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Sample records for plastidic isoprenoid synthesis

  1. Plastidic Isoprenoid Synthesis during Chloroplast Development 1

    PubMed Central

    Heintze, Adolf; Görlach, Jörn; Leuschner, Carola; Hoppe, Petra; Hagelstein, Petra; Schulze-Siebert, Detlef; Schultz, Gernot

    1990-01-01

    The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO2 → pyruvate → acetyl-coenzyme A (C3 → C2) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C3 → C2 metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO2-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH14CO3 decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A. PMID:16667567

  2. Bisphosphonate inhibitors reveal a large elasticity of plastidic isoprenoid synthesis pathway in isoprene-emitting hybrid aspen.

    PubMed

    Rasulov, Bahtijor; Talts, Eero; Kännaste, Astrid; Niinemets, Ülo

    2015-06-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-D-xylulose 5-phosphate (DXP)/2-C-methyl-D-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

  3. Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen1

    PubMed Central

    2015-01-01

    Recently, a feedback inhibition of the chloroplastic 1-deoxy-d-xylulose 5-phosphate (DXP)/2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid synthesis by end products dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) was postulated, but the extent to which DMADP and IDP can build up is not known. We used bisphosphonate inhibitors, alendronate and zoledronate, that inhibit the consumption of DMADP and IDP by prenyltransferases to gain insight into the extent of end product accumulation and possible feedback inhibition in isoprene-emitting hybrid aspen (Populus tremula × Populus tremuloides). A kinetic method based on dark release of isoprene emission at the expense of substrate pools accumulated in light was used to estimate the in vivo pool sizes of DMADP and upstream metabolites. Feeding with fosmidomycin, an inhibitor of DXP reductoisomerase, alone or in combination with bisphosphonates was used to inhibit carbon input into DXP/MEP pathway or both input and output. We observed a major increase in pathway intermediates, 3- to 4-fold, upstream of DMADP in bisphosphonate-inhibited leaves, but the DMADP pool was enhanced much less, 1.3- to 1.5-fold. In combined fosmidomycin/bisphosphonate treatment, pathway intermediates accumulated, reflecting cytosolic flux of intermediates that can be important under strong metabolic pull in physiological conditions. The data suggested that metabolites accumulated upstream of DMADP consist of phosphorylated intermediates and IDP. Slow conversion of the huge pools of intermediates to DMADP was limited by reductive energy supply. These data indicate that the DXP/MEP pathway is extremely elastic, and the presence of a significant pool of phosphorylated intermediates provides an important valve for fine tuning the pathway flux. PMID:25926480

  4. Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; Quennemet, J; d'Harlingue, A; Camara, B

    1996-01-01

    Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals. PMID:8787029

  5. Metabolic Flux Analysis of Plastidic Isoprenoid Biosynthesis in Poplar Leaves Emitting and Nonemitting Isoprene1[W

    PubMed Central

    Ghirardo, Andrea; Wright, Louwrance Peter; Bi, Zhen; Rosenkranz, Maaria; Pulido, Pablo; Rodríguez-Concepción, Manuel; Niinemets, Ülo; Brüggemann, Nicolas; Gershenzon, Jonathan; Schnitzler, Jörg-Peter

    2014-01-01

    The plastidic 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway is one of the most important pathways in plants and produces a large variety of essential isoprenoids. Its regulation, however, is still not well understood. Using the stable isotope 13C-labeling technique, we analyzed the carbon fluxes through the MEP pathway and into the major plastidic isoprenoid products in isoprene-emitting and transgenic isoprene-nonemitting (NE) gray poplar (Populus × canescens). We assessed the dependence on temperature, light intensity, and atmospheric [CO2]. Isoprene biosynthesis was by far (99%) the main carbon sink of MEP pathway intermediates in mature gray poplar leaves, and its production required severalfold higher carbon fluxes compared with NE leaves with almost zero isoprene emission. To compensate for the much lower demand for carbon, NE leaves drastically reduced the overall carbon flux within the MEP pathway. Feedback inhibition of 1-deoxy-d-xylulose-5-phosphate synthase activity by accumulated plastidic dimethylallyl diphosphate almost completely explained this reduction in carbon flux. Our data demonstrate that short-term biochemical feedback regulation of 1-deoxy-d-xylulose-5-phosphate synthase activity by plastidic dimethylallyl diphosphate is an important regulatory mechanism of the MEP pathway. Despite being relieved from the large carbon demand of isoprene biosynthesis, NE plants redirected only approximately 0.5% of this saved carbon toward essential nonvolatile isoprenoids, i.e. β-carotene and lutein, most probably to compensate for the absence of isoprene and its antioxidant properties. PMID:24590857

  6. "One-pot" reductive lactone alkylation provides a concise asymmetric synthesis of chiral isoprenoid targets.

    PubMed

    Cao, Jia; Perlmutter, Patrick

    2013-09-01

    An efficient method, based on nucleophilic addition to lactones followed by modified in situ Clemmensen reduction, provides a short synthetic route to chiral isoprenoid targets. The efficacy of this method has been exemplified through the synthesis of several targets including the commercial fragrance Rosaphen, the side chain of Zaragozic acid C, the cotton leaf sex pheromone, and the side chains of vitamin E. PMID:23957629

  7. Isoprenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Guggisberg, Ann M.; Amthor, Rachel E.

    2014-01-01

    Malaria kills nearly 1 million people each year, and the protozoan parasite Plasmodium falciparum has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in P. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In P. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in P. falciparum has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in P. falciparum, in order to identify valuable directions for future research. PMID:25217461

  8. The prokaryotic pathway of lipid synthesis in oat leaf plastids

    SciTech Connect

    Gillanders, B.; Mackender, R. )

    1990-05-01

    Evidence for the operation of the prokaryotic pathway of acyl lipid synthesis is an 18:3 plant was sought by incubating plastids from 6 sequential segments from oat seedling leaves as described previously except that {sup 14}C-G3P was used. Proplastids were the most biosynthetically active plastids (x2-3) and DAG and PA were the most heavily labelled acyl lipids (>75% of label). Analysis of MGDG and DGDG molecular species (MS) following pre-incubation with {sup 14}C-G3P (40 mins) and subsequently with UDP{sup 3}H-gal (30 mins), showed that only MS 16:0/18:0 and MS 16:0/18:1 were significantly labelled with {sup 14}C whereas all 5 MS were labelled with {sup 3}H-gal. However most of the {sup 3}H label was in the same MS as the {sup 14}C except when 18:2/18:2 DAG or PC was added with the {sup 3}H-gal, in which case considerably additional {sup 3}H-gal (up to 100%) appeared in the 18:2/18:2 MS of both these lipids.

  9. Studies of the isoprenoid-mediated inhibition of mevalonate synthesis applied to cancer chemotherapy and chemoprevention.

    PubMed

    Mo, Huanbiao; Elson, Charles E

    2004-07-01

    Pools of farnesyl diphosphate and other phosphorylated products of the mevalonate pathway are essential to the post-translational processing and physiological function of small G proteins, nuclear lamins, and growth factor receptors. Inhibitors of enzyme activities providing those pools, namely, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and mevalonic acid-pyrophosphate decarboxylase, and of activities requiring substrates from the pools, the prenyl protein transferases, have potential for development as novel chemotherapeutic agents. Their potentials as suggested by the clinical responses recorded in Phase I and II investigations of inhibitors of HMG CoA reductase (the statins), of mevalonic acid-pyrophosphate decarboxylase (sodium phenylacetate and sodium phenylbutyrate), and of farnesyl protein transferase (R115777, SCH66336, BMS-214662, Tipifarnib, L-778,123, and, prematurely, perillyl alcohol) are dimmed by dose-limiting toxicities. These nondiscriminant growth-suppressive agents induce G1 arrest and initiate apoptosis and differentiation, effects attributed to modulation of cell signaling pathways either by modulating gene expression, suppressing the post-translational processing of signaling proteins and growth factor receptors, or altering diacylglycerol signaling. Diverse isoprenoids and the HMG CoA reductase inhibitor, lovastatin, modulate cell growth, induce cell cycle arrest, initiate apoptosis, and suppress cellular signaling activities. Perillyl alcohol, the isoprenoid of greatest clinical interest, initially was considered to inhibit farnesyl protein transferase; follow-up studies revealed that perillyl alcohol suppresses the synthesis of small G proteins and HMG CoA reductase. In sterologenic tissues, sterol feedback control, mediated by sterol regulatory element binding proteins (SREBPs) 1a and 2, exerts the primary regulation on HMG CoA reductase activity at the transcriptional level. Secondary regulation, a nonsterol isoprenoid

  10. Enzymic Capacities of Purified Cauliflower Bud Plastids for Lipid Synthesis and Carbohydrate Metabolism 1

    PubMed Central

    Journet, Etienne-Pascal; Douce, Roland

    1985-01-01

    Isolated cauliflower (Brassica oleracea) bud plastids, purified by isopycnic centrifugation in density gradients of Percoll, were found to be highly intact, to be practically devoid of extraplastidial contaminations, and to retain all the enzymes involved in fatty acid, phosphatidic acid, and monogalactosyldiacylglycerol synthesis. Purified plastids possess all the enzymes needed to convert triose phosphate to starch and vice versa, and are capable of conversion of glycerate 3-phosphate to pyruvate for fatty acid synthesis. They are also capable of oxidation of hexose phosphate and conversion to triose phosphate via the oxidative pentosephosphate pathway. Cauliflower bud plastids prove to be, therefore, biochemically very flexible organelles. Images Fig. 1 PMID:16664432

  11. Helper component-proteinase enhances the activity of 1-deoxy-D-xylulose-5-phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y-infected tobacco.

    PubMed

    Li, Heng; Ma, Dongyuan; Jin, Yongsheng; Tu, Yayi; Liu, Liping; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2015-10-01

    Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships. PMID:25736930

  12. A photoactive isoprenoid diphosphate analogue containing a stable phosphonate linkage: synthesis and biochemical studies with prenyltransferases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A number of biochemical processes rely on isoprenoids, including the post-translational modification of signaling proteins and the biosynthesis of a wide array of compounds. Photoactivatable analogues have been developed to study isoprenoid utilizing enzymes such as the isoprenoid synthases and pren...

  13. In Vitro Antimalarial Activity of Different Inhibitors of the Plasmodial Isoprenoid Synthesis Pathway.

    PubMed

    da Silva, Marcia F; Saito, Alexandre Y; Peres, Valnice J; Oliveira, Antonio C; Katzin, Alejandro M

    2015-08-01

    Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects. PMID:26055383

  14. In Vitro Antimalarial Activity of Different Inhibitors of the Plasmodial Isoprenoid Synthesis Pathway

    PubMed Central

    da Silva, Marcia F.; Saito, Alexandre Y.; Peres, Valnice J.; Oliveira, Antonio C.

    2015-01-01

    Previous studies have shown that fosmidomycin, risedronate, and nerolidol exert antimalarial activity in vitro. We included squalestatin, an inhibitor of the isoprenoid metabolism in Erwinia uredovora, and found that combinations of compounds which act on different targets of the plasmodial isoprenoid pathway possess important supra-additivity effects. PMID:26055383

  15. Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii

    PubMed Central

    Nair, Sethu C.; Brooks, Carrie F.; Goodman, Christopher D.; Strurm, Angelika; McFadden, Geoffrey I.; Sundriyal, Sandeep; Anglin, Justin L.; Song, Yongcheng; Moreno, Silvia N.J.

    2011-01-01

    Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance. PMID:21690250

  16. A cryptic algal group unveiled: a plastid biosynthesis pathway in the oyster parasite Perkinsus marinus.

    PubMed

    Matsuzaki, Motomichi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Kita, Kiyoshi; Nozaki, Hisayoshi

    2008-06-01

    Plastids are widespread in plant and algal lineages. They are also exploited by some nonphotosynthetic protists, including malarial parasites, to support their diverse modes of life. However, cryptic plastids may exist in other nonphotosynthetic protists, which could be important in studies on the diversity and evolution of plastids. The parasite Perkinsus marinus, which causes mass mortality in oyster farms, is a nonphotosynthetic protist that is phylogenetically related to plastid-bearing dinoflagellates and apicomplexans. In this study, we searched for P. marinus methylerythritol phosphate (MEP) pathway genes, responsible for de novo isoprenoid synthesis in plastids, and determined the full-length gene sequences for 6 of 7 of these genes. Phylogenetic analyses revealed that each P. marinus gene clusters with orthologs from plastid-bearing eukaryotes, which have MEP pathway genes with essentially the same mosaic pattern of evolutionary origin. A new analytical method called sliding-window iteration of TargetP was developed to examine the distribution of targeting preferences. This analysis revealed that the sequenced genes encode bipartite targeting peptides that are characteristic of proteins targeted to secondary plastids originating from endosymbiosis of eukaryotic algae. These results support our idea that Perkinsus is a cryptic algal group containing nonphotosynthetic secondary plastids. In fact, immunofluorescent microscopy indicated that 1 of the MEP pathway enzymes, 1-deoxy-D-xylulose 5-phosphate reductoisomerase, was localized to small compartments near mitochondrion, which are possibly plastids. This tiny organelle seems to contain very low quantities of DNA or may even lack DNA entirely. The MEP pathway genes are a useful tool for investigating plastid evolution in both of the photosynthetic and nonphotosynthetic eukaryotes and led us to propose the hypothesis that ancestral "chromalveolates" harbored plastids before a secondary endosymbiotic event

  17. Evidence for a Contribution of ALA Synthesis to Plastid-To-Nucleus Signaling

    SciTech Connect

    Czarnecki, Olaf; Gläßer, Christine; Chen, Jin-Gui; Mayer, Klaus F. X.; Grimm, Bernhard

    2012-01-01

    The formation of 5-aminolevulinic acid (ALA) in tetrapyrrole biosynthesis is widely controlled by environmental and metabolic feedback cues that determine the influx into the entire metabolic path. Because of its central role as the rate-limiting step, we hypothesized a potential role of ALA biosynthesis in tetrapyrrole-mediated retrograde signaling and exploited the direct impact of ALA biosynthesis on nuclear gene expression (NGE) by using two different approaches. Firstly, the Arabidopsis gun1, hy1 (gun2), hy2 (gun3), gun4 mutants showing uncoupled NGE from the physiological state of chloroplasts were thoroughly examined for regulatory modifications of ALA synthesis and transcriptional control in the nucleus. We found that reduced ALA-synthesizing capacity is common to analyzed gun mutants. Inhibition of ALA synthesis by gabaculine (GAB) that inactivates glutamate-1-semialdehyde aminotransferase and ALA feeding of wild-type and mutant seedlings corroborate the expression data of gun mutants. Transcript level of photosynthetic marker genes were enhanced in norflurazon (NF)-treated seedlings upon additional GAB treatment, while enhanced ALA amounts diminish these RNA levels in NF-treated wild-type in comparison to the solely NF-treated seedlings. Secondly, the impact of posttranslationally down-regulated ALA synthesis on NGE was investigated by global transcriptome analysis of GAB-treated Arabidopsis seedlings and the gun4-1 mutant, which is also characterized by reduced ALA formation. A common set of significantly modulated genes was identified indicating ALA synthesis as a potential signal emitter. The over-represented gene ontology categories of genes with decreased or increased transcript abundance highlight a few biological processes and cellular functions, which are remarkably affected in response to plastid-localized ALA biosynthesis. These results support the hypothesis that ALA biosynthesis correlates with retrograde signaling-mediated control of NGE.

