Petals Between Red and White cultivars.

    PubMed

    Deng, Jiao; Fu, Ziyang; Chen, Sha; Damaris, Rebecca Njeri; Wang, Kun; Li, Tingting; Yang, Pingfang

    2015-08-01

    Lotus is a vital aquatic ornamental plant with different flower colors. To explore the flower coloration mechanism in lotus, the constituents and contents of pigments in two lotus cultivars with red and white flowers were analyzed. Although flavones and flavonols were detected in both cultivars, anthocyanins could only be detected in the red cultivar. A comparative proteomics analysis on the flower petals between these two cultivars was conducted. A total of 88 differentially expressed proteins were identified with 36 more abundant and 52 less abundant in the red than in the white cultivar. Among them, four enzymes involved in the anthocyanin pathway were identified, i.e. flavanone 3-hydroxylase, anthocyanidin synthase, anthocyanidin 3-O-glucosyltransferase and glutathione S-transferase. Analysis of the expression patterns of anthocyanin biosynthetic genes indicated that the anthocyanindin synthase (ANS) gene might be the critical gene determining anthocyanin biosynthesis and accumulation in lotus flower. Further analysis showed that different methylation intensities on the promoter sequence of the ANS gene might result in the different flower coloration in the red and white cultivar. This study provides new insights into the mechanism of flower coloration in lotus, and may be helpful in its breeding and germplasm enhancement. PMID:26019267

  14. Prolyl Hydroxylase PHD3 Activates Oxygen-dependent Protein Aggregation

    PubMed Central

    Rantanen, Krista; Pursiheimo, Juha; Högel, Heidi; Himanen, Virpi; Metzen, Eric

    2008-01-01

    The HIF prolyl hydroxylases (PHDs/EGLNs) are central regulators of the molecular responses to oxygen availability. One isoform, PHD3, is expressed in response to hypoxia and causes apoptosis in oxygenated conditions in neural cells. Here we show that PHD3 forms subcellular aggregates in an oxygen-dependent manner. The aggregation of PHD3 was seen under normoxia and was strongly reduced under hypoxia or by the inactivation of the PHD3 hydroxylase activity. The PHD3 aggregates were dependent on microtubular integrity and contained components of the 26S proteasome, chaperones, and ubiquitin, thus demonstrating features that are characteristic for aggresome-like structures. Forced expression of the active PHD3 induced the aggregation of proteasomal components and activated apoptosis under normoxia in HeLa cells. The apoptosis was seen in cells prone to PHD3 aggregation and the PHD3 aggregation preceded apoptosis. The data demonstrates the cellular oxygen sensor PHD3 as a regulator of protein aggregation in response to varying oxygen availability. PMID:18337469

  15. Transcriptome Analysis Reveals the Mechanism Underlying the Production of a High Quantity of Chlorogenic Acid in Young Leaves of Lonicera macranthoides Hand.-Mazz

    PubMed Central

    Chen, Zexiong; Tang, Ning; You, Yuming; Lan, Jianbin; Liu, Yiqing; Li, Zhengguo

    2015-01-01

    Lonicera macranthoides Hand.-Mazz (L. macranthoides) is a medicinal herb that is widely distributed in southern China. The biosynthetic and metabolic pathways for a core secondary metabolite in L. macranthoides, chlorogenic acid (CGA), have been elucidated in many species. However, the mechanisms of CGA biosynthesis and the related gene regulatory network in L. macranthoides are still not well understood. In this study, CGA content was quantified by high performance liquid chromatography (HPLC), and CGA levels differed significantly among three tissues; specifically, the CGA content in young leaves (YL) was greater than that in young stems (YS), which was greater than that in mature flowers (MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads (average length 90 bp) and 76,453 unigenes (average length 703 bp). A total of 3,767 unigenes were involved in biosynthesis pathways of secondary metabolites. Of these unigenes, 80 were possibly related to CGA biosynthesis. Furthermore, differentially expressed genes (DEGs) were screened in different tissues including YL, MF and YS. In these tissues, 24 DEGs were found to be associated with CGA biosynthesis, including six phenylalanine ammonia lyase (PAL) genes, six 4-coumarate coenzyme A ligase (4CL) genes, four cinnamate 4-Hydroxylase (C4H) genes, seven hydroxycinnamoyl transferase/hydroxycinnamoyl-CoA quinate transferase HCT/HQT genes and one coumarate 3-hydroxylase (C3H) gene.These results further the understanding of CGA biosynthesis and the related regulatory network in L. macranthoides. PMID:26381882

  16. Metabolic engineering of Escherichia coli to produce zeaxanthin.

    PubMed

    Li, Xi-Ran; Tian, Gui-Qiao; Shen, Hong-Jie; Liu, Jian-Zhong

    2015-04-01

    Zeaxanthin is a high-value carotenoid that is used in nutraceuticals, cosmetics, food, and animal feed industries. Zeaxanthin is chemically synthesized or purified from microorganisms as a natural product; however, increasing demand requires development of alternative sources such as heterologous biosynthesis by recombinant bacteria. For this purpose, we molecularly engineered Escherichia coli to optimize the synthesis of zeaxanthin from lycopene using fusion protein-mediated substrate channeling as well as by the introduction of tunable intergenic regions. The tunable intergenic regions approach was more efficient compared with protein fusion for coordinating expression of lycopene β-cyclase gene crtY and β-carotene 3-hydroxylase gene crtZ. The influence of the substrate channeling effect suggests that the reaction catalyzed by CrtZ is the rate-limiting step in zeaxanthin biosynthesis. Then Pantoea ananatis, Pantoea agglomerans and Haematococcus pluvialis crtZ were compared. Because P. ananatis crtZ is superior to that of P. agglomerans or H. pluvialis for zeaxanthin production, we used it to generate a recombinant strain of E. coli BETA-1 containing pZSPBA-2(P37-crtZPAN) that produced higher amounts of zeaxanthin (11.95 ± 0.21 mg/g dry cell weight) than other engineered E. coli strains described in the literature. PMID:25533633

  17. Carotenoid β-ring hydroxylase and ketolase from marine bacteria-promiscuous enzymes for synthesizing functional xanthophylls.

    PubMed

    Misawa, Norihiko

    2011-01-01

    Marine bacteria belonging to genera Paracoccus and Brevundimonas of the α-Proteobacteria class can produce C₄₀-type dicyclic carotenoids containing two β-end groups (β rings) that are modified with keto and hydroxyl groups. These bacteria produce astaxanthin, adonixanthin, and their derivatives, which are ketolated by carotenoid β-ring 4(4')-ketolase (4(4')-oxygenase; CrtW) and hydroxylated by carotenoid β-ring 3(3')-hydroxylase (CrtZ). In addition, the genus Brevundimonas possesses a gene for carotenoid β-ring 2(2')-hydroxylase (CrtG). This review focuses on these carotenoid β-ring-modifying enzymes that are promiscuous for carotenoid substrates, and pathway engineering for the production of xanthophylls (oxygen-containing carotenoids) in Escherichia coli, using these enzyme genes. Such pathway engineering researches are performed towards efficient production not only of commercially important xanthophylls such as astaxanthin, but also of xanthophylls minor in nature (e.g., β-ring(s)-2(2')-hydroxylated carotenoids). PMID:21673887

  18. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity

    PubMed Central

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the β-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of β-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  19. Genetic engineering of novel bluer-colored chrysanthemums produced by accumulation of delphinidin-based anthocyanins.

    PubMed

    Noda, Naonobu; Aida, Ryutaro; Kishimoto, Sanae; Ishiguro, Kanako; Fukuchi-Mizutani, Masako; Tanaka, Yoshikazu; Ohmiya, Akemi

    2013-10-01

    Chrysanthemums (Chrysanthemum morifolium Ramat.) have no purple-, violet- or blue-flowered cultivars because they lack delphinidin-based anthocyanins. This deficiency is due to the absence of the flavonoid 3',5'-hydroxylase gene (F3'5'H), which encodes the key enzyme for delphinidin biosynthesis. In F3'5'H-transformed chrysanthemums, unpredictable and unstable expression levels have hampered successful production of delphinidin and reduced desired changes in flower color. With the aim of achieving delphinidin production in chrysanthemum petals, we found that anthocyanin biosynthetic gene promoters combined with a translational enhancer increased expression of some F3'5'H genes and accompanying delphinidin-based anthocyanin accumulation in transgenic chrysanthemums. Dramatic accumulation of delphinidin (up to 95%) was achieved by simple overexpression of Campanula F3'5'H controlled by a petal-specific flavanone 3-hydroxylase promoter from chrysanthemum combined with the 5'-untranslated region of the alcohol dehydrogenase gene as a translational enhancer. The flower colors of transgenic lines producing delphinidin-based anthocyanins changed from a red-purple to a purple-violet hue in the Royal Horticultural Society Colour Charts. This result represents a promising step toward molecular breeding of blue chrysanthemums. PMID:23926063

  20. Production of curcuminoids from tyrosine by a metabolically engineered Escherichia coli using caffeic acid as an intermediate.

    PubMed

    Rodrigues, Joana L; Araújo, Rafael G; Prather, Kristala L J; Kluskens, Leon D; Rodrigues, Ligia R

    2015-04-01

    Curcuminoids are phenylpropanoids with high pharmaceutical potential. Herein, we report an engineered artificial pathway in Escherichia coli to produce natural curcuminoids through caffeic acid. Arabidopsis thaliana 4-coumaroyl-CoA ligase and Curcuma longa diketide-CoA synthase (DCS) and curcumin synthase (CURS1) were used to produce curcuminoids and 70 mg/L of curcumin was obtained from ferulic acid. Bisdemethoxycurcumin and demethoxycurcumin were also produced, but in lower concentrations, by feeding p-coumaric acid or a mixture of p-coumaric acid and ferulic acid, respectively. Additionally, curcuminoids were produced from tyrosine through the caffeic acid pathway. To produce caffeic acid, tyrosine ammonia lyase from Rhodotorula glutinis and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis were used. Caffeoyl-CoA 3-O-methyltransferase from Medicago sativa was used to convert caffeoyl-CoA to feruloyl-CoA. Using caffeic acid, p-coumaric acid or tyrosine as a substrate, 3.9, 0.3, and 0.2 mg/L of curcumin were produced, respectively. This is the first time DCS and CURS1 were used in vivo to produce curcuminoids and that curcumin was produced by feeding tyrosine. We have shown that curcumin can be produced using a pathway involvoing caffeic acid. This alternative pathway represents a step forward in the heterologous production of curcumin using E. coli. PMID:25641677

  1. Heterologous production of caffeic acid from tyrosine in Escherichia coli.

    PubMed

    Rodrigues, J L; Araújo, R G; Prather, K L J; Kluskens, L D; Rodrigues, L R

    2015-04-01

    Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62mM (472mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56mM (280mg/L) and 1mM (180mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids. PMID:25765308

  2. Alterations of cytochrome P450-dependent monooxygenase activities in Eriocheir japonicus in response to water pollution.

    PubMed Central

    Ishizuka, M; Hoshi, H; Minamoto, N; Masuda, M; Kazusaka, A; Fujita, S

    1996-01-01

    Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the field studies, crabs collected from the river with the highest concentration of PAHs exhibited the highest levels of CYP, the highest activities of benzo[a]pyrene 3-hydroxylase, imipramine 2-hydroxylase, bunitrolol 4-hydroxylase, ethoxycoumarin O-deethylase, and the ability to metabolically activate benzo[a]pyrene, but erythromycin N-demethylase activity was not induced. The correlation between PAH levels and drug-metabolizing enzyme activities in female crabs were not as marked as in male crabs. The levels and activities of CYP did not appear to reflect the concentrations of organochlorines and polychlorinated biphenyl congeners (PCBs) studied in the fat of crab hepatopancreas. Images Figure 1. Figure 2. A Figure 2. B Figure 2. C Figure 3. Figure 4. A Figure 4. B Figure 5. A Figure 5. B Figure 5. C Figure 5. D Figure 5. E Figure 5. F Figure 6. PMID:8841764

  3. Enhancing cytochrome P450-mediated conversions in P. pastoris through RAD52 over-expression and optimizing the cultivation conditions.

    PubMed

    Wriessnegger, Tamara; Moser, Sandra; Emmerstorfer-Augustin, Anita; Leitner, Erich; Müller, Monika; Kaluzna, Iwona; Schürmann, Martin; Mink, Daniel; Pichler, Harald

    2016-04-01

    Cytochrome P450 enzymes (CYPs) play an essential role in the biosynthesis of various natural compounds by catalyzing regio- and stereospecific hydroxylation reactions. Thus, CYP activities are of great interest in the production of fine chemicals, pharmaceutical compounds or flavors and fragrances. Industrial applicability of CYPs has driven extensive research efforts aimed at improving the performance of these enzymes to generate robust biocatalysts. Recently, our group has identified CYP-mediated hydroxylation of (+)-valencene as a major bottleneck in the biosynthesis of trans-nootkatol and (+)-nootkatone in Pichia pastoris. In the current study, we aimed at enhancing CYP-mediated (+)-valencene hydroxylation by over-expressing target genes identified through transcriptome analysis in P. pastoris. Strikingly, over-expression of the DNA repair and recombination gene RAD52 had a distinctly positive effect on trans-nootkatol formation. Combining RAD52 over-expression with optimization of whole-cell biotransformation conditions, i.e. optimized media composition and cultivation at higher pH value, enhanced trans-nootkatol production 5-fold compared to the initial strain and condition. These engineering approaches appear to be generally applicable for enhanced hydroxylation of hydrophobic compounds in P. pastoris as confirmed here for two additional membrane-attached CYPs, namely the limonene-3-hydroxylase from Mentha piperita and the human CYP2D6. PMID:26898115

  4. Interaction of moderate UV-B exposure and temperature on the formation of structurally different flavonol glycosides and hydroxycinnamic acid derivatives in kale (Brassica oleracea var. sabellica).

    PubMed

    Neugart, Susanne; Fiol, Michaela; Schreiner, Monika; Rohn, Sascha; Zrenner, Rita; Kroh, Lothar W; Krumbein, Angelika

    2014-05-01

    Kale has a high number of structurally different flavonol glycosides and hydroxycinnamic acid derivatives. In this study we investigated the interaction of moderate UV-B radiation and temperature on these compounds. Kale plants were grown at daily mean temperatures of 5 or 15 °C and were exposed to five subsequent daily doses (each 0.25 kJ m(-2) d(-1)) of moderate UV-B radiation at 1 d intervals. Of 20 phenolic compounds, 11 were influenced by an interaction of UV-B radiation and temperature, e.g., monoacylated quercetin glycosides. Concomitantly, enhanced mRNA expression of flavonol 3'- hydroxylase showed an interaction of UV-B and temperature, highest at 0.75 kJ m(-2) and 15 °C. Kaempferol glycosides responded diversely and dependent on, e.g., the hydroxycinnamic acid residue. Compounds containing a catechol structure seem to be favored in the response to UV-B. Taken together, subsequent exposure to moderate UV-B radiation is a successful tool for enhancing the flavonoid profile of plants, and temperature should be considered. PMID:24655223

  5. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification

    NASA Technical Reports Server (NTRS)

    Blee, Kristopher A.; Choi, Joon W.; O'Connell, Ann P.; Schuch, Wolfgang; Lewis, Norman G.; Bolwell, G. Paul

    2003-01-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

  6. Construction of transplastomic lettuce (Lactuca sativa) dominantly producing astaxanthin fatty acid esters and detailed chemical analysis of generated carotenoids.

    PubMed

    Harada, Hisashi; Maoka, Takashi; Osawa, Ayako; Hattan, Jun-Ichiro; Kanamoto, Hirosuke; Shindo, Kazutoshi; Otomatsu, Toshihiko; Misawa, Norihiko

    2014-04-01

    The plastid genome of lettuce (Lactuca sativa L.) cv. Berkeley was site-specifically modified with the addition of three transgenes, which encoded β,β-carotenoid 3,3'-hydroxylase (CrtZ) and β,β-carotenoid 4,4'-ketolase (4,4'-oxygenase; CrtW) from a marine bacterium Brevundimonas sp. strain SD212, and isopentenyl diphosphate isomerase from a marine bacterium Paracoccus sp. strain N81106. Constructed transplastomic lettuce plants were able to grow on soil at a growth rate similar to that of non-transformed lettuce cv. Berkeley and generate flowers and seeds. The germination ratio of the lettuce transformants (T0) (98.8%) was higher than that of non-transformed lettuce (93.1 %). The transplastomic lettuce (T1) leaves produced the astaxanthin fatty acid (myristate or palmitate) diester (49.2% of total carotenoids), astaxanthin monoester (18.2%), and the free forms of astaxanthin (10.0%) and the other ketocarotenoids (17.5%), which indicated that artificial ketocarotenoids corresponded to 94.9% of total carotenoids (230 μg/g fresh weight). Native carotenoids were there lactucaxanthin (3.8%) and lutein (1.3 %) only. This is the first report to structurally identify the astaxanthin esters biosynthesized in transgenic or transplastomic plants producing astaxanthin. The singlet oxygen-quenching activity of the total carotenoids extracted from the transplastomic leaves was similar to that of astaxanthin (mostly esterified) from the green algae Haematococcus pluvialis. PMID:24287848

  7. The trans-anethole degradation pathway in an Arthrobacter sp.

    PubMed

    Shimoni, Eyal; Baasov, Timor; Ravid, Uzi; Shoham, Yuval

    2002-04-01

    A bacterial strain (TA13) capable of utilizing t-anethole as the sole carbon source was isolated from soil. The strain was identified as Arthrobacter aurescens based on its 16 S rRNA gene sequence. Key steps of the degradation pathway of t-anethole were identified by the use of t-anethole-blocked mutants and specific inducible enzymatic activities. In addition to t-anethole, strain TA13 is capable of utilizing anisic acid, anisaldehyde, and anisic alcohol as the sole carbon source. t-Anethole-blocked mutants were obtained following mutagenesis and penicillin enrichment. Some of these blocked mutants, accumulated in the presence of t-anethole quantitative amounts of t-anethole-diol, anisic acid, and 4,6-dicarboxy-2-pyrone and traces of anisic alcohol and anisaldehyde. Enzymatic activities induced by t-anethole included: 4-methoxybenzoate O-demethylase, p-hydroxybenzoate 3-hydroxylase, and protocatechuate-4,5-dioxygenase. These findings indicate that t-anethole is metabolized to protocatechuic acid through t-anethole-diol, anisaldehyde, anisic acid, and p-hydroxybenzoic acid. The protocatechuic acid is then cleaved by protocatechuate-4,5-dioxygenase to yield 2-hydroxy-4-carboxy muconate-semialdehyde. Results from inducible uptake ability and enzymatic assays indicate that at least three regulatory units are involved in the t-anethole degradation pathway. These findings provide new routes for environmental friendly production processes of valuable aromatic chemicals via bioconversion of phenylpropenoids. PMID:11805095

  8. Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

    PubMed

    Whitehill, Justin G A; Henderson, Hannah; Schuetz, Mathias; Skyba, Oleksandr; Yuen, Macaire Man Saint; King, John; Samuels, A Lacey; Mansfield, Shawn D; Bohlmann, Jörg

    2016-08-01

    Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. PMID:26474726

  9. Anion channels and the stimulation of anthocyanin accumulation by blue light in Arabidopsis seedlings

    NASA Technical Reports Server (NTRS)

    Noh, B.; Spalding, E. P.; Evans, M. H. (Principal Investigator)

    1998-01-01

    Activation of anion channels by blue light begins within seconds of irradiation in seedlings and is related to the ensuing growth inhibition. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) is a potent, selective, and reversible blocker of these anion channels in Arabidopsis thaliana. Here we show that 20 microM NPPB blocked 72% of the blue-light-induced accumulation of anthocyanin pigments in seedlings. Feeding biosynthetic intermediates to wild-type and tt5 seedlings provided evidence that NPPB prevented blue light from up-regulating one or more steps between and including phenylalanine ammonia lyase and chalcone isomerase. NPPB was found to have no significant effect on the blue-light-induced increase in transcript levels of PAL1, CHS, CHI, or DFR, which are genes that encode anthocyanin-biosynthetic enzymes. Immunoblots revealed that NPPB also did not inhibit the accumulation of the chalcone synthase, chalcone isomerase, or flavanone-3-hydroxylase proteins. This is in contrast to the reduced anthocyanin accumulation displayed by a mutant lacking the HY4 blue-light receptor, as hy4 displayed reduced expression of the above enzymes. Taken together, the data indicate that blue light acting through HY4 leads to an increase in the amount of biosynthetic enzymes but blue light must also act through a separate, anion-channel-dependent system to create a fully functional biosynthetic pathway.

  10. Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells.

    PubMed

    Ikezawa, Nobuhiro; Tanaka, Masaru; Nagayoshi, Masanori; Shinkyo, Raku; Sakaki, Toshiyuki; Inouye, Kuniyo; Sato, Fumihiko

    2003-10-01

    Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of (S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3'-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes. PMID:12732624

  11. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  12. A lignin-specific peroxidase in tobacco whose antisense suppression leads to vascular tissue modification.

    PubMed

    Blee, Kristopher A; Choi, Joon W; O'Connell, Ann P; Schuch, Wolfgang; Lewis, Norman G; Bolwell, G Paul

    2003-09-01

    A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified. PMID:12946415

  13. Generalized Connective Tissue Disease in Crtap-/- Mouse

    PubMed Central

    Baldridge, Dustin; Lennington, Jennifer; Weis, MaryAnn; Homan, Erica P.; Jiang, Ming-Ming; Munivez, Elda; Keene, Douglas R.; Hogue, William R.; Pyott, Shawna; Byers, Peter H.; Krakow, Deborah; Cohn, Daniel H.; Eyre, David R.; Lee, Brendan; Morello, Roy

    2010-01-01

    Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in α1(I) and α1(II) chains, there was also loss of 3Hyp at proline 986 in α2(V) chains. In contrast, at two of the known 3Hyp sites in α1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin. PMID:20485499

  14. Artificial miRNA-mediated down-regulation of two monolignoid biosynthetic genes (C3H and F5H) cause reduction in lignin content in jute.

    PubMed

    Shafrin, Farhana; Das, Sudhanshu Sekhar; Sanan-Mishra, Neeti; Khan, Haseena

    2015-11-01

    Artificial microRNAs (amiRNA) provide a new feature in the gene silencing era. Concomitantly, reducing the amount of lignin in fiber-yielding plants such as jute holds significant commercial and environmental potential, since this amount is inversely proportional to the quality of the fiber. The present study aimed at reducing the lignin content in jute, by introducing amiRNA based vectors for down-regulation of two monolignoid biosynthetic genes of jute, coumarate 3-hydroxylase (C3H) and ferulate 5-hydroxylase (F5H). The transgenic lines of F5H-amiRNA and C3H-amiRNA showed a reduced level of gene expression, which resulted in about 25% reduction in acid insoluble lignin content for whole stem and 12-15% reduction in fiber lignin as compared to the non-transgenic plants. The results indicate successful F5H-amiRNA and C3H-amiRNA transgenesis for lignin reduction in jute. This is likely to have far-reaching commercial implications and economic acceleration for jute producing countries. PMID:26453352

  15. Enhancement of alkaloid production in opium and California poppy by transactivation using heterologous regulatory factors.

    PubMed

    Apuya, Nestor R; Park, Joon-Hyun; Zhang, Liping; Ahyow, Maurice; Davidow, Patricia; Van Fleet, Jennifer; Rarang, Joel C; Hippley, Matthew; Johnson, Thomas W; Yoo, Hye-Dong; Trieu, Anthony; Krueger, Shannon; Wu, Chuan-yin; Lu, Yu-ping; Flavell, Richard B; Bobzin, Steven C

    2008-02-01

    Genes encoding regulatory factors isolated from Arabidopsis, soybean and corn have been screened to identify those that modulate the expression of genes encoding for enzymes involved in the biosynthesis of morphinan alkaloids in opium poppy (Papaver somniferum) and benzophenanthridine alkaloids in California poppy (Eschscholzia californica). In opium poppy, the over-expression of selected regulatory factors increased the levels of PsCOR (codeinone reductase), Ps4'OMT (S-adenosyl-l-methionine:3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase) and Ps6OMT [(R,S)-norcoclaurine 6-O-methyltransferase] transcripts by 10- to more than 100-fold. These transcriptional activations translated into an enhancement of alkaloid production in opium poppy of up to at least 10-fold. In California poppy, the transactivation effect of regulatory factor WRKY1 resulted in an increase of up to 60-fold in the level of EcCYP80B1 [(S)-N-methylcoclaurine 3'-hydroxylase] and EcBBE (berberine bridge enzyme) transcripts. As a result, the accumulations of selected alkaloid intermediates were enhanced up to 30-fold. The transactivation effects of other regulatory factors led to the accumulation of the same intermediates. These regulatory factors also led to the production of new alkaloids in California poppy callus culture. PMID:17961129

  16. A functional 4-hydroxybenzoate degradation pathway in the phytopathogen Xanthomonas campestris is required for full pathogenicity.

    PubMed

    Wang, Jia-Yuan; Zhou, Lian; Chen, Bo; Sun, Shuang; Zhang, Wei; Li, Ming; Tang, Hongzhi; Jiang, Bo-Le; Tang, Ji-Liang; He, Ya-Wen

    2015-01-01

    Plants contain significant levels of natural phenolic compounds essential for reproduction and growth, as well as defense mechanisms against pathogens. Xanthomonas campestris pv. campestris (Xcc) is the causal agent of crucifers black rot. Here we showed that genes required for the synthesis, utilization, transportation, and degradation of 4-hydroxybenzoate (4-HBA) are present in Xcc. Xcc rapidly degrades 4-HBA, but has no effect on 2-hydroxybenzoate and 3-hydroxybenzoate when grown in XOLN medium. The genes for 4-HBA degradation are organized in a superoperonic cluster. Bioinformatics, biochemical, and genetic data showed that 4-HBA is hydroxylated by 4-HBA 3-hydroxylase (PobA), which is encoded by Xcc0356, to yield PCA. The resulting PCA is further metabolized via the PCA branches of the β-ketoadipate pathway, including Xcc0364, Xcc0365, and PcaFHGBDCR. Xcc0364 and Xcc0365 encode a new form of β-ketoadipate succinyl-coenzyme A transferase that is required for 4-HBA degradation. pobA expression was induced by 4-HBA via the transcriptional activator, PobR. Radish and cabbage hydrolysates contain 2-HBA, 3-HBA, 4-HBA, and other phenolic compounds. Addition of radish and cabbage hydrolysates to Xcc culture significantly induced the expression of pobA via PobR. The 4-HBA degradation pathway is required for full pathogenicity of Xcc in radish. PMID:26672484

  17. Transgenic rice seed synthesizing diverse flavonoids at high levels: a new platform for flavonoid production with associated health benefits.

    PubMed

    Ogo, Yuko; Ozawa, Kenjiro; Ishimaru, Tsutomu; Murayama, Tsugiya; Takaiwa, Fumio

    2013-08-01

    Flavonoids possess diverse health-promoting benefits but are nearly absent from rice, because most of the genes encoding enzymes for flavonoid biosynthesis are not expressed in rice seeds. In the present study, a transgenic rice plant producing several classes of flavonoids in seeds was developed by introducing multiple genes encoding enzymes involved in flavonoid synthesis, from phenylalanine to the target flavonoids, into rice. Rice accumulating naringenin was developed by introducing phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes. Rice producing other classes of flavonoids, kaempferol, genistein, and apigenin, was developed by introducing, together with PAL and CHS, genes encoding flavonol synthase/flavanone-3-hydroxylase, isoflavone synthase, and flavone synthases, respectively. The endosperm-specific GluB-1 promoter or embryo- and aleurone-specific 18-kDa oleosin promoters were used to express these biosynthetic genes in seed. The target flavonoids of naringenin, kaempferol, genistein, and apigenin were highly accumulated in each transgenic rice, respectively. Furthermore, tricin was accumulated by introducing hydroxylase and methyltransferase, demonstrating that modification to flavonoid backbones can be also well manipulated in rice seeds. The flavonoids accumulated as both aglycones and several types of glycosides, and flavonoids in the endosperm were deposited into PB-II-type protein bodies. Therefore, these rice seeds provide an ideal platform for the production of particular flavonoids due to efficient glycosylation, the presence of appropriate organelles for flavonoid accumulation, and the small effect of endogenous enzymes on the production of flavonoids by exogenous enzymes. PMID:23551455

  18. Benzothiadiazole affects the leaf proteome in arctic bramble (Rubus arcticus).

    PubMed

    Hukkanen, Anne; Kokko, Harri; Buchala, Antony; Häyrinen, Jukka; Kärenlampi, Sirpa

    2008-11-01

    Benzothiadiazole (BTH) induces resistance to the downy mildew pathogen, Peronospora sparsa, in arctic bramble, but the basis for the BTH-induced resistance is unknown. Arctic bramble cv. Mespi was treated with BTH to study the changes in leaf proteome and to identify proteins with a putative role in disease resistance. First, BTH induced strong expression of one PR-1 protein isoform, which was also induced by salicylic acid (SA). The PR-1 was responsive to BTH and exogenous SA despite a high endogenous SA content (20-25 microg/g fresh weight), which increased to an even higher level after treatment with BTH. Secondly, a total of 792 protein spots were detected in two-dimensional gel electrophoresis, eight proteins being detected solely in the BTH-treated plants. BTH caused up- or down-regulation of 72 and 31 proteins, respectively, of which 18 were tentatively identified by mass spectrometry. The up-regulation of flavanone-3-hydroxylase, alanine aminotransferase, 1-aminocyclopropane-1-carboxylate oxidase, PR-1 and PR-10 proteins may partly explain the BTH-induced resistance against P. sparsa. Other proteins with changes in intensity appear to be involved in, for example, energy metabolism and protein processing. The decline in ATP synthase, triosephosphate isomerase, fructose bisphosphate aldolase and glutamine synthetase suggests that BTH causes significant changes in primary metabolism, which provides one possible explanation for the decreased vegetative growth of foliage and rhizome observed in BTH-treated plants. PMID:19019008

  19. Activation of isocyanate ligands in Ru25+ complexes

    NASA Astrophysics Data System (ADS)

    Barral, M. Carmen; Herrero, Santiago; Jiménez-Aparicio, Reyes; Priego, José L.; Torres, M. Rosario; Urbanos, Francisco A.