  18. De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

    PubMed

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-04-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

  19. Enabling technologies to advance microbial isoprenoid production.

    PubMed

    Chen, Yun; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens

    2015-01-01

    Microbial production of isoprenoids provides an attractive alternative to biomass extraction and chemical synthesis. Although widespread research aims for isoprenoid biosynthesis, it is still in its infancy in terms of delivering commercial products. Large barriers remain in realizing a cost-competitive process, for example, developing an optimal microbial cell factory. Here, we summarize the many tools and methods that have been developed in the metabolic engineering of isoprenoid production, with the advent of systems biology and synthetic biology, and discuss how these technologies advance to accelerate the design-build-test engineering cycle to obtain optimum microbial systems. It is anticipated that innovative combinations of new and existing technologies will continue to emerge, which will enable further development of microbial cell factories for commercial isoprenoid production. PMID:25549781

  20. The apicoplast: a review of the derived plastid of apicomplexan parasites.

    PubMed

    Waller, Ross F; McFadden, Geoffrey I

    2005-01-01

    The apicoplast is a plastid organelle, homologous to chloroplasts of plants, that is found in apicomplexan parasites such as the causative agents of Malaria Plasmodium spp. It occurs throughout the Apicomplexa and is an ancient feature of this group acquired by the process of endosymbiosis. Like plant chloroplasts, apicoplasts are semi-autonomous with their own genome and expression machinery. In addition, apicoplasts import numerous proteins encoded by nuclear genes. These nuclear genes largely derive from the endosymbiont through a process of intracellular gene relocation. The exact role of a plastid in parasites is uncertain but early clues indicate synthesis of lipids, heme and isoprenoids as possibilities. The various metabolic processes of the apicoplast are potentially excellent targets for drug therapy. PMID:15580780

  1. Heme Synthesis by Plastid Ferrochelatase I Regulates Nuclear Gene Expression in Plants

    PubMed Central

    Woodson, Jesse D.; Perez-Ruiz, Juan M.; Chory, Joanne

    2016-01-01

    Summary Chloroplast signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of plastid-to-nucleus (retrograde) signaling [1, 2]. In Arabidopsis thaliana, genetic studies using norflurazon (NF), an inhibitor of carotenoid biosynthesis, have identified five GUN (genomes uncoupled) genes, implicating the tetrapyrrole pathway as a source of a retrograde signal. Loss of function of any of these GUN genes leads to increased expression of photosynthesis-associated nuclear genes (PhANGs) when chloroplast development has been blocked by NF [3, 4]. Here we present a new Arabidopsis gain-of-function mutant, gun6-1D, with a similar phenotype. The gun6-1Dmutant overexpresses the conserved plastid ferrochelatase 1 (FC1, heme synthase). Genetic and biochemical experiments demonstrate that increased flux through the heme branch of the plastid tetrapyrrole biosynthetic pathway increases PhANG expression. The second conserved plant ferrochelatase, FC2, colocalizes with FC1, but FC2 activity is unable to increase PhANG expression in undeveloped plastids. These data suggest a model in which heme, specifically produced by FC1, may be used as a retrograde signal to coordinate PhANG expression with chloroplast development. PMID:21565502

  2. Isoprenoid biosynthesis in eukaryotic phototrophs: A spotlight on algae

    SciTech Connect

    Lohr M.; Schwender J.; Polle, J. E. W.

    2012-04-01

    Isoprenoids are one of the largest groups of natural compounds and have a variety of important functions in the primary metabolism of land plants and algae. In recent years, our understanding of the numerous facets of isoprenoid metabolism in land plants has been rapidly increasing, while knowledge on the metabolic network of isoprenoids in algae still lags behind. Here, current views on the biochemistry and genetics of the core isoprenoid metabolism in land plants and in the major algal phyla are compared and some of the most pressing open questions are highlighted. Based on the different evolutionary histories of the various groups of eukaryotic phototrophs, we discuss the distribution and regulation of the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways in land plants and algae and the potential consequences of the loss of the MVA pathway in groups such as the green algae. For the prenyltransferases, serving as gatekeepers to the various branches of terpenoid biosynthesis in land plants and algae, we explore the minimal inventory necessary for the formation of primary isoprenoids and present a preliminary analysis of their occurrence and phylogeny in algae with primary and secondary plastids. The review concludes with some perspectives on genetic engineering of the isoprenoid metabolism in algae.

  3. Terpenoid Metabolism in Plastids 1

    PubMed Central

    Camara, Bilal

    1984-01-01

    The biosynthesis of phytoene from prephytoene pyrophosphate has been localized exclusively in the plastid compartment of ruptured protoplasts derived from Triticum leaves and Capsicum fruits. The phytoene synthetase activity in Triticum leaves deficient in plastid ribosomes was comparable to those obtained in normal leaves. In addition, the stimulation of phytoene synthetase activity observed in green Capsicum fruit after 2-(4-chlorophenylthio)triethylamine hydrochloride treatment was not abolished by chlororamphenicol and lincomycin, in contrast to the inhibition observed after cycloheximide treatment. These data conclusively show that phytoene synthetase is localized exclusively in the plastid compartment in higher plants and that its synthesis is not performed on 70S ribosomes. PMID:16663362

  4. Isoprenoid drugs, biofuels, and chemicals--artemisinin, farnesene, and beyond.

    PubMed

    George, Kevin W; Alonso-Gutierrez, Jorge; Keasling, Jay D; Lee, Taek Soon

    2015-01-01

    Isoprenoids have been identified and used as natural pharmaceuticals, fragrances, solvents, and, more recently, advanced biofuels. Although isoprenoids are most commonly found in plants, researchers have successfully engineered both the eukaryotic and prokaryotic isoprenoid biosynthetic pathways to produce these valuable chemicals in microorganisms at high yields. The microbial synthesis of the precursor to artemisinin--an important antimalarial drug produced from the sweet wormwood Artemisia annua--serves as perhaps the most successful example of this approach. Through advances in synthetic biology and metabolic engineering, microbial-derived semisynthetic artemisinin may soon replace plant-derived artemisinin as the primary source of this valuable pharmaceutical. The richness and diversity of isoprenoid structures also make them ideal candidates for advanced biofuels that may act as "drop-in" replacements for gasoline, diesel, and jet fuel. Indeed, the sesquiterpenes farnesene and bisabolene, monoterpenes pinene and limonene, and hemiterpenes isopentenol and isopentanol have been evaluated as fuels or fuel precursors. As in the artemisinin project, these isoprenoids have been produced microbially through synthetic biology and metabolic engineering efforts. Here, we provide a brief review of the numerous isoprenoid compounds that have found use as pharmaceuticals, flavors, commodity chemicals, and, most importantly, advanced biofuels. In each case, we highlight the metabolic engineering strategies that were used to produce these compounds successfully in microbial hosts. In addition, we present a current outlook on microbial isoprenoid production, with an eye towards the many challenges that must be addressed to achieve higher yields and industrial-scale production. PMID:25577395

  5. Arabidopsis GERANYLGERANYL DIPHOSPHATE SYNTHASE 11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids.

    PubMed

    Ruiz-Sola, M Águila; Coman, Diana; Beck, Gilles; Barja, M Victoria; Colinas, Maite; Graf, Alexander; Welsch, Ralf; Rütimann, Philipp; Bühlmann, Peter; Bigler, Laurent; Gruissem, Wilhelm; Rodríguez-Concepción, Manuel; Vranová, Eva

    2016-01-01

    Most plastid isoprenoids, including photosynthesis-related metabolites such as carotenoids and the side chain of chlorophylls, tocopherols (vitamin E), phylloquinones (vitamin K), and plastoquinones, derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. Seven out of 10 functional GGPPS isozymes in Arabidopsis thaliana reside in plastids. We aimed to address the function of different GGPPS paralogues for plastid isoprenoid biosynthesis. We constructed a gene co-expression network (GCN) using GGPPS paralogues as guide genes and genes from the upstream and downstream pathways as query genes. Furthermore, knock-out and/or knock-down ggpps mutants were generated and their growth and metabolic phenotypes were analyzed. Also, interacting protein partners of GGPPS11 were searched for. Our data showed that GGPPS11, encoding the only plastid isozyme essential for plant development, functions as a hub gene among GGPPS paralogues and is required for the production of all major groups of plastid isoprenoids. Furthermore, we showed that the GGPPS11 protein physically interacts with enzymes that use GGPP for the production of carotenoids, chlorophylls, tocopherols, phylloquinone, and plastoquinone. GGPPS11 is a hub isozyme required for the production of most photosynthesis-related isoprenoids. Both gene co-expression and protein-protein interaction likely contribute to the channeling of GGPP by GGPPS11. PMID:26224411

  6. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants. PMID:27485226

  7. Plant Plastid Engineering

    PubMed Central

    Wani, Shabir H.; Haider, Nadia; Kumar, Hitesh; Singh, N.B.

    2010-01-01

    Genetic material in plants is distributed into nucleus, plastids and mitochondria. Plastid has a central role of carrying out photosynthesis in plant cells. Plastid transformation is becoming more popular and an alternative to nuclear gene transformation because of various advantages like high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission. Recently, much progress in plastid engineering has been made. In addition to model plant tobacco, many transplastomic crop plants have been generated which possess higher resistance to biotic and abiotic stresses and molecular pharming. In this mini review, we will discuss the features of the plastid DNA and advantages of plastid transformation. We will also present some examples of transplastomic plants developed so far through plastid engineering, and the various applications of plastid transformation. PMID:21532834

  8. Plastids and carotenoid accumulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastids are ubiquitously in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids except proplastids can synth...

  9. Cellular Localization of Isoprenoid Biosynthetic Enzymes in Marchantia polymorpha. Uncovering a New Role of Oil Bodies

    PubMed Central

    Suire, Claude; Bouvier, Florence; Backhaus, Ralph A.; Bégu, Dominique; Bonneu, Marc; Camara, Bilal

    2000-01-01

    Like seed plants, liverworts synthesize and accumulate a myriad of isoprenoid compounds. Using antibodies raised against several isoprenoid biosynthetic enzymes, we investigated their intracellular compartmentation by in situ immunolocalization from Marchantia polymorpha. The enzymes examined were deoxy-xylulose phosphate synthase, geranyl diphosphate synthase, farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, monoterpene synthase, geranylgeranyl diphosphate reductase, phytoene synthase, and phytoene desaturase. Our results show that liverwort oil bodies, which are organelles bound by a single unit membrane, possess isoprenoid biosynthetic enzymes similar to those found in plastids and the cytosol. We postulate that oil bodies play a dynamic role in cell metabolism in addition to their role as sites of essential oil accumulation and sequestration. The occurrence of such enzymes in different cellular compartments might be due to multiple targeting of gene products to various organelles. PMID:11080275

  10. Glycerolipid biosynthesis in isolated pea root plastids

    SciTech Connect

    Xue, Lingru; Sparace, S.A. )

    1990-05-01

    Plastids have been isolated from germinating pea (Pisum sativum L.) roots by differential centrifugation and purified on Percoll gradients. Marker enzymes (NADPH: cytochrome c reductase, fumarase and fatty acid synthesis) indicate that greater than 50% of the plastids are recovered essentially free from mitochondrial and endoplasmic reticulum contamination. Fatty acids synthesized from ({sup 14}C)acetate by Percoll-purified plastids are primarily 16:0, 16:1 and 18:1. ({sup 14}C)Acetate-labelled fatty acids and ({sup 14}C)glycerol-3-phosphate are both readily incorporated into glycerolipid. Approximately 12% of the total activity for glycerolipid biosynthesis from glycerol-3-phosphate is recovered in the purified plastid fraction. Glycerolipids synthesized from these precursors are primarily TAG, DAG, PE, PG, PC, PI and PA. Acyl-CoA's also accumulate when acetate is the precursor.

  11. Factors mediating plastid dependency and the origins of parasitism in apicomplexans and their close relatives

    PubMed Central

    Janouškovec, Jan; Tikhonenkov, Denis V.; Burki, Fabien; Howe, Alexis T.; Kolísko, Martin; Mylnikov, Alexander P.; Keeling, Patrick J.