    2008-11-01

    The reaction of [Ru 2(O 2CMe)(DPhF) 3(H 2O)]BF 4 · 0.5CH 2Cl 2 (DphF dbnd N, N'-diphenylformidinate) with sodium cyanate leads to the substitution of the H 2O ligand giving Ru 2(NCO)(O 2CMe)(DPhF) 3 ( 1). In contrast, in the similar reaction of Ru 2Cl 2(DPhF) 3 with NaOCN one of the cyanate groups undergoes the addition of a MeOH molecule leading to the carbamate complex Ru 2(NCO)(NH(O)COMe)(DPhF) 3 ( 2). The spectroscopic properties of 1 and 2 are studied. Both complexes are paramagnetic showing the presence of three unpaired electrons with an important zero-field splitting and a small intermolecular antiferromagnetic interaction. The crystal structures of 1 · 3CHCl 3 and 2 · C 7H 8 · 0.5MeOH are also reported. Compound 2 represents the first example of a ruthenium paddlewheel compound with a carbamate ligand.

  20. The hot plasma spectrometers on Freja

    NASA Astrophysics Data System (ADS)

    Norberg, O.; Eliasson, L.

    1991-11-01

    The hot plasma instrumentation F3H on the Swedish-German Freja satellite due for launch in 1992 will consist of electron and ion spectrometers. The spectrometer Magnetic imaging Two dimensional Electron (MATE) will measure the two dimensional electron distribution in the spin plane in the energy range 0.1 to 120 keV. The ion mass spectrometer Three dimensional Ion Composition Spectrometer (TICS) measures a full three dimensional distribution in the energy range 0.5 to 15000 eV/q with high mass resolution. The instruments use a particle 'imaging' detector technique based on a large diameter microchannel plate with position sensitive anode. The topics to be studied with the Freja hot plasma spectrometers include auroral particle acceleration, heating and acceleration of ionospheric ions, and the dynamics of auroral arc systems. Of special importance to the scientific objectives is the high data rate from the Freja instrumentation, the MATE and TICS spectrometers will be sampled every 10 ms, corresponding to a spatial resolution better than 70 m at ionospheric heights. The design, simulation, and calibration of the spectrometers are discussed.

  1. Identification of Vitis vinifera L. grape berry skin color mutants and polyphenolic profile.

    PubMed

    Ferreira, Vanessa; Fernandes, Fátima; Pinto-Carnide, Olinda; Valentão, Patrícia; Falco, Virgílio; Martín, Juan Pedro; Ortiz, Jesús María; Arroyo-García, Rosa; Andrade, Paula B; Castro, Isaura

    2016-03-01

    A germplasm set of twenty-five grapevine accessions, forming eleven groups of possible berry skin color mutants, were genotyped with twelve microsatellite loci, being eleven of them identified as true color mutants. The polyphenolic profiling of the confirmed mutant cultivars revealed a total of twenty-four polyphenols, comprising non-colored compounds (phenolic acids, flavan-3-ols, flavonols and a stilbene) and anthocyanins. Results showed differences in the contribution of malvidin-3-O-glucoside to the characteristic Pinot Noir anthocyanins profile. Regarding the two Pique-Poul colored variants, the lighter variant was richer than the darker one in all classes of compounds, excepting anthocyanins. In Moscatel Galego Roxo the F3'H pathway seems to be more active than F3'5'H, resulting in higher amounts of cyanidin, precursor of the cyanidin derivatives. As far as we are aware, this is the first time that a relationship between the content of polyphenolic compounds is established in groups of grape berry skin color mutant cultivars. PMID:26471534

  2. Metabolite profiling and expression analysis of flavonoid, vitamin C and tocopherol biosynthesis genes in the antioxidant-rich sea buckthorn (Hippophae rhamnoides L.).

    PubMed

    Fatima, Tahira; Kesari, Vigya; Watt, Ian; Wishart, David; Todd, James F; Schroeder, William R; Paliyath, Gopinadhan; Krishna, Priti

    2015-10-01

    In this study, phenolic compounds were analyzed in developing berries of four Canadian grown sea buckthorn (Hippophae rhamnoides L.) cultivars ('RC-4', 'E6590', 'Chuyskaya' and 'Golden Rain') and in leaves of two of these cultivars. Among phenolic acids, p-coumaric acid was the highest in berries, while gallic acid was predominant in leaves. In the flavonoid class of compounds, myricetin/rutin, kaempferol, quercetin and isorhamnetin were detected in berries and leaves. Berries of the 'RC-4' cultivar had approximately ⩾ 2-fold higher levels of myricetin and quercetin at 17.5mg and 17.2 mg/100 g FW, respectively, than the other cultivars. The flavonoid content in leaves was considerably more than in berries with rutin and quercetin levels up to 135 mg and 105 mg/100 g FW, respectively. Orthologs of 15 flavonoid biosynthesis pathway genes were identified within the transcriptome of sea buckthorn mature seeds. Semi-quantitative RT-PCR analysis of these genes in developing berries indicated relatively higher expression of genes such as CHS, F3'H, DFR and LDOX in the 'RC-4' cultivar than in the 'Chuyskaya' cultivar. Vitamin C levels in ripened berries of the Canadian cultivars were on the high end of the concentration range reported for most other sea buckthorn cultivars. Orthologs of genes involved in vitamins C and E biosynthesis were also identified, expanding the genomic resources for this nutritionally important plant. PMID:26318327

  3. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves

    PubMed Central

    Zhang, Zhen; Li, Da-Wei; Jin, Jing-Hao; Yin, Yan-Xu; Zhang, Huai-Xia; Chai, Wei-Guo; Gong, Zhen-Hui

    2015-01-01

    The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3′5′H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens. PMID:26217354

  4. Visualization and targeted disruption of protein interactions in living cells

    PubMed Central

    Herce, Henry D.; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M. Cristina

    2013-01-01

    Protein–protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein–protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53–HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein–protein interactions in practically any cell type and species. PMID:24154492

  5. Visualization and targeted disruption of protein interactions in living cells.

    PubMed

    Herce, Henry D; Deng, Wen; Helma, Jonas; Leonhardt, Heinrich; Cardoso, M Cristina

    2013-01-01

    Protein-protein interactions are the basis of all processes in living cells, but most studies of these interactions rely on biochemical in vitro assays. Here we present a simple and versatile fluorescent-three-hybrid (F3H) strategy to visualize and target protein-protein interactions. A high-affinity nanobody anchors a GFP-fusion protein of interest at a defined cellular structure and the enrichment of red-labelled interacting proteins is measured at these sites. With this approach, we visualize the p53-HDM2 interaction in living cells and directly monitor the disruption of this interaction by Nutlin 3, a drug developed to boost p53 activity in cancer therapy. We further use this approach to develop a cell-permeable vector that releases a highly specific peptide disrupting the p53 and HDM2 interaction. The availability of multiple anchor sites and the simple optical readout of this nanobody-based capture assay enable systematic and versatile analyses of protein-protein interactions in practically any cell type and species. PMID:24154492

  6. Metabolomic analysis and differential expression of anthocyanin biosynthetic genes in white- and red-flowered buckwheat cultivars (Fagopyrum esculentum).

    PubMed

    Kim, Yeon Bok; Park, Soo-Yun; Thwe, Aye Aye; Seo, Jeong Min; Suzuki, Tastsuro; Kim, Sun-Ju; Kim, Jae Kwang; Park, Sang Un

    2013-11-01

    Red-flowered buckwheat ( Fagopyrum esculentum ) is used in the production of tea, juice, and alcohols after the detoxification of fagopyrin. In order to investigate the metabolomics and regulatory of anthocyanin production in red-flowered (Gan-Chao) and white-flowered (Tanno) buckwheat cultivars, quantitative real-time RT-PCR (qRT-PCR), gas chromatography time-of-flight mass spectrometry (GC-TOFMS), and high performance liquid chromatography (HPLC) were conducted. The transcriptions of FePAL, FeC4H, Fe4CL1, FeF3H, FeANS, and FeDFR increased gradually from flowering stage 1 and reached their highest peaks at flowering stage 3 in Gan-Chao flower. In total 44 metabolites, 18 amino acids, 15 organic acids, 7 sugars, 3 sugar alcohols, and 1 amine were detected in Gan-Chao flowers. Two anthocyanins, cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside, were identified in Gan-Chao cultivar. The first component of the partial least-squares to latent structures-discriminate analysis (PLS-DA) indicated that high amounts of phenolic, shikimic, and pyruvic acids were present in Gan-Chao. We suggest that transcriptions of genes involved in anthocyanin biosynthesis, anthocyanin contents, and metabolites have correlation in the red-flowered buckwheat Gan-Chao flowers. Our results may be helpful to understand anthocyanin biosynthesis in red-flowered buckwheat. PMID:24083509

  7. Role of a chalcone isomerase-like protein in flavonoid biosynthesis in Arabidopsis thaliana

    PubMed Central

    Jiang, Wenbo; Yin, Qinggang; Wu, Ranran; Zheng, Guangshun; Liu, Jinyue; Dixon, Richard A.; Pang, Yongzhen

    2015-01-01

    Flavonoids are important natural products for plant defence and human health. Although almost all the flavonoid pathway genes have been well-documented by biochemical and/or genetic approaches, the role of the Arabidopsis chalcone isomerase-like (CHIL) gene remains unclear. Two chil mutants with a seed colour similar to that of wild-type Arabidopsis have been identified here, but in sharp contrast to the characteristic transparent testa seed phenotype associated with other known flavonoid pathway genes. CHIL loss-of-function mutations led to a strong reduction in the proanthocyanidin and flavonol levels in seeds, but not in the anthocyanin levels in leaves. CHIL over-expression could partially recover the mutant phenotype of the chil mutant and increased both proanthocyanidin and flavonol accumulation in wild-type Arabidopsis. However, the CHIL gene could not rescue the mutant phenotype of TT5 that encodes the intrinsic chalcone isomerase in Arabidopsis. Parallel phenotypical and metabolic analyses of the chil, tt5, chs, and f3h mutants revealed that, genetically, CHIL functions at the same step as TT5. Moreover, it is demonstrated that CHIL co-expresses, co-localizes, and interacts with TT5 in Arabidopsis for flavonoid production. Based on these genetic and metabolic studies, it is concluded that CHIL functions with TT5 to promote flavonoid production, which is a unique enhancer in the flavonoid pathway. PMID:26347569

  8. Jasmonic acid involves in grape fruit ripening and resistant against Botrytis cinerea.

    PubMed

    Jia, Haifeng; Zhang, Cheng; Pervaiz, Tariq; Zhao, Pengcheng; Liu, Zhongjie; Wang, Baoju; Wang, Chen; Zhang, Lin; Fang, Jinggui; Qian, Jianpu

    2016-01-01

    Fruit ripening is a complex process that is regulated by a signal network. Whereas the regulatory mechanism of abscisic acid has been studied extensively in non-climacteric fruit, little is know about other signaling pathways involved in this process. In this study, we performed that plant hormone jasmonic acid plays an important role in grape fruit coloring and softening by increasing the transcription levels of several ripening-related genes, such as the color-related genes PAL1, DFR, CHI, F3H, GST, CHS, and UFGT; softening-related genes PG, PL, PE, Cell, EG1, and XTH1; and aroma-related genes Ecar, QR, and EGS. Lastly, the fruit anthocyanin, phenol, aroma, and cell wall materials were changed. Jasmonic acid positively regulated its biosynthesis pathway genes LOS, AOS, and 12-oxophytodienoate reductase (OPR) and signal pathway genes COI1 and JMT. RNA interference of grape jasmonic acid pathway gene VvAOS in strawberry fruit appeared fruit un-coloring phenotypes; exogenous jasmonic acid rescued this phenotypes. On the contrary, overexpression of grape jasmonic acid receptor VvCOI1 in the strawberry fruit accelerated the fruit-ripening process and induced some plant defense-related gene expression level. Furthermore, jasmonic acid treatment or strong jasmonic acid signal pathway in strawberry fruit make the fruit resistance against Botrytis cinerea. PMID:26498957

  9. Analysis of biochemical compounds and differentially expressed genes of the anthocyanin biosynthetic pathway in variegated peach flowers.

    PubMed

    Hassani, D; Liu, H L; Chen, Y N; Wan, Z B; Zhuge, Q; Li, S X

    2015-01-01

    Variegated plants are highly valuable in the floricultural market, yet the genetic mechanism underlying this attractive phenomenon has not been completely elucidated. In this study, we identified and measured different compounds in pink and white flower petals of peach (Prunus persica) by high-performance liquid chromatography and liquid chromatography/mass spectrometry analyses. No cyanidin-based or pelargonidin-based compounds were detected in white petals, but high levels of these compounds were found in pink petals. Additionally, we sequenced and analyzed the expression of six key structural genes in the anthocyanin biosynthesis pathway (CHI, CHS, DFR, F3'H, ANS, and UFGT) in both white and pink petals. Quantitative real-time polymerase chain reaction revealed all six genes to be expressed at greatly reduced levels in white flower petals, relative to pink. No allelic variations were found in the transcribed sequences. However, alignment of transcribed and genomic sequences of the ANS gene detected alternative splicing, resulting in transcripts of 1.071 and 942 bp. Only the longer transcript was observed in white flower petals. Since ANS is the key intermediate enzyme catalyzing the colorless leucopelargonidin and leucocyanidin to substrates required for completion of anthocyanin biosynthesis, the ANS gene is implicated in flower color variegation and should be explored in future studies. This article, together with a previous transcriptome study, elucidates the mechanism underlying peach flower color variegation in terms of the key structural genes involved in anthocyanin biosynthesis. PMID:26535657

  10. VIGS approach reveals the modulation of anthocyanin biosynthetic genes by CaMYB in chili pepper leaves.

    PubMed

    Zhang, Zhen; Li, Da-Wei; Jin, Jing-Hao; Yin, Yan-Xu; Zhang, Huai-Xia; Chai, Wei-Guo; Gong, Zhen-Hui

    2015-01-01

    The purple coloration of pepper leaves arises from the accumulation of anthocyanin. Three regulatory and 12 structural genes have been characterized for their involvement in the anthocyanin biosynthesis. Examination of the abundance of these genes in leaves showed that the majority of them differed between anthocyanin pigmented line Z1 and non-pigmented line A3. Silencing of the R2R3-MYB transcription factor CaMYB in pepper leaves of Z1 resulted in the loss of anthocyanin accumulation. Moreover, the expression of multiple genes was altered in the silenced leaves. The expression of MYC was significantly lower in CaMYB-silenced leaves, whereas WD40 showed the opposite pattern. Most structural genes including CHS, CHI, F3H, F3'5'H, DFR, ANS, UFGT, ANP, and GST were repressed in CaMYB-silenced foliage with the exception of PAL, C4H, and 4CL. These results indicated that MYB plays an important role in the regulation of anthocyanin biosynthetic related genes. Besides CaMYB silenced leaves rendered more sporulation of Phytophthora capsici Leonian indicating that CaMYB might be involved in the defense response to pathogens. PMID:26217354

  11. Induced transcriptional profiling of phenylpropanoid pathway genes increased flavonoid and lignin content in Arabidopsis leaves in response to microbial products

    PubMed Central

    2014-01-01

    Background The production and use of biologically derived soil additives is one of the fastest growing sectors of the fertilizer industry. These products have been shown to improve crop yields while at the same time reducing fertilizer inputs to and nutrient loss from cropland. The mechanisms driving the changes in primary productivity and soil processes are poorly understood and little is known about changes in secondary productivity associated with the use of microbial products. Here we investigate secondary metabolic responses to a biologically derived soil additive by monitoring changes in the phenlypropanoid (PP) pathway in Arabidopsis thaliana. Results This study was designed to test the influence of one of these products (Soil Builder™-AF, SB) on secondary metabolism after being applied at different times. One time (TI) application of SB to Arabidopsis increased the accumulation of flavonoids compared to multiple (TII) applications of the same products. Fourteen phenolic compounds including flavonols and anothocyanins were identified by mass spectrometry. Kaempferol-3,7-O-bis-α-L-rhamnoside and quercetin 3,7-dirhamnoside, the major compounds, increased 3-fold and 4-fold, respectively compared to control in the TI treatment. The most abundant anthocyanin was cyanidin 3-rhamnoglucoside, which increased 3-fold and 2-fold in TI compared to the control and TII, respectively. Simultaneously, the expression of genes coding for key enzymes in the PP pathway (phenylalanine ammonia lyase, cinnamate 4-hydroxylase, chalcone synthase, flavonoid-3′-O-hydroxylase, flavonol synthase1 and dihydroflavonol-4-reductase) and regulatory genes (production of anthocyanin pigment2, MYB12, MYB113, MYB114, EGL3, and TT8) were up-regulated in both treatments (TI and TII). Furthermore, application of TI and TII induced expression of the lignin pathway genes (hydroxyl cinamyl transferase, caffeyl-CoA O-methyl transferase, cinnamyl alcohol dehydrogenase, cinnamyl-CoA reductase

  12. The Plant Short-Chain Dehydrogenase (SDR) superfamily: genome-wide inventory and diversification patterns

    PubMed Central

    2012-01-01

    Background Short-chain dehydrogenases/reductases (SDRs) form one of the largest and oldest NAD(P)(H) dependent oxidoreductase families. Despite a conserved ‘Rossmann-fold’ structure, members of the SDR superfamily exhibit low sequence similarities, which constituted a bottleneck in terms of identification. Recent classification methods, relying on hidden-Markov models (HMMs), improved identification and enabled the construction of a nomenclature. However, functional annotations of plant SDRs remain scarce. Results Wide-scale analyses were performed on ten plant genomes. The combination of hidden Markov model (HMM) based analyses and similarity searches led to the construction of an exhaustive inventory of plant SDR. With 68 to 315 members found in each analysed genome, the inventory confirmed the over-representation of SDRs in plants compared to animals, fungi and prokaryotes. The plant SDRs were first classified into three major types — ‘classical’, ‘extended’ and ‘divergent’ — but a minority (10% of the predicted SDRs) could not be classified into these general types (‘unknown’ or ‘atypical’ types). In a second step, we could categorize the vast majority of land plant SDRs into a set of 49 families. Out of these 49 families, 35 appeared early during evolution since they are commonly found through all the Green Lineage. Yet, some SDR families — tropinone reductase-like proteins (SDR65C), ‘ABA2-like’-NAD dehydrogenase (SDR110C), ‘salutaridine/menthone-reductase-like’ proteins (SDR114C), ‘dihydroflavonol 4-reductase’-like proteins (SDR108E) and ‘isoflavone-reductase-like’ (SDR460A) proteins — have undergone significant functional diversification within vascular plants since they diverged from Bryophytes. Interestingly, these diversified families are either involved in the secondary metabolism routes (terpenoids, alkaloids, phenolics) or participate in developmental processes (hormone biosynthesis or catabolism, flower

  13. A R2R3-MYB Transcription Factor from Epimedium sagittatum Regulates the Flavonoid Biosynthetic Pathway

    PubMed Central

    Lv, Haiyan; Luo, Ming; Zeng, Shaohua; Pattanaik, Sitakanta; Yuan, Ling; Wang, Ying

    2013-01-01

    Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. The bioactive components in herba epimedii are mainly prenylated flavonol glycosides, end-products of the flavonoid pathway. Epimedium species are also used as garden plants due to the colorful flowers and leaves. Many R2R3-MYB transcription factors (TFs) have been identified to regulate the flavonoid and anthocyanin biosynthetic pathways. However, little is known about the R2R3-MYB TFs involved in regulation of the flavonoid pathway in Epimedium. Here, we reported the isolation and functional characterization of the first R2R3-MYB TF (EsMYBA1) from Epimedium sagittatum (Sieb. Et Zucc.) Maxim. Conserved domains and phylogenetic analysis showed that EsMYBA1 belonged to the subgroup 6 clade (anthocyanin-related MYB clade) of R2R3-MYB family, which includes Arabidopsis AtPAP1, apple MdMYB10 and legume MtLAP1. EsMYBA1 was preferentially expressed in leaves, especially in red leaves that contain higher content of anthocyanin. Alternative splicing of EsMYBA1 resulted in three transcripts and two of them encoded a MYB-related protein. Yeast two-hybrid and transient luciferase expression assay showed that EsMYBA1 can interact with several bHLH regulators of the flavonoid pathway and activate the promoters of dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS). In both transgenic tobacco and Arabidopsis, overexpression of EsMYBA1 induced strong anthocyanin accumulation in reproductive and/or vegetative tissues via up-regulation of the main flavonoid-related genes. Furthermore, transient expression of EsMYBA1 in E. sagittatum leaves by Agrobacterium infiltration also induced anthocyanin accumulation in the wounded area. This first functional characterization of R2R3-MYB TFs in Epimedium species will promote further studies of the flavonoid biosynthesis and regulation in medicinal plants. PMID:23936468

  14. Flower colour and cytochromes P450†

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  15. Arabidopsis FHY3 and FAR1 Regulate Light-Induced myo-Inositol Biosynthesis and Oxidative Stress Responses by Transcriptional Activation of MIPS1.

    PubMed

    Ma, Lin; Tian, Tian; Lin, Rongcheng; Deng, Xing-Wang; Wang, Haiyang; Li, Gang

    2016-04-01

    myo-Inositol-1-phosphate synthase (MIPS) catalyzes the limiting step of inositol biosynthesis and has crucial roles in plant growth and development. In response to stress, the transcription of MIPS1 is induced and the biosynthesis of inositol or inositol derivatives is promoted by unknown mechanisms. Here, we found that the light signaling protein FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) regulate light-induced inositol biosynthesis and oxidative stress responses by activating the transcription of MIPS1. Disruption of FHY3 and FAR1 caused light-induced cell death after dark-light transition, precocious leaf senescence, and increased sensitivity to oxidative stress. Reduction of salicylic acid (SA) accumulation by overexpression of SALICYLIC ACID 3-HYDROXYLASE largely suppressed the cell death phenotype of fhy3 far1 mutant plants, suggesting that FHY3- and FAR1-mediated cell death is dependent on SA. Furthermore, comparative analysis of chromatin immunoprecipitation sequencing and microarray results revealed that FHY3 and FAR1 directly target both MIPS1 and MIPS2. The fhy3 far1 mutant plants showed severely decreased MIPS1/2 transcript levels and reduced inositol levels. Conversely, constitutive expression of MIPS1 partially rescued the inositol contents, caused reduced transcript levels of SA-biosynthesis genes, and prevented oxidative stress in fhy3 far1. Taken together, our results indicate that the light signaling proteins FHY3 and FAR1 directly bind the promoter of MIPS1 to activate its expression and thereby promote inositol biosynthesis to prevent light-induced oxidative stress and SA-dependent cell death. PMID:26714049

  16. Molecular and Biochemical Basis for Stress-Induced Accumulation of Free and Bound p-Coumaraldehyde in Cucumber1[W][OA

    PubMed Central

    Varbanova, Marina; Porter, Katie; Lu, Fachuang; Ralph, John; Hammerschmidt, Ray; Jones, A. Daniel; Day, Brad

    2011-01-01

    To elucidate the genetic and biochemical regulation of elicitor-induced p-coumaraldehyde accumulation in plants, we undertook a multifaceted approach to characterize the metabolic flux through the phenylpropanoid pathway via the characterization and chemical analysis of the metabolites in the p-coumaryl, coniferyl, and sinapyl alcohol branches of this pathway. Here, we report the identification and characterization of four cinnamyl alcohol dehydrogenases (CADs) from cucumber (Cucumis sativus) with low activity toward p-coumaraldehyde yet exhibiting significant activity toward other phenylpropanoid hydroxycinnamaldehydes. As part of this analysis, we identified and characterized the activity of a hydroxycinnamoyl-coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) capable of utilizing shikimate and p-coumaroyl-coenzyme A to generate p-coumaroyl shikimate. Following pectinase treatment of cucumber, we observed the rapid accumulation of p-coumaraldehyde, likely the result of low aldehyde reductase activity (i.e. alcohol dehydrogenase in the reverse reaction) of CsCAD enzymes on p-coumaraldehyde. In parallel, we noted a concomitant reduction in the activity of CsHCT. Taken together, our findings support the hypothesis that the up-regulation of the phenylpropanoid pathway upon abiotic stress greatly enhances the overall p-coumaryl alcohol branch of the pathway. The data presented here point to a role for CsHCT (as well as, presumably, p-coumarate 3-hydroxylase) as a control point in the regulation of the coniferyl and sinapyl alcohol branches of this pathway. This mechanism represents a potentially evolutionarily conserved process to efficiently and quickly respond to biotic and abiotic stresses in cucurbit plants, resulting in the rapid lignification of affected tissues. PMID:21940999

  17. Bone Collagen: New Clues to its Mineralization Mechanism From Recessive Osteogenesis Imperfecta

    PubMed Central

    Eyre, David R.; Ann Weis, Mary

    2013-01-01

    Until 2006 the only mutations known to cause osteogenesis imperfecta (OI) were in the two genes coding for type I collagen chains. These dominant mutations affecting the expression or primary sequence of collagen α1(I) and α2(I) chains account for over 90% of OI cases. Since then a growing list of mutant genes causing the 5–10% of recessive cases has rapidly emerged. They include CRTAP, LEPRE1 and PPIB, which encode three proteins forming the prolyl 3-hydroxylase complex; PLOD2 and FKBP10, which encode respectively lysyl hydroxylase 2 and a foldase required for its activity in forming mature cross-links in bone collagen; SERPIN H1, which encodes the collagen chaperone HSP47; SERPIN F1, which encodes pigment epithelium-derived factor required for osteoid mineralization; and BMP1, which encodes the type I procollagen C-propeptidase. All cause fragile bone in infancy, which can include over-mineralization or under-mineralization defects as well as abnormal collagen post-translational modifications. Consistently both dominant and recessive variants lead to abnormal cross-linking chemistry in bone collagen. These recent discoveries strengthen the potential for a common pathogenic mechanism of misassembled collagen fibrils. Of the new genes identified, eight encode proteins required for collagen post-translational modification, chaperoning of newly synthesized collagen chains into native molecules or transport through the endoplasmic reticulum and Golgi for polymerization, cross-linking and mineralization. In reviewing these findings, we conclude that a common theme is emerging in the pathogenesis of brittle bone disease of mishandled collagen assembly with important insights on post-translational features of bone collagen that have evolved to optimize it as a biomineral template. PMID:23508630

  18. Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide

    PubMed Central

    Chung, Hak Suk; Raetz, Christian R. H.