    2015-01-01

    Apicomplexans are a major lineage of parasites, including causative agents of malaria and toxoplasmosis. How such highly adapted parasites evolved from free-living ancestors is poorly understood, particularly because they contain nonphotosynthetic plastids with which they have a complex metabolic dependency. Here, we examine the origin of apicomplexan parasitism by resolving the evolutionary distribution of several key characteristics in their closest free-living relatives, photosynthetic chromerids and predatory colpodellids. Using environmental sequence data, we describe the diversity of these apicomplexan-related lineages and select five species that represent this diversity for transcriptome sequencing. Phylogenomic analysis recovered a monophyletic lineage of chromerids and colpodellids as the sister group to apicomplexans, and a complex distribution of retention versus loss for photosynthesis, plastid genomes, and plastid organelles. Reconstructing the evolution of all plastid and cytosolic metabolic pathways related to apicomplexan plastid function revealed an ancient dependency on plastid isoprenoid biosynthesis, predating the divergence of apicomplexan and dinoflagellates. Similarly, plastid genome retention is strongly linked to the retention of two genes in the plastid genome, sufB and clpC, altogether suggesting a relatively simple model for plastid retention and loss. Lastly, we examine the broader distribution of a suite of molecular characteristics previously linked to the origins of apicomplexan parasitism and find that virtually all are present in their free-living relatives. The emergence of parasitism may not be driven by acquisition of novel components, but rather by loss and modification of the existing, conserved traits. PMID:25717057

  12. The potential of the mevalonate pathway for enhanced isoprenoid production.

    PubMed

    Liao, Pan; Hemmerlin, Andréa; Bach, Thomas J; Chye, Mee-Len

    2016-01-01

    The cytosol-localised mevalonic acid (MVA) pathway delivers the basic isoprene unit isopentenyl diphosphate (IPP). In higher plants, this central metabolic intermediate is also synthesised by the plastid-localised methylerythritol phosphate (MEP) pathway. Both MVA and MEP pathways conspire through exchange of intermediates and regulatory interactions. Products downstream of IPP such as phytosterols, carotenoids, vitamin E, artemisinin, tanshinone and paclitaxel demonstrate antioxidant, cholesterol-reducing, anti-ageing, anticancer, antimalarial, anti-inflammatory and antibacterial activities. Other isoprenoid precursors including isoprene, isoprenol, geraniol, farnesene and farnesol are economically valuable. An update on the MVA pathway and its interaction with the MEP pathway is presented, including the improvement in the production of phytosterols and other isoprenoid derivatives. Such attempts are for instance based on the bioengineering of microbes such as Escherichia coli and Saccharomyces cerevisiae, as well as plants. The function of relevant genes in the MVA pathway that can be utilised in metabolic engineering is reviewed and future perspectives are presented. PMID:26995109

  13. Coordination of plastid and nuclear gene expression.

    PubMed Central

    Gray, John C; Sullivan, James A; Wang, Jun-Hui; Jerome, Cheryl A; MacLean, Daniel

    2003-01-01

    The coordinated expression of genes distributed between the nuclear and plastid genomes is essential for the assembly of functional chloroplasts. Although the nucleus has a pre-eminent role in controlling chloroplast biogenesis, there is considerable evidence that the expression of nuclear genes encoding photosynthesis-related proteins is regulated by signals from plastids. Perturbation of several plastid-located processes, by inhibitors or in mutants, leads to decreased transcription of a set of nuclear photosynthesis-related genes. Characterization of arabidopsis gun (genomes uncoupled) mutants, which express nuclear genes in the presence of norflurazon or lincomycin, has provided evidence for two separate signalling pathways, one involving tetrapyrrole biosynthesis intermediates and the other requiring plastid protein synthesis. In addition, perturbation of photosynthetic electron transfer produces at least two different redox signals, as part of the acclimation to altered light conditions. The recognition of multiple plastid signals requires a reconsideration of the mechanisms of regulation of transcription of nuclear genes encoding photosynthesis-related proteins. PMID:12594922

  14. Isoprenoids, Small GTPases and Alzheimer’s Disease

    PubMed Central

    Hooff, Gero P.; Wood, W. Gibson; Müller, Walter E.; Eckert, Gunter P.

    2010-01-01

    The mevalonate-pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased, but that dolichyl-phosphate and ubiquinone are elevated in brains of Alzheimer´s diseased (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post translational modification of certain proteins which function as molecular switches in numerous, signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction. PMID:20382260

  15. Crystal structure of 1-deoxy-D-xylulose 5-phosphate synthase, a crucial enzyme for isoprenoids biosynthesis.

    PubMed

    Xiang, Song; Usunow, Gerlinde; Lange, Gudrun; Busch, Marco; Tong, Liang

    2007-01-26

    Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies. PMID:17135236

  16. Crystal Structure of 1-Deoxy-D-xylulose 5-Phosphate Synthase, A Crucial Enzyme for Isoprenoids Biosynthesis

    SciTech Connect

    Xiang,S.; Usunow, G.; Busch, G.; Tong, L.

    2007-01-01

    Isopentenyl pyrophosphate (IPP) is a common precursor for the synthesis of all isoprenoids, which have important functions in living organisms. IPP is produced by the mevalonate pathway in archaea, fungi, and animals. In contrast, IPP is synthesized by a mevalonate-independent pathway in most bacteria, algae, and plant plastids. 1-Deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the first and the rate-limiting step of the mevalonate-independent pathway and is an attractive target for the development of novel antibiotics, antimalarials, and herbicides. We report here the first structural information on DXS, from Escherichia coli and Deinococcus radiodurans, in complex with the coenzyme thiamine pyrophosphate (TPP). The structure contains three domains (I, II, and III), each of which bears homology to the equivalent domains in transketolase and the E1 subunit of pyruvate dehydrogenase. However, DXS has a novel arrangement of these domains as compared with the other enzymes, such that the active site of DXS is located at the interface of domains I and II in the same monomer, whereas that of transketolase is located at the interface of the dimer. The coenzyme TPP is mostly buried in the complex, but the C-2 atom of its thiazolium ring is exposed to a pocket that is the substrate-binding site. The structures identify residues that may have important roles in catalysis, which have been confirmed by our mutagenesis studies.

  17. Cholesterol synthesis is the trigger and isoprenoid dependent interleukin-6 mediated inflammation is the common causative factor and therapeutic target for atherosclerotic vascular disease and age-related disorders including osteoporosis and type 2 diabetes.

    PubMed

    Omoigui, Sota

    2005-01-01

    This is a unifying theory that cholesterol metabolites (isoprenoids) are an integral component of the signaling pathway for interleukin-6 (IL-6) mediated inflammation. IL-6 inflammation is the common causative origin for atherosclerosis, peripheral vascular disease, coronary artery disease, and age-related disorders including osteoporosis, dementia, Alzheimer's disease and type 2 diabetes. Therapeutic effects of bisphosphonates and statins are mediated by isoprenoid depletion. Statins and bisphosphonates act in the cholesterol pathway to deplete isoprenoids. Anti-inflammatory properties of statins and bisphosphonates are due to isoprenoid depletion with subsequent inhibition of IL-6 mediated inflammation. Therapeutic targets for the prevention and control of all the above diseases should focus on cholesterol metabolites and IL-6 mediated inflammation. Prevention of atherosclerotic vascular disease and age-related disorders will be by utilization of cholesterol lowering agents or techniques and/or treatment with statins and/or bisphosphonates to inhibit IL-6 inflammation through regulation of cholesterol metabolism. PMID:15935563

  18. Plastid transformation in soybean.

    PubMed

    Dubald, Manuel; Tissot, Ghislaine; Pelissier, Bernard

    2014-01-01

    The biotechnological potential of plastid genetic engineering has been illustrated in a limited number of higher plant species. We have developed a reproducible method to generate plastid transformants in soybean (Glycine max), a crop of major agronomic importance. The transformation vectors are delivered to embryogenic cultures by the particle gun method and selection performed using the aadA antibiotic resistance gene. Homoplasmy is established rapidly in the selected events without the need for further selection or regeneration cycles, and genes of interest can be expressed at a high level in green tissues. This is a significant step toward the commercial application of this technology. PMID:24599865

  19. Functional Characterization of the Xanthophyllomyces dendrorhous Farnesyl Pyrophosphate Synthase and Geranylgeranyl Pyrophosphate Synthase Encoding Genes That Are Involved in the Synthesis of Isoprenoid Precursors

    PubMed Central

    Niklitschek, Mauricio; Sepúlveda, Dionisia; Rojas, María Cecilia; Baeza, Marcelo; Cifuentes, Víctor

    2014-01-01

    The yeast Xanthophyllomyces dendrorhous synthesizes the carotenoid astaxanthin, which has applications in biotechnology because of its antioxidant and pigmentation properties. However, wild-type strains produce too low amounts of carotenoids to be industrially competitive. Considering this background, it is indispensable to understand how the synthesis of astaxanthin is controlled and regulated in this yeast. In this work, the steps leading to the synthesis of the carotenoid precursor geranylgeranyl pyrophosphate (GGPP, C20) in X. dendrorhous from isopentenyl pyrophosphate (IPP, C5) and dimethylallyl pyrophosphate (DMAPP, C5) was characterized. Two prenyl transferase encoding genes, FPS and crtE, were expressed in E. coli. The enzymatic assays using recombinant E. coli protein extracts demonstrated that FPS and crtE encode a farnesyl pyrophosphate (FPP, C15) synthase and a GGPP-synthase, respectively. X. dendrorhous FPP-synthase produces geranyl pyrophosphate (GPP, C10) from IPP and DMAPP and FPP from IPP and GPP, while the X. dendrorhous GGPP-synthase utilizes only FPP and IPP as substrates to produce GGPP. Additionally, the FPS and crtE genes were over-expressed in X. dendrorhous, resulting in an increase of the total carotenoid production. Because the parental strain is diploid, the deletion of one of the alleles of these genes did not affect the total carotenoid production, but the composition was significantly altered. These results suggest that the over-expression of these genes might provoke a higher carbon flux towards carotenogenesis, most likely involving an earlier formation of a carotenogenic enzyme complex. Conversely, the lower carbon flux towards carotenogenesis in the deletion mutants might delay or lead to a partial formation of a carotenogenic enzyme complex, which could explain the accumulation of astaxanthin carotenoid precursors in these mutants. In conclusion, the FPS and the crtE genes represent good candidates to manipulate to favor

  20. From flavors and pharmaceuticals to advanced biofuels: production of isoprenoids in Saccharomyces cerevisiae.

    PubMed

    Tippmann, Stefan; Chen, Yun; Siewers, Verena; Nielsen, Jens

    2013-12-01

    Isoprenoids denote the largest group of chemicals in the plant kingdom and are employed for a wide range of applications in the food and pharmaceutical industry. In recent years, isoprenoids have additionally been recognized as suitable replacements for petroleum-derived fuels and could thus promote the transition towards a more sustainable society. To realize the biofuel potential of isoprenoids, a very efficient production system is required. While complex chemical structures as well as the low abundance in nature demonstrate the shortcomings of chemical synthesis and plant extraction, isoprenoids can be produced by genetically engineered microorganisms from renewable carbon sources. In this article, we summarize the development of isoprenoid applications from flavors and pharmaceuticals to advanced biofuels and review the strategies to design microbial cell factories, focusing on Saccharomyces cerevisiae for the production of these compounds. While the high complexity of biosynthetic pathways and the toxicity of certain isoprenoids still denote challenges that need to be addressed, metabolic engineering has enabled large-scale production of several terpenoids and thus, the utilization of these compounds is likely to expand in the future. PMID:24227704

  1. Plastid markers in Carya

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated 8 "universal" plastid primers and qualified 3 as polymorphic and informative within Carya. Pecans of known lineage and geographic origin, representatives of other Carya species and interspecific hybrids were profiled. In an analysis of 169 Carya accessions, 21 alleles were observed am...

  2. A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway.

    PubMed

    Lange, B M; Wildung, M R; McCaskill, D; Croteau, R

    1998-03-01

    Isopentenyl diphosphate, the common precursor of all isoprenoids, has been widely assumed to be synthesized by the acetate/mevalonate pathway in all organisms. However, based on in vivo feeding experiments, isopentenyl diphosphate formation in several eubacteria, a green alga, and plant chloroplasts has been demonstrated very recently to originate via a mevalonate-independent route from pyruvate and glyceraldehyde 3-phosphate as precursors. Here we describe the cloning from peppermint (Mentha x piperita) and heterologous expression in Escherichia coli of 1-deoxy-D-xylulose-5-phosphate synthase, the enzyme that catalyzes the first reaction of this pyruvate/glyceraldehyde 3-phosphate pathway. This synthase gene contains an ORF of 2,172 base pairs. When the proposed plastid targeting sequence is excluded, the deduced amino acid sequence indicates the peppermint synthase to be about 650 residues in length, corresponding to a native size of roughly 71 kDa. The enzyme appears to represent a novel class of highly conserved transketolases and likely plays a key role in the biosynthesis of plastid-derived isoprenoids essential for growth, development, and defense in plants. PMID:9482845

  3. Substrate analogues for isoprenoid enzymes

    SciTech Connect

    Stremler, K.E.

    1987-01-01

    Diphosphonate analogues of geranyl diphosphate, resistant to degradation by phosphatases, were found to be alternate substrates for the reaction with farnesyl diphosphate synthetase isolated from avian liver. The difluoromethane analogue was shown to be the better alternate substrate, in agreement with solvolysis results which indicate that the electronegativity of the difluoromethylene unit more closely approximates that of the normal bridging oxygen. The usefulness of the C/sub 10/ difluoro analogue, for detecting low levels of isoprenoid enzymes in the presence of high levels of phosphatase activity, was demonstrated with a cell-free preparation from lemon peel. A series of C/sub 5/ through C/sub 15/ homoallylic and allylic diphosphonates, as well as two 5'-nucleotide diphosphonates, was prepared in high overall yield using the activation-displacement sequence. Radiolabeled samples of several of the allylic diphosphonates were prepared with tritium located at C1. A series of geraniols, stereospecifically deuterated at C1, was prepared. The enantiomeric purities and absolute configurations were determined by derivatization as the mandelate esters for analysis by /sup 1/H NMR. The stereochemistry of the activation-displacement sequence was examined using C1-deuterated substrates.

  4. Inhibition of IspH, a [4Fe-4S]2+ enzyme involved in the biosynthesis of isoprenoids via the methylerythritol phosphate pathway.

    PubMed

    Janthawornpong, Karnjapan; Krasutsky, Sergiy; Chaignon, Philippe; Rohmer, Michel; Poulter, C Dale; Seemann, Myriam

    2013-02-01

    The MEP pathway, which is absent in animals but present in most pathogenic bacteria, in the parasite responsible for malaria and in plant plastids, is a target for the development of antimicrobial drugs. IspH, an oxygen-sensitive [4Fe-4S] enzyme, catalyzes the last step of this pathway and converts (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A crucial step in the mechanism of this enzyme is the binding of the C4 hydroxyl of HMBPP to the unique fourth iron site in the [4Fe-4S](2+) moiety. Here, we report the synthesis and the kinetic investigations of two new extremely potent inhibitors of E. coli IspH where the OH group of HMBPP is replaced by an amino and a thiol group. (E)-4-Mercapto-3-methylbut-2-en-1-yl diphosphate is a reversible tight-binding inhibitor of IspH with K(i) = 20 ± 2 nM. A detailed kinetic analysis revealed that (E)-4-amino-3-methylbut-2-en-1-yl diphosphate is a reversible slow-binding inhibitor of IspH with K(i) = 54 ± 19 nM. The slow binding behavior of this inhibitor is best described by a one-step mechanism with the slow step consisting of the formation of the enzyme-inhibitor (EI) complex. PMID:23316732

  5. Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system.

    PubMed Central

    Kruse, E; Mock, H P; Grimm, B

    1995-01-01

    A full-length cDNA sequence encoding coproporphyrinogen oxidase was inserted in inverse orientation behind a CaMV promoter and transferred to tobacco (Nicotiana tabacum) by standard transformation techniques. Transformants showed reduced coproporphyrinogen oxidase activity and accumulation of photosensitive coproporphyrin(ogen), indicating antisense RNA expression. An inverse correlation was observed between the level of coproporphyrinogen oxidase and transformant phenotype. The latter is characterized by a broad range of growth retardation and necrosis, indicating oxidative leaf damage. Coproporphyrinogen is an apparent chromophore and its excitation finally leads to the production of reactive oxygen. Evidence is presented that indicates a direct correlation between the accumulation of non-metabolized coproporphyrinogen and oxidative damage to cellular structural components. Enzymatic and non-enzymatic antioxidants were investigated. Whereas superoxide dismutase activity increased in transgenic plants, catalase and ascorbate peroxidase activity remained constant. Tocopherol, rather than carotene or zeaxanthin, seemed to be involved in detoxification, indicating the putative localization and allocation of coproporphyrinogen. Expression of coproporphyrinogen oxidase antisense RNA did not significantly influence the level of other enzymes in the chlorophyll metabolic pathway, but deregulated gene expression of nuclear encoded plastid proteins. Accumulation of coproporphyrinogen and/or the resulting effects, such as oxidative stress, impairs a plastid/nuclear signal which may adapt gene expression to the plastid state. Images PMID:7641690

  6. Biodegradation of acyclic isoprenoids by Pseudomonas species.

    PubMed Central

    Cantwell, S G; Lau, E P; Watt, D S; Fall, R R

    1978-01-01

    The ability of various pseudomonads to utilize acyclic isoprenoids as a sole carbon source was investigated. Tests for utilization of acyclic isoprenols such as citronellol and geraniol were complicated by toxic effects of these alcohols, and most species tested were killed by exposure to citronellol or geraniol (0.1%, vol/vol) in liquid culture. In the case of Pseudomonas citronellolis, sensitivity to isoprenols is reduced by prior induction of the isoprenoid degradative pathway via either growth on succinate in the presence of citronellol or growth on citronellic acid. For this species, citronellic acid proved to be the best isoprenoid growth substrate tested. Geraniol utilization as a taxonomic indicator for different subgroups of pseudomonads is discussed. Only a few of the species tested were able to utilize acyclic isoprenoids. Two species which utilize C10 acyclic isoprenoids, P. aeruginosa and P. mendocina, were shown to contain the inducible enzyme geranyl-coenzyme A carboxylase, one of the unique enzymes in the isoprenol degradative pathway known to occur in P. citronellolis. Of the species which utilized geranitol, none showed definite growth on the homologous C15 and C20 isoprenols. PMID:681275

  7. Plastid microsatellite markers in Carya

    Technology Transfer Automated Retrieval System (TEKTRAN)

    PCR primers capable of amplifying polymorphic regions of the plastid genome in Carya have broad applications for characterizing the genus and for conservation and improvement of pecan. We evaluated 8 "universal" plastid primers and qualified 3 as polymorphic and informative. Pecans of known lineag...

  8. Respiratory processes in non-photosynthetic plastids

    PubMed Central

    Renato, Marta; Boronat, Albert; Azcón-Bieto, Joaquín

    2015-01-01

    Chlororespiration is a respiratory process located in chloroplast thylakoids which consists in an electron transport chain from NAD(P)H to oxygen. This respiratory chain involves the NAD(P)H dehydrogenase complex, the plastoquinone pool and the plastid terminal oxidase (PTOX), and it probably acts as a safety valve to prevent the over-reduction of the photosynthetic machinery in stress conditions. The existence of a similar respiratory activity in non-photosynthetic plastids has been less studied. Recently, it has been reported that tomato fruit chromoplasts present an oxygen consumption activity linked to ATP synthesis. Etioplasts and amyloplasts contain several electron carriers and some subunits of the ATP synthase, so they could harbor a similar respiratory process. This review provides an update on the study about respiratory processes in chromoplasts, identifying the major gaps that need to be addressed in future research. It also reviews the proteomic data of etioplasts and amyloplasts, which suggest the presence of a respiratory electron transport chain in these plastids. PMID:26236317

  9. Plastid genomics in horticultural species: importance and applications for plant population genetics, evolution, and biotechnology

    PubMed Central

    Rogalski, Marcelo; do Nascimento Vieira, Leila; Fraga, Hugo P.; Guerra, Miguel P.

    2015-01-01

    During the evolution of the eukaryotic cell, plastids, and mitochondria arose from an endosymbiotic process, which determined the presence of three genetic compartments into the incipient plant cell. After that, these three genetic materials from host and symbiont suffered several rearrangements, bringing on a complex interaction between nuclear and organellar gene products. Nowadays, plastids harbor a small genome with ∼130 genes in a 100–220 kb sequence in higher plants. Plastid genes are mostly highly conserved between plant species, being useful for phylogenetic analysis in higher taxa. However, intergenic spacers have a relatively higher mutation rate and are important markers to phylogeographical and plant population genetics analyses. The predominant uniparental inheritance of plastids is like a highly desirable feature for phylogeny studies. Moreover, the gene content and genome rearrangements are efficient tools to capture and understand evolutionary events between different plant species. Currently, genetic engineering of the plastid genome (plastome) offers a number of attractive advantages as high-level of foreign protein expression, marker gene excision, gene expression in operon and transgene containment because of maternal inheritance of plastid genome in most crops. Therefore, plastid genome can be used for adding new characteristics related to synthesis of metabolic compounds, biopharmaceutical, and tolerance to biotic and abiotic stresses. Here, we describe the importance and applications of plastid genome as tools for genetic and evolutionary studies, and plastid transformation focusing on increasing the performance of horticultural species in the field. PMID:26284102

  10. Comparative analysis of predicted plastid-targeted proteomes of sequenced higher plant genomes.

    PubMed

    Schaeffer, Scott; Harper, Artemus; Raja, Rajani; Jaiswal, Pankaj; Dhingra, Amit

    2014-01-01

    Plastids are actively involved in numerous plant processes critical to growth, development and adaptation. They play a primary role in photosynthesis, pigment and monoterpene synthesis, gravity sensing, starch and fatty acid synthesis, as well as oil, and protein storage. We applied two complementary methods to analyze the recently published apple genome (Malus × domestica) to identify putative plastid-targeted proteins, the first using TargetP and the second using a custom workflow utilizing a set of predictive programs. Apple shares roughly 40% of its 10,492 putative plastid-targeted proteins with that of the Arabidopsis (Arabidopsis thaliana) plastid-targeted proteome as identified by the Chloroplast 2010 project and ∼57% of its entire proteome with Arabidopsis. This suggests that the plastid-targeted proteomes between apple and Arabidopsis are different, and interestingly alludes to the presence of differential targeting of homologs between the two species. Co-expression analysis of 2,224 genes encoding putative plastid-targeted apple proteins suggests that they play a role in plant developmental and intermediary metabolism. Further, an inter-specific comparison of Arabidopsis, Prunus persica (Peach), Malus × domestica (Apple), Populus trichocarpa (Black cottonwood), Fragaria vesca (Woodland Strawberry), Solanum lycopersicum (Tomato) and Vitis vinifera (Grapevine) also identified a large number of novel species-specific plastid-targeted proteins. This analysis also revealed the presence of alternatively targeted homologs across species. Two separate analyses revealed that a small subset of proteins, one representing 289 protein clusters and the other 737 unique protein sequences, are conserved between seven plastid-targeted angiosperm proteomes. Majority of the novel proteins were annotated to play roles in stress response, transport, catabolic processes, and cellular component organization. Our results suggest that the current state of knowledge regarding

  11. Over-expression of Arabidopsis thaliana SFD1/GLY1, the gene encoding plastid localized glycerol-3-phosphate dehydrogenase, increases plastidic lipid content in transgenic rice plants.

    PubMed

    Singh, Vijayata; Singh, Praveen Kumar; Siddiqui, Adnan; Singh, Subaran; Banday, Zeeshan Zahoor; Nandi, Ashis Kumar

    2016-03-01

    Lipids are the major constituents of all membranous structures in plants. Plants possess two pathways for lipid biosynthesis: the prokaryotic pathway (i.e., plastidic pathway) and the eukaryotic pathway (i.e., endoplasmic-reticulum (ER) pathway). Whereas some plants synthesize galactolipids from diacylglycerol assembled in the plastid, others, including rice, derive their galactolipids from diacylglycerols assembled by the eukaryotic pathway. Arabidopsis thaliana glycerol-3-phosphate dehydrogenase (G3pDH), coded by SUPPRESSOR OF FATTY ACID DESATURASE 1 (SFD1; alias GLY1) gene, catalyzes the formation of glycerol 3-phosphate (G3p), the backbone of many membrane lipids. Here SFD1 was introduced to rice as a transgene. Arabidopsis SFD1 localizes in rice plastids and its over-expression increases plastidic membrane lipid content in transgenic rice plants without any major impact on ER lipids. The results suggest that over-expression of plastidic G3pDH enhances biosynthesis of plastid-localized lipids in rice. Lipid composition in the transgenic plants is consistent with increased phosphatidylglycerol synthesis in the plastid and increased galactolipid synthesis from diacylglycerol produced via the ER pathway. The transgenic plants show a higher photosynthetic assimilation rate, suggesting a possible application of this finding in crop improvement. PMID:26747130

  12. Plastids and gravitropic sensing

    NASA Technical Reports Server (NTRS)

    Sack, F. D.

    1997-01-01

    Data and theories about the identity of the mass that acts in gravitropic sensing are reviewed. Gravity sensing may have evolved several times in plants and algae in processes such as gravitropism of organs and tip-growing cells, gravimorphism, gravitaxis, and the regulation of cytoplasmic streaming in internodal cells of Chara. In the latter and in gravitaxis, the mass of the entire cell may function in sensing. But gravitropic sensing appears to rely upon the mass of amyloplasts that sediment since (i) the location of cells with sedimentation is highly regulated, (ii) such cells contain other morphological specializations favoring sedimentation, (iii) sedimentation always correlates with gravitropic competence in wild-type plants, (iv) magnetophoretic movement of rootcap amyloplasts mimics gravitropism, and (v) starchless and intermediate starch mutants show reduced gravitropic sensitivity. The simplest interpretation of these data is that gravitropic sensing is plastid-based.

  13. The Metabolite Transporters of the Plastid Envelope: An Update

    PubMed Central

    Facchinelli, Fabio; Weber, Andreas P. M.

    2011-01-01

    The engulfment of a photoautotrophic cyanobacterium by a primitive mitochondria-bearing eukaryote traces back to more than 1.2 billion years ago. This single endosymbiotic event not only provided the early petroalgae with the metabolic capacity to perform oxygenic photosynthesis, but also introduced a plethora of other metabolic routes ranging from fatty acids and amino acids biosynthesis, nitrogen and sulfur assimilation to secondary compounds synthesis. This implicated the integration and coordination of the newly acquired metabolic entity with the host metabolism. The interface between the host cytosol and the plastidic stroma became of crucial importance in sorting precursors and products between the plastid and other cellular compartments. The plastid envelope membranes fulfill different tasks: they perform important metabolic functions, as they are involved in the synthesis of carotenoids, chlorophylls, and galactolipids. In addition, since most genes of cyanobacterial origin have been transferred to the nucleus, plastidial proteins encoded by nuclear genes are post-translationally transported across the envelopes through the TIC–TOC import machinery. Most importantly, chloroplasts supply the photoautotrophic cell with photosynthates in form of reduced carbon. The innermost bilayer of the plastidic envelope represents the permeability barrier for the metabolites involved in the carbon cycle and is literally stuffed with transporter proteins facilitating their transfer. The intracellular metabolite transporters consist of polytopic proteins containing membrane spans usually in the number of four or more α-helices. Phylogenetic analyses revealed that connecting the plastid with the host metabolism was mainly a process driven by the host cell. In Arabidopsis, 58% of the metabolite transporters are of host origin, whereas only 12% are attributable to the cyanobacterial endosymbiont. This review focuses on the metabolite transporters of the inner envelope

  14. Fatty acid biosynthesis in pea root plastids

    SciTech Connect

    Stahl, R.J.; Sparace, S.A. )

    1989-04-01

    Fatty acid biosynthesis from (1-{sup 14}C)acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 {mu}M acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl{sub 2}, 1 mM each of the MnCl{sub 2} and glycerol-3-phosphate, 15 mM KHCO{sub 3}, and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 {mu}g/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO{sub 3}, divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg{sup 2+} and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor.

  15. Expression of poly-3-(R)-hydroxyalkanoate (PHA) polymerase and acyl-CoA-transacylase in plastids of transgenic potato leads to the synthesis of a hydrophobic polymer, presumably medium-chain-length PHAs.

    PubMed

    Romano, Andrea; van der Plas, Linus H W; Witholt, Bernard; Eggink, Gerrit; Mooibroek, Hans

    2005-01-01

    Medium-chain-length poly-3-(R)-hydroxyalkanoates (mcl-PHAs) belong to the group of microbial polyesters. The minimum gene-set for the accumulation of mcl-PHAs from de novo fatty acid biosynthesis has been identified in prokaryotes as consisting of the Pha-C1 polymerase and the ACP-CoA-transacylase. In this paper, the synthesis of mcl-PHAs has been attempted in transgenic potato (Solanum tuberosum L.) using the same set of genes that were introduced into potato by particle bombardment. Polymer contents of transgenic lines were analysed by gas chromatography and by a new simple method employing a size-exclusion filter column. The expression of the Pha-C1 polymerase and the ACP-CoA-transacylase in the plastids of transgenic potato led to the synthesis of a hydrophobic polymer composed of mcl-hydroxy-fatty acids with carbon chain lengths ranging from C-6 to C-12 in leaves of the selected transgenic lines. We strongly suggest that the polymer observed consists of mcl-PHAs and that this report establishes for the first time a possible route for the production of mcl-PHAs from de novo fatty acid biosynthesis in plants. PMID:15351883

  16. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  17. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts.