    2011-01-01

    Several Gram-negative pathogens, including Yersinia pestis, Burkholderia cepacia, and Acinetobacter haemolyticus, synthesize an isosteric analog of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), known as d-glycero-d-talo-oct-2-ulosonic acid (Ko), in which the axial hydrogen atom at the Kdo 3-position is replaced with OH. Here we report a unique Kdo 3-hydroxylase (KdoO) from Burkholderia ambifaria and Yersinia pestis, encoded by the bamb_0774 (BakdoO) and the y1812 (YpkdoO) genes, respectively. When expressed in heptosyl transferase-deficient Escherichia coli, these genes result in conversion of the outer Kdo unit of Kdo2-lipid A to Ko in an O2-dependent manner. KdoO contains the putative iron-binding motif, HXDXn>40H. Reconstitution of KdoO activity in vitro with Kdo2-lipid A as the substrate required addition of Fe2+, α-ketoglutarate, and ascorbic acid, confirming that KdoO is a Fe2+/α-ketoglutarate/O2-dependent dioxygenase. Conversion of Kdo to Ko in Kdo2-lipid A conferred reduced susceptibility to mild acid hydrolysis. Although two enzymes that catalyze Fe2+/α-ketoglutarate/O2-dependent hydroxylation of deoxyuridine in fungal extracts have been reported previously, kdoO is the first example of a gene encoding a deoxy-sugar hydroxylase. Homologues of KdoO are found exclusively in Gram-negative bacteria, including the human pathogens Burkholderia mallei, Yersinia pestis, Klebsiella pneumoniae, Legionella longbeachae, and Coxiella burnetii, as well as the plant pathogen Ralstonia solanacearum. PMID:21178073

  19. Elucidating the genetic basis of antioxidant status in lettuce (Lactuca sativa)

    PubMed Central

    Damerum, Annabelle; Selmes, Stacey L; Biggi, Gaia F; Clarkson, Graham JJ; Rothwell, Steve D; Truco, Maria José; Michelmore, Richard W; Hancock, Robert D; Shellcock, Connie; Chapman, Mark A; Taylor, Gail

    2015-01-01

    A diet rich in phytonutrients from fruit and vegetables has been acknowledged to afford protection against a range of human diseases, but many of the most popular vegetables are low in phytonutrients. Wild relatives of crops may contain allelic variation for genes determining the concentrations of these beneficial phytonutrients, and therefore understanding the genetic basis of this variation is important for breeding efforts to enhance nutritional quality. In this study, lettuce recombinant inbred lines, generated from a cross between wild and cultivated lettuce (Lactuca serriola and Lactuca sativa, respectively), were analysed for antioxidant (AO) potential and important phytonutrients including carotenoids, chlorophyll and phenolic compounds. When grown in two environments, 96 quantitative trait loci (QTL) were identified for these nutritional traits: 4 for AO potential, 2 for carotenoid content, 3 for total chlorophyll content and 87 for individual phenolic compounds (two per compound on average). Most often, the L. serriola alleles conferred an increase in total AOs and metabolites. Candidate genes underlying these QTL were identified by BLASTn searches; in several cases, these had functions suggesting involvement in phytonutrient biosynthetic pathways. Analysis of a QTL on linkage group 3, which accounted for >30% of the variation in AO potential, revealed several candidate genes encoding multiple MYB transcription factors which regulate flavonoid biosynthesis and flavanone 3-hydroxylase, an enzyme involved in the biosynthesis of the flavonoids quercetin and kaempferol, which are known to have powerful AO activity. Follow-up quantitative RT-PCR of these candidates revealed that 5 out of 10 genes investigated were significantly differentially expressed between the wild and cultivated parents, providing further evidence of their potential involvement in determining the contrasting phenotypes. These results offer exciting opportunities to improve the nutritional

  20. Membrane protein complexes catalyze both 4- and 3-hydroxylation of cinnamic acid derivatives in monolignol biosynthesis.

    PubMed

    Chen, Hsi-Chuan; Li, Quanzi; Shuford, Christopher M; Liu, Jie; Muddiman, David C; Sederoff, Ronald R; Chiang, Vincent L

    2011-12-27

    The hydroxylation of 4- and 3-ring carbons of cinnamic acid derivatives during monolignol biosynthesis are key steps that determine the structure and properties of lignin. Individual enzymes have been thought to catalyze these reactions. In stem differentiating xylem (SDX) of Populus trichocarpa, two cinnamic acid 4-hydroxylases (PtrC4H1 and PtrC4H2) and a p-coumaroyl ester 3-hydroxylase (PtrC3H3) are the enzymes involved in these reactions. Here we present evidence that these hydroxylases interact, forming heterodimeric (PtrC4H1/C4H2, PtrC4H1/C3H3, and PtrC4H2/C3H3) and heterotrimeric (PtrC4H1/C4H2/C3H3) membrane protein complexes. Enzyme kinetics using yeast recombinant proteins demonstrated that the enzymatic efficiency (V(max)/k(m)) for any of the complexes is 70-6,500 times greater than that of the individual proteins. The highest increase in efficiency was found for the PtrC4H1/C4H2/C3H3-mediated p-coumaroyl ester 3-hydroxylation. Affinity purification-quantitative mass spectrometry, bimolecular fluorescence complementation, chemical cross-linking, and reciprocal coimmunoprecipitation provide further evidence for these multiprotein complexes. The activities of the recombinant and SDX plant proteins demonstrate two protein-complex-mediated 3-hydroxylation paths in monolignol biosynthesis in P. trichocarpa SDX; one converts p-coumaric acid to caffeic acid and the other converts p-coumaroyl shikimic acid to caffeoyl shikimic acid. Cinnamic acid 4-hydroxylation is also mediated by the same protein complexes. These results provide direct evidence for functional involvement of membrane protein complexes in monolignol biosynthesis. PMID:22160716

  1. Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli.

    PubMed

    Makino, Takuya; Harada, Hisashi; Ikenaga, Hiroshi; Matsuda, Satoru; Takaichi, Shinichi; Shindo, Kazutoshi; Sandmann, Gerhard; Ogata, Takehiko; Misawa, Norihiko

    2008-12-01

    The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin. PMID:18987067

  2. Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy.

    PubMed

    Desgagné-Penix, Isabel; Facchini, Peter J

    2012-10-01

    Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)-norcoclaurine to papaverine involves 3'-hydroxylation, four O-methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)-reticuline, and the enzymes involved. We used virus-induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N-methyltransferase dramatically increased papaverine levels at the expense of N-methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N-desmethylated compounds rather than through (S)-reticuline. Suppression of genes encoding (S)-3'-hydroxy-N-methylcoclaurine 4-O-methyltransferase and norreticuline 7-O-methyltransferase, which accept certain N-desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N-methylcoclaurine 3'-hydroxylase or reticuline 7-O-methyltransferase, which are specific for N-methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6-O-methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)-coclaurine as a key branch-point intermediate. The differential detection of N-desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine. PMID:22725256

  3. Gene expression and metabolite profiling of developing highbush blueberry fruit indicates transcriptional regulation of flavonoid metabolism and activation of abscisic acid metabolism.

    PubMed

    Zifkin, Michael; Jin, Alena; Ozga, Jocelyn A; Zaharia, L Irina; Schernthaner, Johann P; Gesell, Andreas; Abrams, Suzanne R; Kennedy, James A; Constabel, C Peter

    2012-01-01

    Highbush blueberry (Vaccinium corymbosum) fruits contain substantial quantities of flavonoids, which are implicated in a wide range of health benefits. Although the flavonoid constituents of ripe blueberries are known, the molecular genetics underlying their biosynthesis, localization, and changes that occur during development have not been investigated. Two expressed sequence tag libraries from ripening blueberry fruit were constructed as a resource for gene identification and quantitative real-time reverse transcription-polymerase chain reaction primer design. Gene expression profiling by quantitative real-time reverse transcription-polymerase chain reaction showed that flavonoid biosynthetic transcript abundance followed a tightly regulated biphasic pattern, and transcript profiles were consistent with the abundance of the three major classes of flavonoids. Proanthocyanidins (PAs) and corresponding biosynthetic transcripts encoding anthocyanidin reductase and leucoanthocyanidin reductase were most concentrated in young fruit and localized predominantly to the inner fruit tissue containing the seeds and placentae. Mean PA polymer length was seven to 8.5 subunits, linked predominantly via B-type linkages, and was relatively constant throughout development. Flavonol accumulation and localization patterns were similar to those of the PAs, and the B-ring hydroxylation pattern of both was correlated with flavonoid-3'-hydroxylase transcript abundance. By contrast, anthocyanins accumulated late in maturation, which coincided with a peak in flavonoid-3-O-glycosyltransferase and flavonoid-3'5'-hydroxylase transcripts. Transcripts of VcMYBPA1, which likely encodes an R2R3-MYB transcriptional regulator of PA synthesis, were prominent in both phases of development. Furthermore, the initiation of ripening was accompanied by a substantial rise in abscisic acid, a growth regulator that may be an important component of the ripening process and contribute to the regulation of

  4. Mutations in FKBP10 Cause Recessive Osteogenesis Imperfecta and Bruck Syndrome

    PubMed Central

    Kelley, Brian P; Malfait, Fransiska; Bonafe, Luisa; Baldridge, Dustin; Homan, Erica; Symoens, Sofie; Willaert, Andy; Elcioglu, Nursel; Van Maldergem, Lionel; Verellen-Dumoulin, Christine; Gillerot, Yves; Napierala, Dobrawa; Krakow, Deborah; Beighton, Peter; Superti-Furga, Andrea; De Paepe, Anne; Lee, Brendan

    2011-01-01

    Osteogenesis imperfecta (OI) is a genetic disorder of connective tissue characterized by bone fragility and alteration in synthesis and posttranslational modification of type I collagen. Autosomal dominant OI is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Bruck syndrome is a recessive disorder featuring congenital contractures in addition to bone fragility; Bruck syndrome type 2 is caused by mutations in PLOD2 encoding collagen lysyl hydroxylase, whereas Bruck syndrome type 1 has been mapped to chromosome 17, with evidence suggesting region 17p12, but the gene has remained elusive so far. Recently, the molecular spectrum of OI has been expanded with the description of the basis of a unique posttranslational modification of type I procollagen, that is, 3-prolyl-hydroxylation. Three proteins, cartilage-associated protein (CRTAP), prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene), and the prolyl cis-trans isomerase cyclophilin-B (PPIB), form a complex that is required for fibrillar collagen 3-prolyl-hydroxylation, and mutations in each gene have been shown to cause recessive forms of OI. Since then, an additional putative collagen chaperone complex, composed of FKBP10 (also known as FKBP65) and SERPINH1 (also known as HSP47), also has been shown to be mutated in recessive OI. Here we describe five families with OI-like bone fragility in association with congenital contractures who all had FKBP10 mutations. Therefore, we conclude that FKBP10 mutations are a cause of recessive osteogenesis imperfecta and Bruck syndrome, possibly Bruck syndrome Type 1 since the location on chromosome 17 has not been definitely localized. © 2011 American Society for Bone and Mineral Research. PMID:20839288

  5. CRTAP AND LEPRE1 MUTATIONS IN RECESSIVE OSTEOGENESIS IMPERFECTA

    PubMed Central

    Baldridge, Dustin; Schwarze, Ulrike; Morello, Roy; Lennington, Jennifer; Bertin, Terry K.; Pace, James M.; Pepin, Melanie G.; Weis, MaryAnn; Eyre, David R.; Walsh, Jennifer; Lambert, Deborah; Green, Andrew; Robinson, Haynes; Michelson, Melonie; Houge, Gunnar; Lindman, Carl; Martin, Judith; Ward, Jewell; Lemyre, Emmanuelle; Mitchell, John J.; Krakow, Deborah; Rimoin, David L.; Cohn, Daniel H.; Byers, Peter H.; Lee, Brendan

    2009-01-01

    Autosomal dominant osteogenesis imperfecta (OI) is caused by mutations in the genes (COL1A1 or COL1A2) encoding the chains of type I collagen. Recently, dysregulation of hydroxylation of a single proline residue at position 986 of both the triple-helical domains of type I collagen α1(I) and type II collagen α1(II) chains has been implicated in the pathogenesis of recessive forms of OI. Two proteins, CRTAP, or cartilage-associated protein, and prolyl-3-hydroxylase-1 (P3H1, encoded by the LEPRE1 gene) form a complex that performs the hydroxylation and brings the prolyl cis-trans isomerase cyclophilin-B (CYPB) to the unfolded collagen. In our screen of 78 subjects diagnosed with OI type II or III, we identified three probands with mutations in CRTAP and sixteen with mutations in LEPRE1. The latter group includes a mutation in patients from the Irish Traveller population, a genetically isolated community with increased incidence of OI. The clinical features resulting from CRTAP or LEPRE1 loss of function mutations were difficult to distinguish at birth. Infants in both groups had multiple fractures, decreased bone modeling (affecting especially the femurs), and extremely low bone mineral density. Interestingly, “popcorn” epiphyses may reflect underlying cartilaginous and bone dysplasia in this form of OI. These results expand the range of CRTAP/LEPRE1 mutations that result in recessive OI and emphasize the importance of distinguishing recurrence of severe OI of recessive inheritance from those that result from parental germline mosaicism for COL1A1 or COL1A2 mutations. PMID:18566967

  6. Mutations in PPIB (cyclophilin B) delay type I procollagen chain association and result in perinatal lethal to moderate osteogenesis imperfecta phenotypes

    PubMed Central

    Pyott, Shawna M.; Schwarze, Ulrike; Christiansen, Helena E.; Pepin, Melanie G.; Leistritz, Dru F.; Dineen, Richard; Harris, Catharine; Burton, Barbara K.; Angle, Brad; Kim, Katherine; Sussman, Michael D.; Weis, MaryAnn; Eyre, David R.; Russell, David W.; McCarthy, Kevin J.; Steiner, Robert D.; Byers, Peter H.

    2011-01-01

    Recessive mutations in the cartilage-associated protein (CRTAP), leucine proline-enriched proteoglycan 1 (LEPRE1) and peptidyl prolyl cis–trans isomerase B (PPIB) genes result in phenotypes that range from lethal in the perinatal period to severe deforming osteogenesis imperfecta (OI). These genes encode CRTAP (encoded by CRTAP), prolyl 3-hydroxylase 1 (P3H1; encoded by LEPRE1) and cyclophilin B (CYPB; encoded by PPIB), which reside in the rough endoplasmic reticulum (RER) and can form a complex involved in prolyl 3-hydroxylation in type I procollagen. CYPB, a prolyl cis–trans isomerase, has been thought to drive the prolyl-containing peptide bonds to the trans configuration needed for triple helix formation. Here, we describe mutations in PPIB identified in cells from three individuals with OI. Cultured dermal fibroblasts from the most severely affected infant make some overmodified type I procollagen molecules. Proα1(I) chains are slow to assemble into trimers, and abnormal procollagen molecules concentrate in the RER, and bind to protein disulfide isomerase (PDI) and prolyl 4-hydroxylase 1 (P4H1). These findings suggest that although CYPB plays a role in helix formation another effect is on folding of the C-terminal propeptide and trimer formation. The extent of procollagen accumulation and PDI/P4H1 binding differs among cells with mutations in PPIB, CRTAP and LEPRE1 with the greatest amount in PPIB-deficient cells and the least in LEPRE1-deficient cells. These findings suggest that prolyl cis–trans isomerase may be required to effectively fold the proline-rich regions of the C-terminal propeptide to allow proα chain association and suggest an order of action for CRTAP, P3H1 and CYPB in procollagen biosynthesis and pathogenesis of OI. PMID:21282188

  7. Osteogenesis imperfecta caused by PPIB mutation with severe phenotype and congenital hearing loss

    PubMed Central

    Rush, Eric T.; Caldwell, Kathleen S.; Kreikemeier, Rose M.; Lutz, Richard E.; Esposito, Paul W.