    PubMed

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  18. Plastid ribosomal protein S5 plays a critical role in photosynthesis, plant development, and cold stress tolerance in arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plastid ribosomal proteins (RPs) are essential components for protein synthesis machinery and exert diverse roles in plant growth and development. Mutations in plastid RPs lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood and th...

  19. Isoprenoid modification permits 2',3'-cyclic nucleotide 3'-phosphodiesterase to bind to membranes.

    PubMed

    Braun, P E; De Angelis, D; Shtybel, W W; Bernier, L

    1991-11-01

    The myelination-related enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a relatively abundant protein in the CNS possesses the C-terminal isoprenylation consensus domain found in a small family that includes the ras oncoproteins and their relatives, some G-proteins, and nuclear lamins. We found that CNP, like these other proteins, is modified posttranslationally by an isoprenoid derived from mevalonic acid. It appears that only the smaller of the two CNP isoforms (CNP1) is isoprenylated, but similar modification of CNP2 cannot be excluded. Inhibition of isoprenoid synthesis by Lovastatin blocks the binding of newly synthesized CNP to cell membranes; binding is restored upon addition of mevalonate to the culture medium. This shows that isoprenylation is permissive for the well-known avid association of CNP with membranes. PMID:1666129

  20. Human Isoprenoid Synthase Enzymes as Therapeutic Targets

    NASA Astrophysics Data System (ADS)

    Park, Jaeok; Matralis, Alexios; Berghuis, Albert; Tsantrizos, Youla

    2014-07-01

    The complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids in the human body, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently, pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

  1. Human isoprenoid synthase enzymes as therapeutic targets

    PubMed Central

    Park, Jaeok; Matralis, Alexios N.; Berghuis, Albert M.; Tsantrizos, Youla S.

    2014-01-01

    In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies. PMID:25101260

  2. Codon Adaptation of Plastid Genes.

    PubMed

    Suzuki, Haruo; Morton, Brian R

    2016-01-01

    Codon adaptation is codon usage bias that results from selective pressure to increase the translation efficiency of a gene. Codon adaptation has been studied across a wide range of genomes and some early analyses of plastids have shown evidence for codon adaptation in a limited set of highly expressed plastid genes. Here we study codon usage bias across all fully sequenced plastid genomes which includes representatives of the Rhodophyta, Alveolata, Cryptophyta, Euglenozoa, Glaucocystophyceae, Rhizaria, Stramenopiles and numerous lineages within the Viridiplantae, including Chlorophyta and Embryophyta. We show evidence that codon adaptation occurs in all genomes except for two, Theileria parva and Heicosporidium sp., both of which have highly reduced gene contents and no photosynthesis genes. We also show evidence that selection for codon adaptation increases the representation of the same set of codons, which we refer to as the adaptive codons, across this wide range of taxa, which is probably due to common features descended from the initial endosymbiont. We use various measures to estimate the relative strength of selection in the different lineages and show that it appears to be fairly strong in certain Stramenopiles and Chlorophyta lineages but relatively weak in many members of the Rhodophyta, Euglenozoa and Embryophyta. Given these results we propose that codon adaptation in plastids is widespread and displays the same general features as adaptation in eubacterial genomes. PMID:27196606

  3. Codon Adaptation of Plastid Genes

    PubMed Central

    Suzuki, Haruo; Morton, Brian R.

    2016-01-01

    Codon adaptation is codon usage bias that results from selective pressure to increase the translation efficiency of a gene. Codon adaptation has been studied across a wide range of genomes and some early analyses of plastids have shown evidence for codon adaptation in a limited set of highly expressed plastid genes. Here we study codon usage bias across all fully sequenced plastid genomes which includes representatives of the Rhodophyta, Alveolata, Cryptophyta, Euglenozoa, Glaucocystophyceae, Rhizaria, Stramenopiles and numerous lineages within the Viridiplantae, including Chlorophyta and Embryophyta. We show evidence that codon adaptation occurs in all genomes except for two, Theileria parva and Heicosporidium sp., both of which have highly reduced gene contents and no photosynthesis genes. We also show evidence that selection for codon adaptation increases the representation of the same set of codons, which we refer to as the adaptive codons, across this wide range of taxa, which is probably due to common features descended from the initial endosymbiont. We use various measures to estimate the relative strength of selection in the different lineages and show that it appears to be fairly strong in certain Stramenopiles and Chlorophyta lineages but relatively weak in many members of the Rhodophyta, Euglenozoa and Embryophyta. Given these results we propose that codon adaptation in plastids is widespread and displays the same general features as adaptation in eubacterial genomes. PMID:27196606

  4. Genome evolution of a tertiary dinoflagellate plastid.

    PubMed

    Gabrielsen, Tove M; Minge, Marianne A; Espelund, Mari; Tooming-Klunderud, Ave; Patil, Vishwanath; Nederbragt, Alexander J; Otis, Christian; Turmel, Monique; Shalchian-Tabrizi, Kamran; Lemieux, Claude; Jakobsen, Kjetill S

    2011-01-01

    The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata. PMID:21541332

  5. Genome Evolution of a Tertiary Dinoflagellate Plastid

    PubMed Central

    Espelund, Mari; Tooming-Klunderud, Ave; Patil, Vishwanath; Nederbragt, Alexander J.; Otis, Christian; Turmel, Monique; Shalchian-Tabrizi, Kamran; Lemieux, Claude; Jakobsen, Kjetill S.

    2011-01-01

    The dinoflagellates have repeatedly replaced their ancestral peridinin-plastid by plastids derived from a variety of algal lineages ranging from green algae to diatoms. Here, we have characterized the genome of a dinoflagellate plastid of tertiary origin in order to understand the evolutionary processes that have shaped the organelle since it was acquired as a symbiont cell. To address this, the genome of the haptophyte-derived plastid in Karlodinium veneficum was analyzed by Sanger sequencing of library clones and 454 pyrosequencing of plastid enriched DNA fractions. The sequences were assembled into a single contig of 143 kb, encoding 70 proteins, 3 rRNAs and a nearly full set of tRNAs. Comparative genomics revealed massive rearrangements and gene losses compared to the haptophyte plastid; only a small fraction of the gene clusters usually found in haptophytes as well as other types of plastids are present in K. veneficum. Despite the reduced number of genes, the K. veneficum plastid genome has retained a large size due to expanded intergenic regions. Some of the plastid genes are highly diverged and may be pseudogenes or subject to RNA editing. Gene losses and rearrangements are also features of the genomes of the peridinin-containing plastids, apicomplexa and Chromera, suggesting that the evolutionary processes that once shaped these plastids have occurred at multiple independent occasions over the history of the Alveolata. PMID:21541332

  6. Corrected sequence of the wheat plastid genome.

    PubMed

    Bahieldin, Ahmed; Al-Kordy, Magdy A; Shokry, Ahmed M; Gadalla, Nour O; Al-Hejin, Ahmed M M; Sabir, Jamal S M; Hassan, Sabah M; Al-Ahmadi, Ahlam A; Schwarz, Erika N; Eissa, Hala F; El-Domyati, Fotouh M; Jansen, Robert K

    2014-09-01

    Wheat is the most important cereal in the world in terms of acreage and productivity. We sequenced and assembled the plastid genome of one Egyptian wheat cultivar using next-generation sequence data. The size of the plastid genome is 133,873 bp, which is 672 bp smaller than the published plastid genome of "Chinese Spring" cultivar, due mainly to the presence of three sequences from the rice plastid genome. The difference in size between the previously published wheat plastid genome and the sequence reported here is due to contamination of the published genome with rice plastid DNA, most of which is present in three sequences of 332, 131 and 131 bp. The corrected plastid genome of wheat has been submitted to GenBank (accession number KJ592713) and can be used in future comparisons. PMID:25242688

  7. Emerging facets of plastid division regulation.

    PubMed

    Basak, Indranil; Møller, Simon Geir

    2013-02-01

    Plastids are complex organelles that are integrated into the plant host cell where they differentiate and divide in tune with plant differentiation and development. In line with their prokaryotic origin, plastid division involves both evolutionary conserved proteins and proteins of eukaryotic origin where the host has acquired control over the process. The plastid division apparatus is spatially separated between the stromal and the cytosolic space but where clear coordination mechanisms exist between the two machineries. Our knowledge of the plastid division process has increased dramatically during the past decade and recent findings have not only shed light on plastid division enzymology and the formation of plastid division complexes but also on the integration of the division process into a multicellular context. This review summarises our current knowledge of plastid division with an emphasis on biochemical features, the functional assembly of protein complexes and regulatory features of the overall process. PMID:22965912

  8. Cytokinin membrane receptors participate in regulation of plastid genome expression in the skotomorphogenesis.

    PubMed

    Doroshenko, A S; Danilova, M N; Kudryakova, N V; Soloviev, A A; Kusnetsov, V V

    2016-07-01

    Analysis by real-time PCR of single and double insertion mutants of A. thaliana with inactivated cytokinin receptor genes showed that the level of transcripts of some of plastid genes during skotomorphogenesis depended on the state of functionally active receptor AHK3. The cytokinin-regulated plastid encoded genes involved the housekeeping genes (rpoB and accD) and the genes for photosynthetic proteins (ndhA, psbA, atpB, and psaA). However, the absence of hormone activation of plastid encoded genes engaged in the translation of plastid proteins, rRNA (rrn16), and tRNA (trnE), indirectly indicate the disruption of the synthesis of chloroplast proteins in the dark. PMID:27599515

  9. Hepatic Isoprenoid Metabolism in a Rat Model of Smith-Lemli-Opitz Syndrome

    PubMed Central

    Keller, R. Kennedy; Mitchell, David A.; Goulah, Christopher C.

    2013-01-01

    Elevated (4 to 7-fold) levels of urinary dolichol and coenzyme Q and substantially longer chain lengths for urinary dolichols have been reported in Smith-Lemli-Opitz Syndrome (SLOS) patients, compared to normal subjects. We investigated the possibility of similar alterations in hepatic, nonsterol isoprenoids in a well-established rat model of SLOS. In this model, the ratio of 7-dehydrocholesterol (7DHC) to cholesterol (Chol) in serum approached 15:1; however, total sterol mass in serum decreased by >80 %. Livers from treated rats had 7DHC/Chol ratios of ~32:1, but the steady-state levels of total sterols were >40 % those of livers from age-matched (3-month-old) control animals. No significant differences in the levels of LDL receptor or HMG-CoA reductase were observed. The levels of dolichol and coenzyme Q were elevated only modestly (by 64 and 31 %, respectively; p < 0.05, N = 6) in the livers of the SLOS rat model compared to controls; moreover, the chain lengths of these isoprenoids were not different in the two groups. We conclude that hepatic isoprenoid synthesis is marginally elevated in this animal model of SLOS, but without preferential shunting to the nonsterol branches (dolichol and coenzyme Q) of the pathway and without alteration of normal dolichol chain lengths. PMID:23361583

  10. Isoprenoid metabolism is required for stimulation of the respiratory burst oxidase of HL-60 cells.

    PubMed Central

    Bokoch, G M; Prossnitz, V

    1992-01-01

    The formation of oxygen radicals by phagocytic cells occurs through the activation of a multiple-component NADPH oxidase system. An unidentified low molecular weight GTP-binding protein has been proposed to modulate the activity of the NADPH oxidase. The low molecular weight GTP-binding proteins undergo posttranslational processing, including an initial covalent incorporation of an isoprenyl group. To test whether such an isoprenylation reaction might be required for the activity of the oxidase, we utilized compactin and lovastatin as inhibitors of the isoprenylation pathway. Treatment of DMSO-differentiated HL-60 cells with compactin produced a concentration-dependent inhibition of O2- formation in response to FMLP or phorbol myristate acetate. Cell viability was not affected nor was normal differentiation of the HL-60 cells into a neutrophil-like cell. The inhibitory effect of compactin was specifically prevented by addition of exogenous mevalonic acid to the HL-60 cells, indicating that the inhibitory effects of the drug were due to blockade of the pathway leading to isoprenoid synthesis. Addition of cholesterol, ubiquinone, or dolichol, which are also downstream products of the isoprenoid pathway, did not override the inhibitory effects of the drug. Subcellular fractions were prepared from compactin-treated cells, and the location of the compactin-sensitive factor was determined by complementation analysis in a cell-free NADPH oxidase system. The inhibited factor was localized to the HL-60 cytosol. These data suggest that an isoprenoid pathway intermediate is necessary for activation of the phagocyte NADPH oxidase. This is likely to represent the requirement for an isoprenoid moiety in the posttranslational modification of a low molecular weight GTP-binding protein. Our studies provide support for the involvement of such a low molecular weight GTP-binding protein in NADPH oxidase activation. Images PMID:1310693

  11. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis.

    PubMed

    Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

    2016-04-01

    Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

  12. Plastid ribosomal protein S5 is involved in photosynthesis, plant development, and cold stress tolerance in Arabidopsis

    PubMed Central

    Zhang, Junxiang; Yuan, Hui; Yang, Yong; Fish, Tara; Lyi, Sangbom M.; Thannhauser, Theodore W; Zhang, Lugang; Li, Li

    2016-01-01

    Plastid ribosomal proteins are essential components of protein synthesis machinery and have diverse roles in plant growth and development. Mutations in plastid ribosomal proteins lead to a range of developmental phenotypes in plants. However, how they regulate these processes is not fully understood, and the functions of some individual plastid ribosomal proteins remain unknown. To identify genes responsible for chloroplast development, we isolated and characterized a mutant that exhibited pale yellow inner leaves with a reduced growth rate in Arabidopsis. The mutant (rps5) contained a missense mutation of plastid ribosomal protein S5 (RPS5), which caused a dramatically reduced abundance of chloroplast 16S rRNA and seriously impaired 16S rRNA processing to affect ribosome function and plastid translation. Comparative proteomic analysis revealed that the rps5 mutation suppressed the expression of a large number of core components involved in photosystems I and II as well as many plastid ribosomal proteins. Unexpectedly, a number of proteins associated with cold stress responses were greatly decreased in rps5, and overexpression of the plastid RPS5 improved plant cold stress tolerance. Our results indicate that RPS5 is an important constituent of the plastid 30S subunit and affects proteins involved in photosynthesis and cold stress responses to mediate plant growth and development. PMID:27006483

  13. Metabolic engineering for isoprenoid-based biofuel production.

    PubMed

    Gupta, P; Phulara, S C

    2015-09-01

    Sustainable economic and industrial growth is the need of the hour and it requires renewable energy resources having better performance and compatibility with existing fuel infrastructure from biological routes. Isoprenoids (C ≥ 5) can be a potential alternative due to their diverse nature and physiochemical properties similar to that of petroleum based fuels. In the past decade, extensive research has been done to utilize metabolic engineering strategies in micro-organisms primarily, (i) to overcome the limitations associated with their natural and non-natural production and (ii) to develop commercially competent microbial strain for isoprenoid-based biofuel production. This review briefly describes the engineered isoprenoid biosynthetic pathways in well-characterized microbial systems for the production of several isoprenoid-based biofuels and fuel precursors. PMID:26095690

  14. Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

    PubMed Central

    Jarchow-Choy, Sarah K; Koppisch, Andrew T; Fox, David T

    2014-01-01

    Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C5-precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2C-methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined. PMID:25009443

  15. A chemical rescue screen identifies a Plasmodium falciparum apicoplast inhibitor targeting MEP isoprenoid precursor biosynthesis.

    PubMed

    Wu, Wesley; Herrera, Zachary; Ebert, Danny; Baska, Katie; Cho, Seok H; DeRisi, Joseph L; Yeh, Ellen

    2015-01-01

    The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action. PMID:25367906

  16. Plastid-LCGbase: a collection of evolutionarily conserved plastid-associated gene pairs.

    PubMed

    Wang, Dapeng; Yu, Jun

    2015-01-01

    Plastids carry their own genetic material that encodes a variable set of genes that are limited in number but functionally important. Aside from orthology, the lineage-specific order and orientation of these genes are also relevant. Here, we develop a database, Plastid-LCGbase (http://lcgbase.big.ac.cn/plastid-LCGbase/), which focuses on organizational variability of plastid genes and genomes from diverse taxonomic groups. The current Plastid-LCGbase contains information from 470 plastid genomes and exhibits several unique features. First, through a genome-overview page generated from OrganellarGenomeDRAW, it displays general arrangement of all plastid genes (circular or linear). Second, it shows patterns and modes of all paired plastid genes and their physical distances across user-defined lineages, which are facilitated by a step-wise stratification of taxonomic groups. Third, it divides the paired genes into three categories (co-directionally-paired genes or CDPGs, convergently-paired genes or CPGs and divergently-paired genes or DPGs) and three patterns (separation, overlap and inclusion) and provides basic statistics for each species. Fourth, the gene pairing scheme is expandable, where neighboring genes can also be included in species-/lineage-specific comparisons. We hope that Plastid-LCGbase facilitates gene variation (insertion-deletion, translocation and rearrangement) and transcription-level studies of plastid genomes. PMID:25378306

  17. Negative Feedbacks by Isoprenoids on a Mevalonate Kinase Expressed in the Corpora Allata of Mosquitoes

    PubMed Central

    Noriega, Fernando G.

    2015-01-01

    Background Juvenile hormones (JH) regulate development and reproductive maturation in insects. JHs are synthesized through the mevalonate pathway (MVAP), an ancient metabolic pathway present in the three domains of life. Mevalonate kinase (MVK) is a key enzyme in the MVAP. MVK catalyzes the synthesis of phosphomevalonate (PM) by transferring the γ-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid (MA). Despite the importance of MVKs, these enzymes have been poorly characterized in insects. Results We functionally characterized an Aedes aegypti MVK (AaMVK) expressed in the corpora allata (CA) of the mosquito. AaMVK displayed its activity in the presence of metal cofactors. Different nucleotides were used by AaMVK as phosphoryl donors. In the presence of Mg2+, the enzyme has higher affinity for MA than ATP. The activity of AaMVK was regulated by feedback inhibition from long-chain isoprenoids, such as geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). Conclusions AaMVK exhibited efficient inhibition by GPP and FPP (Ki less than 1 μM), and none by isopentenyl pyrophosphate (IPP) and dimethyl allyl pyrophosphate (DPPM). These results suggest that GPP and FPP might act as physiological inhibitors in the synthesis of isoprenoids in the CA of mosquitoes. Changing MVK activity can alter the flux of precursors and therefore regulate juvenile hormone biosynthesis. PMID:26566274

  18. Metabolic engineering of volatile isoprenoids in plants and microbes.

    PubMed

    Vickers, Claudia E; Bongers, Mareike; Liu, Qing; Delatte, Thierry; Bouwmeester, Harro

    2014-08-01

    The chemical properties and diversity of volatile isoprenoids lends them to a broad variety of biological roles. It also lends them to a host of biotechnological applications, both by taking advantage of their natural functions and by using them as industrial chemicals/chemical feedstocks. Natural functions include roles as insect attractants and repellents, abiotic stress protectants in pathogen defense, etc. Industrial applications include use as pharmaceuticals, flavours, fragrances, fuels, fuel additives, etc. Here we will examine the ways in which researchers have so far found to exploit volatile isoprenoids using biotechnology. Production and/or modification of volatiles using metabolic engineering in both plants and microorganisms are reviewed, including engineering through both mevalonate and methylerythritol diphosphate pathways. Recent advances are illustrated using several case studies (herbivores and bodyguards, isoprene, and monoterpene production in microbes). Systems and synthetic biology tools with particular utility for metabolic engineering are also reviewed. Finally, we discuss the practical realities of various applications in modern biotechnology, explore possible future applications, and examine the challenges of moving these technologies forward so that they can deliver tangible benefits. While this review focuses on volatile isoprenoids, many of the engineering approaches described here are also applicable to non-isoprenoid volatiles and to non-volatile isoprenoids. PMID:24588680

  19. Plastid-Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae.

    PubMed

    Weng, Mao-Lun; Ruhlman, Tracey A; Jansen, Robert K

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid-nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  20. Structure-activity analysis and antiprion mechanism of isoprenoid compounds.

    PubMed

    Hamanaka, Taichi; Nishizawa, Keiko; Sakasegawa, Yuji; Teruya, Kenta; Doh-ura, Katsumi

    2015-12-01

    The prion strain-specific mechanism by which normal prion protein is converted to abnormal prion protein remains largely unknown. This study found that insect juvenile hormone III reduced abnormal prion protein levels only in cells infected with the RML prion. We conducted a structure-activity analysis using juvenile hormone III biosynthetic intermediates in the isoprenoid pathway. Both farnesol and geranylgeraniol, the most potent inhibitors of abnormal prion protein formation, behaved in an RML prion-dependent fashion. Neither of them modified cellular and cell surface prion protein levels. Events downstream of this pathway include cholesterol biosynthesis and protein prenylation. However, neither of these isoprenoid compounds modified lipid raft microdomains and cellular cholesterol levels and neither affected the representative prenylated protein expression levels of prenylation pathways. Therefore, these isoprenoid compounds are a new class of prion strain-dependent antiprion compounds. They are useful for exploring strain-specific prion biology. PMID:26402376

  1. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae.

    PubMed

    Huerlimann, Roger; Zenger, Kyall R; Jerry, Dean R; Heimann, Kirsten

    2015-01-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  2. Phylogenetic Analysis of Nucleus-Encoded Acetyl-CoA Carboxylases Targeted at the Cytosol and Plastid of Algae

    PubMed Central

    Huerlimann, Roger; Zenger, Kyall R.; Jerry, Dean R.; Heimann, Kirsten

    2015-01-01

    The understanding of algal phylogeny is being impeded by an unknown number of events of horizontal gene transfer (HGT), and primary and secondary/tertiary endosymbiosis. Through these events, previously heterotrophic eukaryotes developed photosynthesis and acquired new biochemical pathways. Acetyl-CoA carboxylase (ACCase) is a key enzyme in the fatty acid synthesis and elongation pathways in algae, where ACCase exists in two locations (cytosol and plastid) and in two forms (homomeric and heteromeric). All algae contain nucleus-encoded homomeric ACCase in the cytosol, independent of the origin of the plastid. Nucleus-encoded homomeric ACCase is also found in plastids of algae that arose from a secondary/tertiary endosymbiotic event. In contrast, plastids of algae that arose from a primary endosymbiotic event contain heteromeric ACCase, which consists of three nucleus-encoded and one plastid-encoded subunits. These properties of ACCase provide the potential to inform on the phylogenetic relationships of hosts and their plastids, allowing different hypothesis of endosymbiotic events to be tested. Alveolata (Dinoflagellata and Apicomplexa) and Chromista (Stramenopiles, Haptophyta and Cryptophyta) have traditionally been grouped together as Chromalveolata, forming the red lineage. However, recent genetic evidence groups the Stramenopiles, Alveolata and green plastid containing Rhizaria as SAR, excluding Haptophyta and Cryptophyta. Sequences coding for plastid and cytosol targeted homomeric ACCases were isolated from Isochrysis aff. galbana (TISO), Chromera velia and Nannochloropsis oculata, representing three taxonomic groups for which sequences were lacking. Phylogenetic analyses show that cytosolic ACCase strongly supports the SAR grouping. Conversely, plastidial ACCase groups the SAR with the Haptophyta, Cryptophyta and Prasinophyceae (Chlorophyta). These two ACCase based, phylogenetic relationships suggest that the plastidial homomeric ACCase was acquired by the

  3. Mutation of Archaeal Isopentenyl Phosphate Kinase Highlights Mechanism and Guides Phosphorylation of Additional Isoprenoid Monophosphates

    PubMed Central

    2010-01-01

    The biosynthesis of isopentenyl diphosphate (IPP) from either the mevalonate (MVA) or the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway provides the key metabolite for primary and secondary isoprenoid biosynthesis. Isoprenoid metabolism plays crucial roles in membrane stability, steroid biosynthesis, vitamin production, protein localization, defense and communication, photoprotection, sugar transport, and glycoprotein biosynthesis. Recently, an alternative branch of the MVA pathway was discovered in the archaeon Methanocaldococcus jannaschii involving a small molecule kinase, isopentenyl phosphate kinase (IPK). IPK belongs to the amino acid kinase (AAK) superfamily. In vitro, IPK phosphorylates isopentenyl monophosphate (IP) in an ATP and Mg2+-dependent reaction producing IPP. Here, we describe crystal structures of IPK from M. jannaschii refined to nominal resolutions of 2.0−2.8 Å. Notably, an active site histidine residue (His60) forms a hydrogen bond with the terminal phosphate of both substrate and product. This His residue serves as a marker for a subset of the AAK family that catalyzes phosphorylation of phosphate or phosphonate functional groups; the larger family includes carboxyl-directed kinases, which lack this active site residue. Using steady-state kinetic analysis of H60A, H60N, and H60Q mutants, the protonated form of the Nε2 nitrogen of His60 was shown to be essential for catalysis, most likely through hydrogen bond stabilization of the transition state accompanying transphosphorylation. Moreover, the structures served as the starting point for the engineering of IPK mutants capable of the chemoenzymatic synthesis of longer chain isoprenoid diphosphates from monophosphate precursors. PMID:20392112

  4. A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway.

    PubMed

    Yang, Tianhong; Fang, Lingling; Rimando, Agnes M; Sobolev, Victor; Mockaitis, Keithanne; Medina-Bolivar, Fabricio

    2016-08-01

    Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. PMID:27356974

  5. A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway1[OPEN

    PubMed Central

    2016-01-01

    Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. PMID:27356974

  6. Pleiotropic Effects of a Schweinfurthin on Isoprenoid Homeostasis

    PubMed Central

    Holstein, Sarah A.; Kuder, Craig H.; Tong, Huaxiang

    2013-01-01

    The schweinfurthins, a family of natural products derived from the isoprenoid biosynthetic pathway (IBP), have marked growth inhibitory activity. However, the biochemical basis for the schweinfurthins cellular effects has remained ill-defined. Here, the effects of the synthetic schweinfurthin, 3-deoxyschweinfurthin (3dSB) on multiple aspects of isoprenoid homeostasis are explored. Cytotoxicity assays demonstrate a synergistic interaction between 3dSB and the HMG-CoA reductase inhibitor lovastatin but not with other IBP inhibitors in a variety of human cancer cell lines. The cytotoxic effects of 3dSB were enhanced in cells incubated in lipid-depleted serum. 3dSB was found to enhance the lovastatin-induced decrease in protein prenylation. In addition, 3dSB decreases intracellular farnesyl pyrophosphate and geranylgeranyl pyrophosphate levels in both established cell lines and primary cells. To determine whether 3dSB alters the regulation of expression of genes involved in isoprenoid homeostasis, real-time PCR studies were performed in human cell lines cultured in either lipid-replete or -deplete conditions. These studies demonstrate that 3dSB abrogates lovastatin-induced upregulation of sterol regulatory element-containing genes and lovastatin-induced downregulation of ABCA1. In aggregate, these studies are the first to demonstrate that a schweinfurthin exerts pleiotropic effects on isoprenoid homeostasis. PMID:21633866

  7. Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid Endosymbiosis?

    PubMed Central

    Cenci, Ugo; Ducatez, Mathieu; Kadouche, Derifa; Colleoni, Christophe; Ball, Steven G.

    2016-01-01

    Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol. We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal. PMID:27446814

  8. Plastids: The Green Frontiers for Vaccine Production

    PubMed Central

    Waheed, Mohammad T.; Ismail, Hammad; Gottschamel, Johanna; Mirza, Bushra; Lössl, Andreas G.