    2014-01-01

    Osteogenesis imperfecta (OI) is an inherited disorder of connective tissue typically caused by defects in either COL1A1 or COL1A2. A number of other genes causative of this disorder have been found, including PPIB, which forms one subunit of the prolyl 3-hydroxylase enzyme complex. Patients with homozygous or compound heterozygous mutations in this gene have OI with a trend toward lethal or severe phenotype. We present a Native American female with prenatal diagnosis of OI. Long bones were shortened with significant rhizomelia. At birth, fractures were present in ribs, humerii, and femurs. She had significant respiratory disease at birth, and required oxygen throughout her life. She also had recurrent pneumonias, one of which ultimately caused her death at age 16 mo. She also had significant bilateral sensorineural hearing loss. Molecular testing showed that the patient was homozygous for a single nucleotide substitution in PPIB (c. 136-2A>G). Patients with OI caused by PPIB mutations have had variable disease, but with majority of either with perinatal lethality or progressively deforming severe disease. Patients with OI due to PPIB mutation have shown some differences in phenotype. There appears to be a trend toward rhizomelic shortening and less severe bowing of the extremities, as compared to patients with comparably severe OI caused by COL1A1 or COL1A2 mutation. Congenital hearing loss may be an inconsistent feature of this condition, or may have co-occurred in our patient for unrelated reasons. Still, patients with OI caused by PPIB mutation should have appropriate early and regular management of their hearing.

  8. 4-Deoxyaurone formation in Bidens ferulifolia (Jacq.) DC.

    PubMed

    Miosic, Silvija; Knop, Katrin; Hölscher, Dirk; Greiner, Jürgen; Gosch, Christian; Thill, Jana; Kai, Marco; Shrestha, Binita Kumari; Schneider, Bernd; Crecelius, Anna C; Schubert, Ulrich S; Svatoš, Aleš; Stich, Karl; Halbwirth, Heidi

    2013-01-01

    The formation of 4-deoxyaurones, which serve as UV nectar guides in Bidens ferulifolia (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6'-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of B. ferulifolia and from plants, which do not accumulate aurones e.g. Arabidopsis thaliana, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in Antirrhinum majus L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general. PMID:23667445

  9. Elucidating the genetic basis of antioxidant status in lettuce (Lactuca sativa).

    PubMed

    Damerum, Annabelle; Selmes, Stacey L; Biggi, Gaia F; Clarkson, Graham Jj; Rothwell, Steve D; Truco, Maria José; Michelmore, Richard W; Hancock, Robert D; Shellcock, Connie; Chapman, Mark A; Taylor, Gail

    2015-01-01

    A diet rich in phytonutrients from fruit and vegetables has been acknowledged to afford protection against a range of human diseases, but many of the most popular vegetables are low in phytonutrients. Wild relatives of crops may contain allelic variation for genes determining the concentrations of these beneficial phytonutrients, and therefore understanding the genetic basis of this variation is important for breeding efforts to enhance nutritional quality. In this study, lettuce recombinant inbred lines, generated from a cross between wild and cultivated lettuce (Lactuca serriola and Lactuca sativa, respectively), were analysed for antioxidant (AO) potential and important phytonutrients including carotenoids, chlorophyll and phenolic compounds. When grown in two environments, 96 quantitative trait loci (QTL) were identified for these nutritional traits: 4 for AO potential, 2 for carotenoid content, 3 for total chlorophyll content and 87 for individual phenolic compounds (two per compound on average). Most often, the L. serriola alleles conferred an increase in total AOs and metabolites. Candidate genes underlying these QTL were identified by BLASTn searches; in several cases, these had functions suggesting involvement in phytonutrient biosynthetic pathways. Analysis of a QTL on linkage group 3, which accounted for >30% of the variation in AO potential, revealed several candidate genes encoding multiple MYB transcription factors which regulate flavonoid biosynthesis and flavanone 3-hydroxylase, an enzyme involved in the biosynthesis of the flavonoids quercetin and kaempferol, which are known to have powerful AO activity. Follow-up quantitative RT-PCR of these candidates revealed that 5 out of 10 genes investigated were significantly differentially expressed between the wild and cultivated parents, providing further evidence of their potential involvement in determining the contrasting phenotypes. These results offer exciting opportunities to improve the nutritional

  10. Berry skin development in Norton grape: Distinct patterns of transcriptional regulation and flavonoid biosynthesis

    PubMed Central

    2011-01-01

    patterns of MYB5A and MYB5B were similar during berry development between the two varieties, but those of MYBPA1 and MYBPA2 were strikingly different, demonstrating that the general flavonoid pathways are regulated under different MYB factors. The data showed that there were higher transcript levels of the genes encoding flavonoid-3'-O-hydroxylase (F3'H), flavonoid-3',5'-hydroxylase (F3'5'H), leucoanthocyanidin dioxygenase (LDOX), UDP-glucose:flavonoid 3'-O-glucosyltransferase (UFGT), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR) 1 and LAR2 in berry skin of Norton than in those of Cabernet Sauvignon. It was also found that the total amount of anthocyanins was markedly higher in Norton than in Cabernet Sauvignon berry skin at harvest, and five anthocyanin derivatives and three PA compounds exhibited distinctive accumulation patterns in Norton berry skin. Conclusions This study provides an overview of the transcriptome changes and the flavonoid profiles in the berry skin of Norton, an important North American wine grape, during berry development. The steady increase of transcripts of PR-1 and stilbene synthase genes likely contributes to the developmentally regulated resistance during ripening of Norton berries. More studies are required to address the precise role of each stilbene synthase gene in berry development and disease resistance. Transcriptional regulation of MYBA1, MYBA2, MYB5A and MYBPA1 as well as expression levels of their putative targets F3'H, F3'5'H, LDOX, UFGT, ANR, LAR1, and LAR2 are highly correlated with the characteristic anthocyanin and PA profiles in Norton berry skin. These results reveal a unique pattern of the regulation of transcription and biosynthesis pathways underlying the viticultural and enological characteristics of Norton grape, and yield new insights into the understanding of the flavonoid pathway in non-vinifera grape varieties. PMID:21219654

  11. Molybdenum(VI) network polymers based on anion-π interaction and hydrogen bonding: Synthesis, crystal structures and oxidation catalytic application

    NASA Astrophysics Data System (ADS)

    Li, Jia; Wang, Ge; Shi, Zhan; Yang, Mu; Luck, Rudy L.

    2009-11-01

    A crystallographic investigation of anion-π interactions and hydrogen bonds on the preferred structural motifs of molybdenum(VI) complexes has been carried out. Two molybdenum(VI) network polymers MoO 2F 4·(Hinca) 2 ( 1) and MoO 2F 3(H 2O)·(Hinpa) ( 2), where inca = isonicotinamide and inpa = isonipecotamide, have been synthesized, crystallographically characterized and successfully applied to alcohol oxidation reaction. Complex 1 crystallizes in the monoclinic space C2/ c: a = 16.832(3) Å, b = 8.8189(15) Å, c = 12.568(2) Å, β = 118.929(3)°, V = 1560.1(5) Å 3, Z = 4. Complex 2 crystallizes in the triclinic space P-1: a = 5.459(2) Å, b = 9.189(4) Å, c = 12.204(5) Å, α = 71.341(6)°, β = 81.712(7)°, γ = 77.705(7)°, V = 564.8(4) Å 3, Z = 2. Complex 1 consists of hydrogen bonding and anion-π interactions, both of which are considered as important factors for controlling the geometric features and packing characteristics of the crystal structure. The geometry of the sandwich complex of [MoO 2F 4] 2- with two pyridine rings indicates that the anion-π interaction is an additive and provides a base for the design and synthesis of new complexes. For complex 2, the anions and the protonated inpa ligands form a 2D supramolecular network by four different types of hydrogen contacts (N-H⋯F, N-H⋯O, O-H⋯F and O-H⋯O). The catalytic ability of complexes 1 and 2 has also been evaluated by applying them to the oxidation of benzyl alcohol with TBHP as oxidant.

  12. Efficient Rutin and Quercetin Biosynthesis through Flavonoids-Related Gene Expression in Fagopyrum tataricum Gaertn. Hairy Root Cultures with UV-B Irradiation

    PubMed Central

    Huang, Xuan; Yao, Jingwen; Zhao, Yangyang; Xie, Dengfeng; Jiang, Xue; Xu, Ziqin

    2016-01-01

    Transformed hairy roots had been efficiently induced from the seedlings of Fagopyrum tataricum Gaertn. due to the infection of Agrobacterium rhizogenes. Hairy roots were able to display active elongation with high root branching in 1/2 MS medium without growth regulators. The stable introduction of rolB and aux1 genes of A. rhizogenes WT strain 15834 into F. tataricum plants was confirmed by PCR analysis. Besides, the absence of virD gene confirmed hairy root was bacteria-free. After six different media and different sources of concentration were tested, the culturing of TB7 hairy root line in 1/2 MS liquid medium supplemented with 30 g l-1 sucrose for 20 days resulted in a maximal biomass accumulation (13.5 g l-1 fresh weight, 1.78 g l-1 dry weight) and rutin content (0.85 mg g-1). The suspension culture of hairy roots led to a 45-fold biomass increase and a 4.11-fold rutin content increase in comparison with the suspension culture of non-transformed roots. The transformation frequency was enhanced through preculturing for 2 days followed by infection for 20 min. The UV-B stress treatment of hairy roots resulted in a striking increase of rutin and quercetin production. Furthermore, the hairy root lines of TB3, TB7, and TB28 were chosen to study the specific effects of UV-B on flavonoid accumulation and flavonoid biosynthetic gene expression by qRT-PCR. This study has demonstrated that the UV-B radiation was an effective elicitor that dramatically changed in the transcript abundance of ftpAL, FtCHI, FtCHS, FtF3H, and FtFLS-1 in F. tataricum hairy roots. PMID:26870075

  13. Efficient Rutin and Quercetin Biosynthesis through Flavonoids-Related Gene Expression in Fagopyrum tataricum Gaertn. Hairy Root Cultures with UV-B Irradiation.

    PubMed

    Huang, Xuan; Yao, Jingwen; Zhao, Yangyang; Xie, Dengfeng; Jiang, Xue; Xu, Ziqin

    2016-01-01

    Transformed hairy roots had been efficiently induced from the seedlings of Fagopyrum tataricum Gaertn. due to the infection of Agrobacterium rhizogenes. Hairy roots were able to display active elongation with high root branching in 1/2 MS medium without growth regulators. The stable introduction of rolB and aux1 genes of A. rhizogenes WT strain 15834 into F. tataricum plants was confirmed by PCR analysis. Besides, the absence of virD gene confirmed hairy root was bacteria-free. After six different media and different sources of concentration were tested, the culturing of TB7 hairy root line in 1/2 MS liquid medium supplemented with 30 g l(-1) sucrose for 20 days resulted in a maximal biomass accumulation (13.5 g l(-1) fresh weight, 1.78 g l(-1) dry weight) and rutin content (0.85 mg g(-1)). The suspension culture of hairy roots led to a 45-fold biomass increase and a 4.11-fold rutin content increase in comparison with the suspension culture of non-transformed roots. The transformation frequency was enhanced through preculturing for 2 days followed by infection for 20 min. The UV-B stress treatment of hairy roots resulted in a striking increase of rutin and quercetin production. Furthermore, the hairy root lines of TB3, TB7, and TB28 were chosen to study the specific effects of UV-B on flavonoid accumulation and flavonoid biosynthetic gene expression by qRT-PCR. This study has demonstrated that the UV-B radiation was an effective elicitor that dramatically changed in the transcript abundance of ftpAL, FtCHI, FtCHS, FtF3H, and FtFLS-1 in F. tataricum hairy roots. PMID:26870075

  14. Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca

    PubMed Central

    Zhang, Junxiang; Jiang, Guihua; Miao, Lixiang; Han, Guofen; Liu, Yuexue; Li, He; Zhang, Zhihong

    2015-01-01

    Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca. PMID:26636322

  15. Transcript Quantification by RNA-Seq Reveals Differentially Expressed Genes in the Red and Yellow Fruits of Fragaria vesca.