    2015-01-01

    Infectious diseases pose an increasing risk to health, especially in developing countries. Vaccines are available to either cure or prevent many of these diseases. However, there are certain limitations related to these vaccines, mainly the costs, which make these vaccines mostly unaffordable for people in resource poor countries. These costs are mainly related to production and purification of the products manufactured from fermenter-based systems. Plastid biotechnology has become an attractive platform to produce biopharmaceuticals in large amounts and cost-effectively. This is mainly due to high copy number of plastids DNA in mature chloroplasts, a characteristic particularly important for vaccine production in large amounts. An additional advantage lies in the maternal inheritance of plastids in most plant species, which addresses the regulatory concerns related to transgenic plants. These and many other aspects of plastids will be discussed in the present review, especially those that particularly make these green biofactories an attractive platform for vaccine production. A summary of recent vaccine antigens against different human diseases expressed in plastids will also be presented. PMID:26635832

  9. Protein transport into secondary plastids and the evolution of primary and secondary plastids.

    PubMed

    Kroth, Peter G

    2002-01-01

    Chloroplasts are key organelles in algae and plants due to their photosynthetic abilities. They are thought to have evolved from prokaryotic cyanobacteria taken up by a eukaryotic host cell in a process termed primary endocytobiosis. In addition, a variety of organisms have evolved by subsequent secondary endocytobioses, in which a heterotrophic host cell engulfed a eukaryotic alga. Both processes dramatically enhanced the complexity of the resulting cells. Since the first version of the endosymbiotic theory was proposed more than 100 years ago, morphological, physiological, biochemical, and molecular data have been collected substantiating the emerging picture about the origin and the relationship of individual organisms with different primary or secondary chloroplast types. Depending on their origin, plastids in different lineages may have two, three, or four envelope membranes. The evolutionary success of endocytobioses depends, among other factors, on the specific exchange of molecules between the host and endosymbiont. This raises questions concerning how targeting of nucleus-encoded proteins into the different plastid types occurs and how these processes may have developed. Most studies of protein translocation into plastids have been performed on primary plastids, but in recent years more complex protein-translocation systems of secondary plastids have been investigated. Analyses of transport systems in different algal lineages with secondary plastids reveal that during evolution existing translocation machineries were recycled or recombined rather than being developed de novo. This review deals with current knowledge about the evolution and function of primary and secondary plastids and the respective protein-targeting systems. PMID:12455749

  10. Control mechanisms of plastid gene expression

    SciTech Connect

    Gruissem, W.; Tonkyn, J.C.

    1993-12-31

    Plastid DNAs of higher plants contain approximately 150 genes that encode RNAs and proteins for genetic and photosynthetic functions of the organelle. Results published in the last few years illustrate that the spatial and temporal expression of these plastid genes is regulated, in part, at the transcriptional level, but that developmentally controlled changes in mRNA stability, translational activity, and protein phosphorylation also have an important role in the control of plastid functions. This comprehensive review summarizes and discusses the mechanisms by which regulation of gene expression is exerted at the transcriptional and post-transcriptional levels. It provides an overview of our current knowledge, but also emphasizes areas that are controversial and in which information on regulatory mechanisms is still incomplete. 455 refs., 3 figs., 3 tabs.

  11. Regulation of chloroplast number and DNA synthesis in higher plants. Final report, August 1995--August 1996

    SciTech Connect

    Mullet, J.E.

    1997-06-17

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focused on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The research focused on the isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  12. Integration of plastids with their hosts: Lessons learned from dinoflagellates.

    PubMed

    Dorrell, Richard G; Howe, Christopher J

    2015-08-18

    After their endosymbiotic acquisition, plastids become intimately connected with the biology of their host. For example, genes essential for plastid function may be relocated from the genomes of plastids to the host nucleus, and pathways may evolve within the host to support the plastid. In this review, we consider the different degrees of integration observed in dinoflagellates and their associated plastids, which have been acquired through multiple different endosymbiotic events. Most dinoflagellate species possess plastids that contain the pigment peridinin and show extreme reduction and integration with the host biology. In some species, these plastids have been replaced through serial endosymbiosis with plastids derived from a different phylogenetic derivation, of which some have become intimately connected with the biology of the host whereas others have not. We discuss in particular the evolution of the fucoxanthin-containing dinoflagellates, which have adapted pathways retained from the ancestral peridinin plastid symbiosis for transcript processing in their current, serially acquired plastids. Finally, we consider why such a diversity of different degrees of integration between host and plastid is observed in different dinoflagellates and how dinoflagellates may thus inform our broader understanding of plastid evolution and function. PMID:25995366

  13. Integration of plastids with their hosts: Lessons learned from dinoflagellates

    PubMed Central

    Dorrell, Richard G.; Howe, Christopher J.

    2015-01-01

    After their endosymbiotic acquisition, plastids become intimately connected with the biology of their host. For example, genes essential for plastid function may be relocated from the genomes of plastids to the host nucleus, and pathways may evolve within the host to support the plastid. In this review, we consider the different degrees of integration observed in dinoflagellates and their associated plastids, which have been acquired through multiple different endosymbiotic events. Most dinoflagellate species possess plastids that contain the pigment peridinin and show extreme reduction and integration with the host biology. In some species, these plastids have been replaced through serial endosymbiosis with plastids derived from a different phylogenetic derivation, of which some have become intimately connected with the biology of the host whereas others have not. We discuss in particular the evolution of the fucoxanthin-containing dinoflagellates, which have adapted pathways retained from the ancestral peridinin plastid symbiosis for transcript processing in their current, serially acquired plastids. Finally, we consider why such a diversity of different degrees of integration between host and plastid is observed in different dinoflagellates and how dinoflagellates may thus inform our broader understanding of plastid evolution and function. PMID:25995366

  14. Microbial Production of Isoprenoids Enabled by Synthetic Biology

    PubMed Central

    Immethun, Cheryl M.; Hoynes-O’Connor, Allison G.; Balassy, Andrea; Moon, Tae Seok

    2013-01-01

    Microorganisms transform inexpensive carbon sources into highly functionalized compounds without toxic by-product generation or significant energy consumption. By redesigning the natural biosynthetic pathways in an industrially suited host, microbial cell factories can produce complex compounds for a variety of industries. Isoprenoids include many medically important compounds such as antioxidants and anticancer and antimalarial drugs, all of which have been produced microbially. While a biosynthetic pathway could be simply transferred to the production host, the titers would become economically feasible when it is rationally designed, built, and optimized through synthetic biology tools. These tools have been implemented by a number of research groups, with new tools pledging further improvements in yields and expansion to new medically relevant compounds. This review focuses on the microbial production of isoprenoids for the health industry and the advancements though synthetic biology. PMID:23577007

  15. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis.

    PubMed

    Kuzuyama, Tomohisa; Seto, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-D-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  16. Two distinct pathways for essential metabolic precursors for isoprenoid biosynthesis

    PubMed Central

    KUZUYAMA, Tomohisa; SETO, Haruo

    2012-01-01

    Isoprenoids are a diverse group of molecules found in all organisms, where they perform such important biological functions as hormone signaling (e.g., steroids) in mammals, antioxidation (e.g., carotenoids) in plants, electron transport (e.g., ubiquinone), and cell wall biosynthesis intermediates in bacteria. All isoprenoids are synthesized by the consecutive condensation of the five-carbon monomer isopentenyl diphosphate (IPP) to its isomer, dimethylallyl diphosphate (DMAPP). The biosynthetic pathway for the formation of IPP from acetyl-CoA (i.e., the mevalonate pathway) had been established mainly in mice and the budding yeast Saccharomyces cerevisiae. Curiously, most prokaryotic microorganisms lack homologs of the genes in the mevalonate pathway, even though IPP and DMAPP are essential for isoprenoid biosynthesis in bacteria. This observation provided an impetus to search for an alternative pathway to synthesize IPP and DMAPP, ultimately leading to the discovery of the mevalonate-independent 2-C-methyl-d-erythritol 4-phosphate pathway. This review article focuses on our significant contributions to a comprehensive understanding of the biosynthesis of IPP and DMAPP. PMID:22450534

  17. A Novel Component of the Disulfide-Reducing Pathway Required for Cytochrome c Assembly in Plastids

    PubMed Central

    Gabilly, Stéphane T.; Kropat, Janette; Karamoko, Mohamed; Page, M. Dudley; Nakamoto, Stacie S.; Merchant, Sabeeha S.; Hamel, Patrice P.

    2011-01-01

    In plastids, the conversion of energy in the form of light to ATP requires key electron shuttles, the c-type cytochromes, which are defined by the covalent attachment of heme to a CXXCH motif. Plastid c-type cytochrome biogenesis occurs in the thylakoid lumen and requires a system for transmembrane transfer of reductants. Previously, CCDA and CCS5/HCF164, found in all plastid-containing organisms, have been proposed as two components of the disulfide-reducing pathway. In this work, we identify a small novel protein, CCS4, as a third component in this pathway. CCS4 was genetically identified in the green alga Chlamydomonas reinhardtii on the basis of the rescue of the ccs4 mutant, which is blocked in the synthesis of holoforms of plastid c-type cytochromes, namely cytochromes f and c6. Although CCS4 does not display sequence motifs suggestive of redox or heme-binding function, biochemical and genetic complementation experiments suggest a role in the disulfide-reducing pathway required for heme attachment to apoforms of cytochromes c. Exogenous thiols partially rescue the growth phenotype of the ccs4 mutant concomitant with recovery of holocytochrome f accumulation, as does expression of an ectopic copy of the CCDA gene, encoding a trans-thylakoid transporter of reducing equivalents. We suggest that CCS4 might function to stabilize CCDA or regulate its activity. PMID:21220358

  18. The Plastid Outer Envelope – A Highly Dynamic Interface between Plastid and Cytoplasm

    PubMed Central

    Breuers, Frederique K. H.; Bräutigam, Andrea; Weber, Andreas P. M.

    2011-01-01

    Plastids are the defining organelles of all photosynthetic eukaryotes. They are the site of photosynthesis and of a large number of other essential metabolic pathways, such as fatty acid and amino acid biosyntheses, sulfur and nitrogen assimilation, and aromatic and terpenoid compound production, to mention only a few examples. The metabolism of plastids is heavily intertwined and connected with that of the surrounding cytosol, thus causing massive traffic of metabolic precursors, intermediates, and products. Two layers of biological membranes that are called the inner (IE) and the outer (OE) plastid envelope membranes bound the plastids of Archaeplastida. While the IE is generally accepted as the osmo-regulatory barrier between cytosol and stroma, the OE was considered to represent an unspecific molecular sieve, permeable for molecules of up to 10 kDa. However, after the discovery of small substrate specific pores in the OE, this view has come under scrutiny. In addition to controlling metabolic fluxes between plastid and cytosol, the OE is also crucial for protein import into the chloroplast. It contains the receptors and translocation channel of the TOC complex that is required for the canonical post-translational import of nuclear-encoded, plastid-targeted proteins. Further, the OE is a metabolically active compartment of the chloroplast, being involved in, e.g., fatty acid metabolism and membrane lipid production. Also, recent findings hint on the OE as a defense platform against several biotic and abiotic stress conditions, such as cold acclimation, freezing tolerance, and phosphate deprivation. Moreover, dynamic non-covalent interactions between the OE and the endomembrane system are thought to play important roles in lipid and non-canonical protein trafficking between plastid and endoplasmic reticulum. While proteomics and bioinformatics has provided us with comprehensive but still incomplete information on proteins localized in the plastid IE, the stroma

  19. A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum

    PubMed Central

    2010-01-01

    Background Plastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates. Results We sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae. Conclusions L. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These

  20. Plastid Proteomic Analysis in Tomato Fruit Development

    PubMed Central

    Kondo, Takanori; Dohra, Hideo; Ito, Yumihiko; Kiriiwa, Yoshikazu; Hayashi, Marina; Kamiya, Shiori; Kato, Masaya; Fujiwara, Masayuki; Fukao, Yoichiro; Kobayashi, Megumi; Nagata, Noriko; Motohashi, Reiko

    2015-01-01

    To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of ‘Micro-Tom’ fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared ‘Micro-Tom’ results with those from two other varieties, ‘Black’ and ‘White Beauty’. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of ‘Micro-Tom’, and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared ‘Micro-Tom’ fruits with ‘Black’ and ‘White Beauty’ using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the ‘Black’ and ‘White Beauty’ varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between ‘Micro-Tom’ and ‘Black’/‘White Beauty’. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast. PMID:26371478

  1. Plastid Proteomic Analysis in Tomato Fruit Development.

    PubMed

    Suzuki, Miho; Takahashi, Sachiko; Kondo, Takanori; Dohra, Hideo; Ito, Yumihiko; Kiriiwa, Yoshikazu; Hayashi, Marina; Kamiya, Shiori; Kato, Masaya; Fujiwara, Masayuki; Fukao, Yoichiro; Kobayashi, Megumi; Nagata, Noriko; Motohashi, Reiko

    2015-01-01

    To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast. PMID:26371478

  2. Plastid Genome-Based Phylogeny Pinpointed the Origin of the Green-Colored Plastid in the Dinoflagellate Lepidodinium chlorophorum

    PubMed Central

    Kamikawa, Ryoma; Tanifuji, Goro; Kawachi, Masanobu; Miyashita, Hideaki; Hashimoto, Tetsuo; Inagaki, Yuji

    2015-01-01

    Unlike many other photosynthetic dinoflagellates, whose plastids contain a characteristic carotenoid peridinin, members of the genus Lepidodinium are the only known dinoflagellate species possessing green alga-derived plastids. However, the precise origin of Lepidodinium plastids has hitherto remained uncertain. In this study, we completely sequenced the plastid genome of Lepidodinium chlorophorum NIES-1868. Our phylogenetic analyses of 52 plastid-encoded proteins unite L. chlorophorum exclusively with a pedinophyte, Pedinomonas minor, indicating that the green-colored plastids in Lepidodinium spp. were derived from an endosymbiotic pedinophyte or a green alga closely related to pedinophytes. Our genome comparison incorporating the origin of the Lepidodinium plastids strongly suggests that the endosymbiont plastid genome acquired by the ancestral Lepidodinium species has lost genes encoding proteins involved in metabolism and biosynthesis, protein/metabolite transport, and plastid division during the endosymbiosis. We further discuss the commonalities and idiosyncrasies in genome evolution between the L. chlorophorum plastid and other plastids acquired through endosymbiosis of eukaryotic photoautotrophs. PMID:25840416

  3. LC-MS method for screening unknown microbial carotenoids and isoprenoid quinones.

    PubMed

    Kaiser, Philipp; Geyer, Roland; Surmann, Peter; Fuhrmann, Herbert

    2012-01-01

    The structure of secondary metabolites from microorganisms provides a useful tool for microbial characterization and chemotaxonomic classification. Microbial isoprenoid quinones, for example, are well described and used to distinguish among photosynthetic microorganism groups. In addition, isoprenoid quinones can also be found, together with carotenoids, in non-photosynthetic microorganisms. The aim of the present study was to develop a LC-MS/MS method which can analyze and identify these microbial isoprenoids. Positive atmospheric pressure chemical ionization (APCI) together with collisionally induced dissociation was applied for generation of informative fragment spectra by mass spectrometry. Enhanced product ion (EPI) scan in a linear ion trap with information dependent data acquisition (IDA) enabled generation of MS fragment data even from minor isoprenoids. The developed liquid chromatography method enabled separation of isoprenoid patterns from their ester derivatives. Discovery and structural characterization of isoprenoid quinones and carotenoids were carried out by comparing characteristics of fragment spectra from unknown compounds with fragment spectra of a range of isoprenoid standard compounds and using published data. Throughout the study 17 microorganisms (e.g., Acremonium butyri, Arthrobacter spp., Brevibacterium linens, Bullera variabilis, Exophiala dermatitidis, Lecythophora hoffmannii, Panthoea agglomerans, Rhodotorula spp., Xanthophyllomyces dendrorhous) were screened and probable structures of isoprenoid quinones and carotenoids were suggested. The method lays some foundations on the analysis of yet unknown isoprenoids in microorganisms by using LCMS/MS techniques. PMID:22036764

  4. Structural analysis, plastid localization, and expression of the biotin carboxylase subunit of acetyl-coenzyme A carboxylase from tobacco.