    PubMed

    Zhang, Yuchao; Li, Weijia; Dou, Yujuan; Zhang, Junxiang; Jiang, Guihua; Miao, Lixiang; Han, Guofen; Liu, Yuexue; Li, He; Zhang, Zhihong

    2015-01-01

    Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow (Yellow Wonder, YW) and red (Ruegen, RG) fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca. PMID:26636322

  16. Identification of differentially expressed genes implicated in peel color (red and green) of Dimocarpus confinis.

    PubMed

    Jiang, Fan; Chen, Xiu-Ping; Hu, Wen-Shun; Zheng, Shao-Quan

    2016-01-01

    Nowadays, there are few reports about regulatory genes implicated in peel color of longan. The basic genetic research of longan has been in stagnation for a long time as a lack of transcriptomic and genetic information. To predict candidate genes associated with peel color, Gene Functional Annotation and Coding Sequence prediction were used to perform functional annotation for our assembled unigenes and investigate differentially expressed genes (DEGs) of fruitlet peels from Longli (Dimocarpus confinis). Finally, a total of 24,044 (44.19 %) unigenes were annotated at least in one database after BLAST search to NCBI non-redundant protein sequence, NCBI non-redundant nucleotide sequences, Kyoto Encyclopedia of Genes and Genomes (KEGG) Ortholog, manually annotated and reviewed protein sequence database (Swiss-Prot), Protein family, Gene Ontology, euKaryotic Ortholog Groups databases. After searching against the KEGG-GENE protein database, a result of 6228 (11.45 %) unigenes were assigned to 245 KEGG pathways. Via comparing the distributions of expression value of all corresponding unigenes from red peel and green peel fruit, it could be intuitively concluded that high similarity was existed in the two distributions; however, on the whole, between two distributions of log RPKM expression value, some differences indicated that expression level in green-peel fruit group is slightly higher than values in red-peel fruit group. Finally, a total of 1349 unigenes were identified as DEGs after blasting the DEGs to public sequence databases, and 32 peel-color-related genes were identified in longan. Our results suggest that a number of unigenes involved in longan metabolic process, including anthocyanin biosynthesis. In addition, DRF, F3H, ANS, CYP75A1 and C1 may be the key ones. The study on key genes related to peel color will be contributed to revealing the molecular mechanisms of regulating peel color in woody plants. PMID:27468388

  17. Synthesis and herbicidal activity evaluation of novel α-amino phosphonate derivatives containing a uracil moiety.

    PubMed

    Che, Jian-yi; Xu, Xiao-yun; Tang, Zi-long; Gu, Yu-cheng; Shi, De-qing

    2016-02-15

    A series of novel α-amino phosphonate derivatives containing a uracil moiety 3a-3l were designed and synthesized by a Lewis acid (magnesium perchlorate) catalyzed the Kabachnik-Fields reaction. The bioassays {in vitro, in vivo [Glass House 1 (GH1) and Glass House 2 (GH2)]} showed that most of compounds 3 exhibited excellent and selective herbicidal activities; for example, in GH1 test, compounds 3b, 3d, 3f, 3h and 3j showed excellent and wide spectrum herbicidal activities at the dose of 1000 g/ha, and compounds 3b and 3j exhibited 100% inhibition activities against the four plants in both post- and pre-emergence treatments. Moreover, most of compounds 3 showed higher inhibition against Amaranthus retroflexus and Digitaria sanguinalis than Glyphosate did in pre-emergence treatment. In GH2 test, the four compounds (3b, 3d, 3h and 3j) exhibited 100% inhibition against Solanum nigrum, Amaranthus retroflexus and Ipomoea hederacea in post-emergence treatment and displayed 100% inhibition against Solanum nigrum, Amaranthus retroflexus in pre-emergence treatment at the rate of 250 g/ha, and compound 3b showed the best and broad spectrum herbicidal activities against the six test plants. However, the four compounds displayed weaker herbicidal activities against Lolium perenne and Echinochloa crus-galli than the other four plants at the rate of 250 g/ha in both pre- and post-emergence treatments. So, compounds 3 can be used as a lead compound for further structure optimization for developing potential selective herbicidal agent. Their preliminary structure-activity relationships were also investigated. PMID:26786699

  18. Flavonols: old compounds for old roles

    PubMed Central

    Pollastri, Susanna; Tattini, Massimiliano

    2011-01-01

    Background New roles for flavonoids, as developmental regulators and/or signalling molecules, have recently been proposed in eukaryotic cells exposed to a wide range of environmental stimuli. In plants, these functions are actually restricted to flavonols, the ancient and widespread class of flavonoids. In mosses and liverworts, the whole set of genes for flavonol biosynthesis – CHS, CHI, F3H, FLS and F3′H – has been detected. The flavonol branch pathway has remained intact for millions of years, and is almost exclusively involved in the responses of plants to a wide array of stressful agents, despite the fact that evolution of flavonoid metabolism has produced >10 000 structures. Scope Here the emerging functional roles of flavonoids in the responses of present-day plants to different stresses are discussed based on early, authoritative views of their primary functions during the colonization of land by plants. Flavonols are not as efficient as other secondary metabolites in absorbing wavelengths in the 290–320 nm spectral region, but display the greatest potential to keep stress-induced changes in cellular reactive oxygen species homeostasis under control, and to regulate the development of individual organs and the whole plant. Very low flavonol concentrations, as probably occurred in early terrestrial plants, may fully accomplish these regulatory functions. Conclusions During the last two decades the routine use of genomic, chromatography/mass spectrometry and fluorescence microimaging techniques has provided new insights into the regulation of flavonol metabolism as well as on the inter- and intracellular distribution of stress-responsive flavonols. These findings offer new evidence on how flavonols may have performed a wide array of functional roles during the colonization of land by plants. In our opinion this ancient flavonoid class is still playing the same old and robust roles in present-day plants. PMID:21880658

  19. Changes in the Chondrocyte and Extracellular Matrix Proteome during Post-natal Mouse Cartilage Development*

    PubMed Central

    Wilson, Richard; Norris, Emma L.; Brachvogel, Bent; Angelucci, Constanza; Zivkovic, Snezana; Gordon, Lavinia; Bernardo, Bianca C.; Stermann, Jacek; Sekiguchi, Kiyotoshi; Gorman, Jeffrey J.; Bateman, John F.

    2012-01-01

    cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head. PMID:21989018

  20. Differential effects of collagen prolyl 3-hydroxylation on skeletal tissues.

    PubMed

    Homan, Erica P; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M; Dawson, Brian; Bertin, Terry K; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H L

    2014-01-01

    Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1(H662A) ). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

  1. Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

    PubMed Central

    Homan, Erica P.; Lietman, Caressa; Grafe, Ingo; Lennington, Jennifer; Morello, Roy; Napierala, Dobrawa; Jiang, Ming-Ming; Munivez, Elda M.; Dawson, Brian; Bertin, Terry K.; Chen, Yuqing; Lua, Rhonald; Lichtarge, Olivier; Hicks, John; Weis, Mary Ann; Eyre, David; Lee, Brendan H. L.

    2014-01-01

    Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity

  2. Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation.

    PubMed

    Król, P; Igielski, R; Pollmann, S; Kępczyńska, E

    2015-05-01

    Methyl jasmonate (MeJA) was tested by seed treatment for its ability to protect tomato seedlings against fusarium wilt caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. lycopersici. Isolated from Solanum lycopersicon L. seeds, cv. Beta fungus was identified as F. oxysporum f.sp. lycopersici Race 3 fungus by using phytopathological and molecular methods. MeJA applied at 0.01, 0.1 and 1 mM reduced spore germination and mycelial growth in vitro. Soaking of tomato seeds in MeJA solution at 0.1 mM for 1 h significantly enhanced the resistance level against the tested fungus in tomato seedlings 4 weeks after inoculation. The extracts from leaves of 15-day-old seedlings obtained from previously MeJA soaked seeds had the ability to inhibit in vitro spore germination of tested fungus. In these seedlings a significant increase in the levels phenolic compounds such as salicylic acid (SA), kaempferol and quercetin was observed. Up-regulation of phenylalanine ammonia-lyase (PAL5) and benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) genes and down-regulation of the isochorysmate synthase (ICS) gene in response to exogenous MeJA application indicate that the phenylalanine ammonia-lyase (PAL), not the isochorismate (IC) pathway, is the primary route for SA production in tomato. Moreover, the increased accumulation of the flavonols quercetin and kaempferol appears closely related to the increase of PAL5, chalcone synthase (CHS) and flavonol synthase/flavanone 3-hydroxylase-like (FLS) genes. Elevated levels of salicylic acid in seedlings raised from MeJA-soaked seeds were simultaneously accompanied by a decrease of jasmonic acid, the precursor of MeJA, and an increase of 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid. The present results indicate that the priming of tomato seeds with 0.1mM MeJA before sowing enables the seedlings grown from these seeds to reduce the attack of the soil-borne fungal pathogen F. oxysporum f.sp. lycopersici

  3. PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics

    PubMed Central

    2013-01-01

    Background Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics. Results A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb (http://urgv.evry.inra.fr/UTILLdb). Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species. Conclusions We have developed a flax mutant population that

  4. De novo characterization of Larix gmelinii (Rupr.) Rupr. transcriptome and analysis of its gene expression induced by jasmonates

    PubMed Central

    2013-01-01

    Background Larix gmelinii is a dominant tree species in China’s boreal forests and plays an important role in the coniferous ecosystem. It is also one of the most economically important tree species in the Chinese timber industry due to excellent water resistance and anti-corrosion of its wood products. Unfortunately, in Northeast China, L. gmelinii often suffers from serious attacks by diseases and insects. The application of exogenous volatile semiochemicals may induce and enhance its resistance against insect or disease attacks; however, little is known regarding the genes and molecular mechanisms related to induced resistance. Results We performed de novo sequencing and assembly of the L. gmelinii transcriptome using a short read sequencing technology (Illumina). Chemical defenses of L. gmelinii seedlings were induced with jasmonic acid (JA) or methyl jasmonate (MeJA) for 6 hours. Transcriptomes were compared between seedlings induced by JA, MeJA and untreated controls using a tag-based digital gene expression profiling system. In a single run, 25,977,782 short reads were produced and 51,157 unigenes were obtained with a mean length of 517 nt. We sequenced 3 digital gene expression libraries and generated between 3.5 and 5.9 million raw tags, and obtained 52,040 reliable reference genes after removing redundancy. The expression of disease/insect-resistance genes (e.g., phenylalanine ammonialyase, coumarate 3-hydroxylase, lipoxygenase, allene oxide synthase and allene oxide cyclase) was up-regulated. The expression profiles of some abundant genes under different elicitor treatment were studied by using real-time qRT-PCR. The results showed that the expression levels of disease/insect-resistance genes in the seedling samples induced by JA and MeJA were higher than those in the control group. The seedlings induced with MeJA elicited the strongest increases in disease/insect-resistance genes. Conclusions Both JA and MeJA induced seedlings of L. gmelinii showed

  5. An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof

    NASA Technical Reports Server (NTRS)

    Costa, Michael A.; Collins, R. Eric; Anterola, Aldwin M.; Cochrane, Fiona C.; Davin, Laurence B.; Lewis, Norman G.

    2003-01-01

    The Arabidopsis genome sequencing in 2000 gave to science the first blueprint of a vascular plant. Its successful completion also prompted the US National Science Foundation to launch the Arabidopsis 2010 initiative, the goal of which is to identify the function of each gene by 2010. In this study, an exhaustive analysis of The Institute for Genomic Research (TIGR) and The Arabidopsis Information Resource (TAIR) databases, together with all currently compiled EST sequence data, was carried out in order to determine to what extent the various metabolic networks from phenylalanine ammonia lyase (PAL) to the monolignols were organized and/or could be predicted. In these databases, there are some 65 genes which have been annotated as encoding putative enzymatic steps in monolignol biosynthesis, although many of them have only very low homology to monolignol pathway genes of known function in other plant systems. Our detailed analysis revealed that presently only 13 genes (two PALs, a cinnamate-4-hydroxylase, a p-coumarate-3-hydroxylase, a ferulate-5-hydroxylase, three 4-coumarate-CoA ligases, a cinnamic acid O-methyl transferase, two cinnamoyl-CoA reductases) and two cinnamyl alcohol dehydrogenases can be classified as having a bona fide (definitive) function; the remaining 52 genes currently have undetermined physiological roles. The EST database entries for this particular set of genes also provided little new insight into how the monolignol pathway was organized in the different tissues and organs, this being perhaps a consequence of both limitations in how tissue samples were collected and in the incomplete nature of the EST collections. This analysis thus underscores the fact that even with genomic sequencing, presumed to provide the entire suite of putative genes in the monolignol-forming pathway, a very large effort needs to be conducted to establish actual catalytic roles (including enzyme versatility), as well as the physiological function(s) for each member

  6. Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence

    PubMed Central

    2010-01-01

    Background Lignification of the fruit endocarp layer occurs in many angiosperms and plays a critical role in seed protection and dispersal. This process has been extensively studied with relationship to pod shatter or dehiscence in Arabidopsis. Dehiscence is controlled by a set of transcription factors that define the fruit tissue layers and whether or not they lignify. In contrast, relatively little is known about similar processes in other plants such as stone fruits which contain an extremely hard lignified endocarp or stone surrounding a single seed. Results Here we show that lignin deposition in peach initiates near the blossom end within the endocarp layer and proceeds in a distinct spatial-temporal pattern. Microarray studies using a developmental series from young fruits identified a sharp and transient induction of phenylpropanoid, lignin and flavonoid pathway genes concurrent with lignification and subsequent stone hardening. Quantitative polymerase chain reaction studies revealed that specific phenylpropanoid (phenylalanine ammonia-lyase and cinnamate 4-hydroxylase) and lignin (caffeoyl-CoA O-methyltransferase, peroxidase and laccase) pathway genes were induced in the endocarp layer over a 10 day time period, while two lignin genes (p-coumarate 3-hydroxylase and cinnamoyl CoA reductase) were co-regulated with flavonoid pathway genes (chalcone synthase, dihydroflavanol 4-reductase, leucoanthocyanidin dioxygen-ase and flavanone-3-hydrosylase) which were mesocarp and exocarp specific. Analysis of other fruit development expression studies revealed that flavonoid pathway induction is conserved in the related Rosaceae species apple while lignin pathway induction is not. The transcription factor expression of peach genes homologous to known endocarp determinant genes in Arabidopsis including SHATTERPROOF, SEEDSTCK and NAC SECONDARY WALL THICENING PROMOTING FACTOR 1 were found to be specifically expressed in the endocarp while the negative regulator FRUITFUL

  7. A new allele of flower color gene W1 encoding flavonoid 3'5'-hydroxylase is responsible for light purple flowers in wild soybean Glycine soja

    PubMed Central

    2010-01-01

    Background Glycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat. Results B09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants. Conclusions B09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be

  8. Transcriptomic Analysis of Paeonia delavayi Wild Population Flowers to Identify Differentially Expressed Genes Involved in Purple-Red and Yellow Petal Pigmentation

    PubMed Central

    Wang, Yan; Li, Kui; Zheng, Baoqiang; Miao, Kun

    2015-01-01

    Tree peony (Paeonia suffruticosa Andrews) is a very famous traditional ornamental plant in China. P. delavayi is a species endemic to Southwest China that has aroused great interest from researchers as a precious genetic resource for flower color breeding. However, the current understanding of the molecular mechanisms of flower pigmentation in this plant is limited, hindering the genetic engineering of novel flower color in tree peonies. In this study, we conducted a large-scale transcriptome analysis based on Illumina HiSeq sequencing of cDNA libraries generated from yellow and purple-red P. delavayi petals. A total of 90,202 unigenes were obtained by de novo assembly, with an average length of 721 nt. Using Blastx, 44,811 unigenes (49.68%) were found to have significant similarity to accessions in the NR, NT, and Swiss-Prot databases. We also examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Further analysis of the two digital transcriptomes revealed that 6,855 unigenes were differentially expressed between yellow and purple-red flower petals, with 3,430 up-regulated and 3,425 down-regulated. According to the RNA-Seq data and qRT-PCR analysis, we proposed that four up-regulated key structural genes, including F3H, DFR, ANS and 3GT, might play an important role in purple-red petal pigmentation, while high co-expression of THC2'GT, CHI and FNS II ensures the accumulation of pigments contributing to the yellow color. We also found 50 differentially expressed transcription factors that might be involved in flavonoid biosynthesis. This study is the first to report genetic information for P. delavayi. The large number of gene sequences produced by transcriptome sequencing and the candidate genes identified using pathway mapping and expression profiles will provide a valuable resource for future association studies aimed at better understanding the molecular mechanisms underlying flower pigmentation in tree peonies. PMID

  9. Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves

    PubMed Central

    2010-01-01

    Background Symptoms of grapevine leafroll disease (GLRD) in red-fruited wine grape (Vitis vinifera L.) cultivars consist of green veins and red and reddish-purple discoloration of inter-veinal areas of leaves. The reddish-purple color of symptomatic leaves may be due to the accumulation of anthocyanins and could reflect an up-regulation of genes involved in their biosynthesis. Results We examined six putative constitutively expressed genes, Ubiquitin, Actin, GAPDH, EF1-a, SAND and NAD5, for their potential as references for normalization of gene expression in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Using the geNorm program, a combination of two genes (Actin and NAD5) was identified as the stable set of reference genes for normalization of gene expression data obtained from grapevine leaves. By using gene-specific RT-qPCR in combination with a reliable normalization factor, we compared relative expression of the flavonoid biosynthetic pathway genes between leaves infected with Grapevine leafroll-associated virus 3 (GLRaV-3) and exhibiting GLRD symptoms and virus-free green leaves obtained from a red-fruited wine grape cultivar (cv. Merlot). The expression levels of these different genes ranged from two- to fifty-fold increase in virus-infected leaves. Among them, CHS3, F3'5'H, F3H1, LDOX, LAR1 and MybA1 showed greater than 10-fold increase suggesting that they were expressed at significantly higher levels in virus-infected symptomatic leaves. HPLC profiling of anthocyanins extracted from leaves indicated the presence of cyanidin-3-glucoside and malvidin-3-glucoside only in virus-infected symptomatic leaves. The results also showed 24% higher levels of flavonols in virus-infected symptomatic leaves than in virus-free green leaves, with quercetin followed by myricetin being the predominant compounds. Proanthocyanidins, estimated as total tannins by protein precipitation method, were 36% higher in virus-infected symptomatic

  10. Synthesis, structure, and reductive elimination in the series Tp'Rh(PR3)(Ar(F))H; determination of rhodium-carbon bond energies of fluoroaryl substituents.

    PubMed

    Tanabe, Taro; Brennessel, William W; Clot, Eric; Eisenstein, Odile; Jones, William D

    2010-11-21

    A series of complexes of the type Tp'Rh(PR(3))(Ar(F))H, where PR(3) = PMe(3) (3) and PMe(2)Ph (9), Ar(F) = C(6)F(5) (a), 2,3,4,5-C(6)F(4)H (b), 2,3,5,6-C(6)F(4)H (c), 2,4,6-C(6)F(3)H(2) (d), 2,3-C(6)F(2)H(3) (e), 2,5-C(6)F(2)H(3) (g), and 2-C(6)FH(4) (h) and Tp' = tris(3,5-dimethylpyrazolyl)borate, has been synthesized as stable crystalline compounds by the reactions of the [Tp'Rh(PR(3))] fragment with the corresponding fluorinated aromatic hydrocarbons, and their structures were characterized by NMR spectroscopy and elemental analysis together with X-ray crystallography. The kinetics of the reductive eliminations of fluoroarenes from complexes 3a-h in benzene-d(6) solutions at 140 °C were investigated, but were complicated by the formation of the rhodium(I) bisphosphine complex, Tp'Rh(PMe(3))(2) (4). On the other hand, thermal reactions of (9) in THF-d(8) solutions at 120 °C resulted in the formation of an intramolecular C-H bond activated complex of the phenyl group on the phosphorus atom, Tp'Rh(κ(2)-C(6)H(4)-2-PMe(2))H (7), which prevents the formation of the corresponding bisphosphine complex. The experimentally determined rates of the reductive eliminations of fluoroarenes from the complexes 9a-h and their kinetic selectivities for formation in competition with the metallacycle have been used to determine relative Rh-CAr(F) bond energies. The Rh-CAr(F) bond energy is found to be dependent on the number of ortho fluorines. A plot of Rh-CAr(F) vs. C-H bond strengths resulted in a line with a slope R(M-C/C-H) of 2.15 that closely matches the DFT calculated value (slope = 2.05). PMID:20924525

  11. Transcriptomic Analysis of Paeonia delavayi Wild Population Flowers to Identify Differentially Expressed Genes Involved in Purple-Red and Yellow Petal Pigmentation.

    PubMed

    Shi, Qianqian; Zhou, Lin; Wang, Yan; Li, Kui; Zheng, Baoqiang; Miao, Kun

    2015-01-01

    Tree peony (Paeonia suffruticosa Andrews) is a very famous traditional ornamental plant in China. P. delavayi is a species endemic to Southwest China that has aroused great interest from researchers as a precious genetic resource for flower color breeding. However, the current understanding of the molecular mechanisms of flower pigmentation in this plant is limited, hindering the genetic engineering of novel flower color in tree peonies. In this study, we conducted a large-scale transcriptome analysis based on Illumina HiSeq sequencing of cDNA libraries generated from yellow and purple-red P. delavayi petals. A total of 90,202 unigenes were obtained by de novo assembly, with an average length of 721 nt. Using Blastx, 44,811 unigenes (49.68%) were found to have significant similarity to accessions in the NR, NT, and Swiss-Prot databases. We also examined COG, GO and KEGG annotations to better understand the functions of these unigenes. Further analysis of the two digital transcriptomes revealed that 6,855 unigenes were differentially expressed between yellow and purple-red flower petals, with 3,430 up-regulated and 3,425 down-regulated. According to the RNA-Seq data and qRT-PCR analysis, we proposed that four up-regulated key structural genes, including F3H, DFR, ANS and 3GT, might play an important role in purple-red petal pigmentation, while high co-expression of THC2'GT, CHI and FNS II ensures the accumulation of pigments contributing to the yellow color. We also found 50 differentially expressed transcription factors that might be involved in flavonoid biosynthesis. This study is the first to report genetic information for P. delavayi. The large number of gene sequences produced by transcriptome sequencing and the candidate genes identified using pathway mapping and expression profiles will provide a valuable resource for future association studies aimed at better understanding the molecular mechanisms underlying flower pigmentation in tree peonies. PMID

  12. Differential effects of environment on potato phenylpropanoid and carotenoid expression

    PubMed Central

    2012-01-01

    Background Plant secondary metabolites, including phenylpropanoids and carotenoids, are stress inducible, have important roles in potato physiology and influence the nutritional value of potatoes. The type and magnitude of environmental effects on tuber phytonutrients is unclear, especially under modern agricultural management that minimizes stress. Understanding factors that influence tuber secondary metabolism could facilitate production of more nutritious crops. Metabolite pools of over forty tuber phenylpropanoids and carotenoids, along with the expression of twenty structural genes, were measured in high-phenylpropanoid purple potatoes grown in environmentally diverse locations in North America (Alaska, Texas and Florida). Results Phenylpropanoids, including chlorogenic acid (CGA), were higher in samples from the northern latitudes, as was the expression of phenylpropanoid genes including phenylalanine ammonia lyase (PAL), which had over a ten-fold difference in relative abundance. Phenylpropanoid gene expression appeared coordinately regulated and was well correlated with metabolite pools, except for hydroxycinnamoyl-CoA:quinatehydroxcinnamoyl transferase (HQT; r = -0.24). In silico promoter analysis identified two cis-acting elements in the HQT promoter not found in the other phenylpropanoid genes. Anthocyanins were more abundant in Alaskan samples and correlated with flavonoid genes including DFR (r = 0.91), UFGT (r = 0.94) and F3H (r = 0.77). The most abundant anthocyanin was petunidin-3-coum-rutinoside-5-glu, which ranged from 4.7 mg g-1 in Alaska to 2.3 mg g-1 in Texas. Positive correlations between tuber sucrose and anthocyanins (r = 0.85), suggested a stimulatory effect of sucrose. Smaller variation was observed in total carotenoids, but marked differences occurred in individual carotenoids, which had over a ten-fold range. Violaxanthin, lutein or zeaxanthin were the predominant carotenoids in tubers from Alaska, Texas and Florida respectively. Unlike

  13. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

    NASA Technical Reports Server (NTRS)

    Anterola, Aldwin M.; Lewis, Norman G.

    2002-01-01

    A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H

  14. Heats of formation of diphosphene, phosphinophosphinidene, diphosphine, and their methyl derivatives, and mechanism of the borane-assisted hydrogen release.

    PubMed

    Matus, Myrna H; Nguyen, Minh Tho; Dixon, David A

    2007-03-01

    The heats of formation of diphosphene (cis- and trans-P2H2), phopshinophosphinidene (singlet and triplet H2PP) and diphosphine (P2H4), as well as those of the P2H and P2H3 radicals resulting from PH bond cleavages, have been calculated by using high-level ab initio electronic structure theory. Energies were calculated using coupled-cluster theory with a perturbative treatment for triple excitations (CCSD(T)) and employing augmented correlation consistent basis sets with additional tight d-functions on P (aug-cc-pV(n+d)Z) up to quadruple- or quintuple-zeta, to perform a complete basis set extrapolation for the energy. Geometries and vibrational frequencies were determined with the CCSD(T) method. Core-valence and scalar relativistic corrections were included, as well as scaled zero-point energies. We find the following heats of formation (kcal/mol) at 298 [0] K: DeltaH(degree)(f)(P2H) = 53.4 [54.4]; DeltaH(degree)(f)(cis-P2H2) = 32.0 [33.9]; DeltaH(degree)(f)(trans-P2H2) = 28.7 [30.6]; DeltaH(degree)(f)(H2PP) = 53.7 [55.6]; DeltaH(degree)(f)(3H2PP) = 56.5 [58.3]; DeltaH(degree)(f)(P2H3) = 32.3 [34.8]; DeltaH(degree)(f)(P2H4) = 5.7 [9.1] (expt, 5.0 +/- 1.0 at 298 K); and DeltaH(degree)(f)(CH3PH2) = -5.0 [-1.4]. We estimate these values to have an accuracy of +/-1.0 kcal/mol. In contrast to earlier results, we found a singlet ground state for phosphinophosphinidene (H2PP) with a singlet-triplet energy gap of 2.8 kcal/mol. We calculated the heats of formation of the methylated derivatives CH3PPH, CH3HPPH2, CH3PPCH3, CH3HPP, (CH3)2PP, (CH3)2PPH2, and CH3HPPHCH3 by using isodesmic reactions at the MP2/CBS level. The calculated results for the hydrogenation reactions RPPR + H2 --> RHPPHR and R2PP + H2 --> R2PPH2 show that substitution of an organic substituent for H improves the energetics, suggesting that secondary diphosphines and diphosphenes are potential candidates for use in a chemical hydrogen storage system. A comparison with the nitrogen analogues is given. The

  15. Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity

    NASA Technical Reports Server (NTRS)

    Anterola, Aldwin M.; Lewis, Norman G.

    2002-01-01

    A comprehensive assessment of lignin configuration in transgenic and mutant plants is long overdue. This review thus undertook the systematic analysis of trends manifested through genetic and mutational manipulations of the various steps associated with monolignol biosynthesis; this included consideration of the downstream effects on organized lignin assembly in the various cell types, on vascular function/integrity, and on plant growth and development. As previously noted for dirigent protein (homologs), distinct and sophisticated monolignol forming metabolic networks were operative in various cell types, tissues and organs, and form the cell-specific guaiacyl (G) and guaiacyl-syringyl (G-S) enriched lignin biopolymers, respectively. Regardless of cell type undergoing lignification, carbon allocation to the different monolignol pools is apparently determined by a combination of phenylalanine availability and cinnamate-4-hydroxylase/"p-coumarate-3-hydroxylase" (C4H/C3H) activities, as revealed by transcriptional and metabolic profiling. Downregulation of either phenylalanine ammonia lyase or cinnamate-4-hydroxylase thus predictably results in reduced lignin levels and impaired vascular integrity, as well as affecting related (phenylpropanoid-dependent) metabolism. Depletion of C3H activity also results in reduced lignin deposition, albeit with the latter being derived only from hydroxyphenyl (H) units, due to both the guaiacyl (G) and syringyl (S) pathways being blocked. Apparently the cells affected are unable to compensate for reduced G/S levels by increasing the amounts of H-components. The downstream metabolic networks for G-lignin enriched formation in both angiosperms and gymnosperms utilize specific cinnamoyl CoA O-methyltransferase (CCOMT), 4-coumarate:CoA ligase (4CL), cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) isoforms: however, these steps neither affect carbon allocation nor H/G designations, this being determined by C4H/C3H

  16. Poster Session A

    PubMed Central

    2014-01-01

    appearance of non-specific associated proteins, and the relatively low data reproducibility across different studies. Here we present a Minkowski distance-based probabilistic scoring method for label-free AP/MS data, which assigns standardized probability scores to interactions in a data-driven manner. We provide a knowledge-driven mode using available AP/MS datasets as controls, and a knowledge-free mode using customized negative controls, to deal with different types and scales of datasets. Comparing with other algorithms, it works more efficiently in both recognizing true positives and eliminating false positives in AP/MS datasets we have tested. Using this method, we filtered TAP/MS data generated by our lab and other labs and achieved high interlaboratory data reproducibility. We hope this new computational tool will be able to direct in-depth functional study of proteins more efficiently, and be helpful for building up a unified human protein interactome. A.11 Large Scale Proteomic Characterisation of Sites of Proline Hydroxylation in Human Proteins Dalila Bensaddek, Sandra C. Moser, Brian Ortmann, Sonia Rocha, Jason R. Swedlow, Angus I. Lamond Centre for Gene Regulation and Expression, Dundee, Scotland, GB Proline hydroxylation is an abundant post-translational modification in humans that is catalysed by prolyl 4- and prolyl 3-hydroxylases to yield 4-hydroxyproline and 3-hydroxyproline, respectively. There are three isoforms of prolyl-4 hydroxylases, designated Prolyl Hydroxylase Domain proteins PHDs (PHD1-3 or EGLN1-3). Hydroxylation of proline residues plays a critical role in sensing molecular oxygen levels. Under normal oxygen levels (normoxia) PHDs catalyse proline hydroxylation within the oxygen-dependent degradation domain (ODDD) of the hypoxia inducible factor one-α (HIF1-α). This promotes its binding to the von Hippel Lindau protein (pVHL), which acts as a targeting component for an E3 ubiquitin ligase complex that mediates rapid degradation of HIF1-α by