    PubMed

    Shorrosh, B S; Roesler, K R; Shintani, D; van de Loo, F J; Ohlrogge, J B

    1995-06-01

    Acetyl-coenzyme A carboxylase (ACCase, EC 6.4.1.2) catalyzes the synthesis of malonyl-coenzyme A, which is utilized in the plastid for de novo fatty acid synthesis and outside the plastid for a variety of reactions, including the synthesis of very long chain fatty acids and flavonoids. Recent evidence for both multifunctional and multisubunit ACCase isozymes in dicot plants has been obtained. We describe here the isolation of a tobacco (Nicotiana tabacum L. cv bright yellow 2 [NT1]) cDNA clone (E3) that encodes a 58.4-kD protein that shares 80% sequence similarity and 65% identity with the Anabaena biotin carboxylase subunit of ACCase. Similar to other biotin carboxylase subunits of acetyl-CoA carboxylase, the E3-encoded protein contains a putative ATP-binding motif but lacks a biotin-binding site (methionine-lysine-methionine or methionine-lysine-leucine). The deduced protein sequence contains a putative transit peptide whose function was confirmed by its ability to direct in vitro chloroplast uptake. The subcellular localization of this biotin carboxylase has also been confirmed to be plastidial by western blot analysis of pea (Pisum sativum), alfalfa (Medicago sativa L.), and castor (Ricinus communis L.) plastid preparations. Northern blot analysis indicates that the plastid biotin carboxylase transcripts are expressed at severalfold higher levels in castor seeds than in leaves. PMID:7610168

  5. Molecular Physiological Analysis of the Two Plastidic ATP/ADP Transporters from Arabidopsis12

    PubMed Central

    Reiser, Jens; Linka, Nicole; Lemke, Lilia; Jeblick, Wolfgang; Neuhaus, H. Ekkehard

    2004-01-01

    Arabidopsis (Arabidopsis thaliana) possesses two isoforms of plastidic ATP/ADP transporters (AtNTT1 and AtNTT2) exhibiting similar biochemical properties. To analyze the function of both isoforms on the molecular level, we examined the expression pattern of both genes by northern-blot analysis and promoter-β-glucuronidase fusions. AtNTT1 represents a sugar-induced gene mainly expressed in stem and roots, whereas AtNTT2 is expressed in several Arabidopsis tissues with highest accumulation in developing roots and young cotyledons. Developing lipid-storing seeds hardly contained AtNTT1 or -2 transcripts. The absence of a functional AtNTT1 gene affected plant development only slightly, whereas AtNTT2∷T-DNA, AtNTT1-2∷T-DNA, and RNA interference (RNAi) plants showed retarded plant development, mainly characterized by a reduced ability to generate primary roots and a delayed chlorophyll accumulation in seedlings. Electron microscopic examination of chloroplast substructure also revealed an impaired formation of thylakoids in RNAi seedlings. Moreover, RNAi- and AtNTT1-2∷T-DNA plants showed reduced accumulation of the nuclear-encoded protein CP24 during deetiolation. Under short-day conditions reduced plastidic ATP import capacity correlates with a substantially reduced plant growth rate. This effect is absent under long-day conditions, strikingly indicating that nocturnal ATP import into chloroplasts is important. Plastidic ATP/ADP transport activity exerts significant control on lipid synthesis in developing Arabidopsis seeds. In total we made the surprising observation that plastidic ATP/ADP transport activity is not required to pass through the complete plant life cycle. However, plastidic ATP/ADP-transporter activity is required for both an undisturbed development of young tissues and a controlled cellular metabolism in mature leaves. PMID:15516503

  6. Membrane Composition and Physiological Activity of Plastids from an Oenothera Plastome Mutator-Induced Chloroplast Mutant 1

    PubMed Central

    Johnson, Ellen M.; Sears, Barbara B.

    1990-01-01

    Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type I) contained in these lines. In these cases, only chlorophyll concentrations differed significantly. PMID:16667256

  7. Biosynthesis of digalactosyldiacylglycerol in plastids from 16:3 and 18:3 plants

    SciTech Connect

    Heemskerk, J.W.M.; Heinz, E. ); Storz, T.; Schmidt, R.R. )

    1990-08-01

    Intact chloroplasts isolated from leaves of eight species of 16:3 and 18:3 plants and chromoplasts isolated from Narcissus pseudonarcissus L. flowers synthesize galactose-labeled mono-, di-, and trigalactosyldiacylglycerol (MGDG, DGDG, and TGDG) when incubated with UDP-(6-{sup 3}H)galactose. In all plastids, galactolipid synthesis, and especially synthesis of DGDG and TGDG, is reduced by treatment of the organelles with the nonpenetrating protease thermolysin. Envelope membranes isolated from thermolysin-treated chloroplasts of Spinacia oleracea L. (16:3 plant) and Pisum sativum L. (18:3 plant) or membranes isolated from thermolysin-treated chromoplasts are strongly reduced in galactolipid:galactolipid galactosyltransferase activity, but not with regard to UDP-Gal:diacylglycerol galactosyltransferase. For the intact plastids, this indicates that thermolysin treatment specifically blocks DGDG (and TGDG) synthesis, whereas MGDG synthesis is not affected. Neither in chloroplast nor in chromoplast membranes is DGDG synthesis stimulated by UDP-Gal. DGDG synthesis in S. oleracea chloroplasts is not stimulated by nucleoside 5{prime}-diphospho digalactosides. Therefore, galactolipid:galactolipid galactosyltransferase is so far the only detectable enzyme synthesizing DGDG.

  8. Plastid control of abaxial-adaxial patterning

    PubMed Central

    Mateo-Bonmatí, Eduardo; Casanova-Sáez, Rubén; Quesada, Víctor; Hricová, Andrea; Candela, Héctor; Micol, José Luis

    2015-01-01

    Translational regulation, exerted by the cytosolic ribosome, has been shown to participate in the establishment of abaxial-adaxial polarity in Arabidopsis thaliana: many hypomorphic and null alleles of genes encoding proteins of the cytosolic ribosome enhance the leaf polarity defects of asymmetric leaves1 (as1) and as2 mutants. Here, we report the identification of the SCABRA1 (SCA1) nuclear gene, whose loss-of-function mutations also enhance the polarity defects of the as2 mutants. In striking contrast to other previously known enhancers of the phenotypes caused by the as1 and as2 mutations, we found that SCA1 encodes a plastid-type ribosomal protein that functions as a structural component of the 70S plastid ribosome and, therefore, its role in abaxial-adaxial patterning was not expected. PMID:26522839

  9. Polar Lipid Composition of a Plastid Ribosome-Deficient Barley Mutant 1

    PubMed Central

    Dorne, Albert-Jean; Carde, Jean-Pierre; Joyard, Jacques; Börner, Thomas; Douce, Roland

    1982-01-01

    Green and white leaves of the barley mutant line `albostrians' were compared for their polar lipid content and fatty acid composition. The mutant plastids of the white leaves have a double-layered envelope, but in contrast with the normal chloroplasts, lack 70 S ribosomes and thylakoids. In the green leaves, the amount of monogalactosyldiacylglycerol (MGDG) consistently exceeds the amount of digalactosyldiacylglycerol (DGDG) and the amount of galactolipids exceeds the amount of phospholipids. In contrast, in white leaves the amount of DGDG exceeds the amount of MGDG and the amount of phospholipids exceeds the amount of galactolipids. In white leaves, the galactolipid composition reflects the plastid envelope composition which is rich in DGDG, whereas in green leaves the galactolipid composition reflects the thylakoid composition which is rich in MGDG. These results demonstrate the likelihood that all the enzymes involved in galactolipid, sulfolipid and fatty acid synthesis are coded by the nuclear genome. Images PMID:16662423

  10. Metabolic engineering of higher plants and algae for isoprenoid production.

    PubMed

    Kempinski, Chase; Jiang, Zuodong; Bell, Stephen; Chappell, Joe

    2015-01-01

    Isoprenoids are a class of compounds derived from the five carbon precursors, dimethylallyl diphosphate, and isopentenyl diphosphate. These molecules present incredible natural chemical diversity, which can be valuable for humans in many aspects such as cosmetics, agriculture, and medicine. However, many terpenoids are only produced in small quantities by their natural hosts and can be difficult to generate synthetically. Therefore, much interest and effort has been directed toward capturing the genetic blueprint for their biochemistry and engineering it into alternative hosts such as plants and algae. These autotrophic organisms are attractive when compared to traditional microbial platforms because of their ability to utilize atmospheric CO2 as a carbon substrate instead of supplied carbon sources like glucose. This chapter will summarize important techniques and strategies for engineering the accumulation of isoprenoid metabolites into higher plants and algae by choosing the correct host, avoiding endogenous regulatory mechanisms, and optimizing potential flux into the target compound. Future endeavors will build on these efforts by fine-tuning product accumulation levels via the vast amount of available "-omic" data and devising metabolic engineering schemes that integrate this into a whole-organism approach. With the development of high-throughput transformation protocols and synthetic biology molecular tools, we have only begun to harness the power and utility of plant and algae metabolic engineering. PMID:25636485

  11. Thermal alteration of organic matter in recent marine sediments. 2: Isoprenoids. [Tanner Basin off Southern California

    NASA Technical Reports Server (NTRS)

    Ikan, R.; Baedecker, M. J.; Kaplan, I. R.

    1974-01-01

    A series of isoprenoid compounds were isolated from a heat treated marine sediment (from Tanner Basin) which were not present in the original sediment. Among the compounds identified were: phytol, dihydrophytol, c-18-isoprenoid ketone, phytanic and pristanic acids, c-19 and c-20-monoolefines, and the alkanes pristane and phytane. The significance and possible routes leading to these compounds is discussed.

  12. FAX1, a novel membrane protein mediating plastid fatty acid export.

    PubMed

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-02-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  13. FAX1, a Novel Membrane Protein Mediating Plastid Fatty Acid Export

    PubMed Central

    Li, Nannan; Gügel, Irene Luise; Giavalisco, Patrick; Zeisler, Viktoria; Schreiber, Lukas; Soll, Jürgen; Philippar, Katrin

    2015-01-01

    Fatty acid synthesis in plants occurs in plastids, and thus, export for subsequent acyl editing and lipid assembly in the cytosol and endoplasmatic reticulum is required. Yet, the transport mechanism for plastid fatty acids still remains enigmatic. We isolated FAX1 (fatty acid export 1), a novel protein, which inserts into the chloroplast inner envelope by α-helical membrane-spanning domains. Detailed phenotypic and ultrastructural analyses of FAX1 mutants in Arabidopsis thaliana showed that FAX1 function is crucial for biomass production, male fertility and synthesis of fatty acid-derived compounds such as lipids, ketone waxes, or pollen cell wall material. Determination of lipid, fatty acid, and wax contents by mass spectrometry revealed that endoplasmatic reticulum (ER)-derived lipids decreased when FAX1 was missing, but levels of several plastid-produced species increased. FAX1 over-expressing lines showed the opposite behavior, including a pronounced increase of triacyglycerol oils in flowers and leaves. Furthermore, the cuticular layer of stems from fax1 knockout lines was specifically reduced in C29 ketone wax compounds. Differential gene expression in FAX1 mutants as determined by DNA microarray analysis confirmed phenotypes and metabolic imbalances. Since in yeast FAX1 could complement for fatty acid transport, we concluded that FAX1 mediates fatty acid export from plastids. In vertebrates, FAX1 relatives are structurally related, mitochondrial membrane proteins of so-far unknown function. Therefore, this protein family might represent a powerful tool not only to increase lipid/biofuel production in plants but also to explore novel transport systems involved in vertebrate fatty acid and lipid metabolism. PMID:25646734

  14. The Plastid Genome of the Cryptomonad Teleaulax amphioxeia

    PubMed Central

    Kim, Jong Im; Yoon, Hwan Su; Yi, Gangman; Kim, Hyung Seop; Yih, Wonho; Shin, Woongghi

    2015-01-01

    Teleaulax amphioxeia is a photosynthetic unicellular cryptophyte alga that is distributed throughout marine habitats worldwide. This alga is an important plastid donor to the dinoflagellate Dinophysis caudata through the ciliate Mesodinium rubrum in the marine food web. To better understand the genomic characteristics of T. amphioxeia, we have sequenced and analyzed its plastid genome. The plastid genome sequence of T. amphioxeia is similar to that of Rhodomonas salina, and they share significant synteny. This sequence exhibits less similarity to that of Guillardia theta, the representative plastid genome of photosynthetic cryptophytes. The gene content and order of the three photosynthetic cryptomonad plastid genomes studied is highly conserved. The plastid genome of T. amphioxeia is composed of 129,772 bp and includes 143 protein-coding genes, 2 rRNA operons and 30 tRNA sequences. The DNA polymerase III gene (dnaX) was most likely acquired via lateral gene transfer (LGT) from a firmicute bacterium, identical to what occurred in R. salina. On the other hand, the psbN gene was independently encoded by the plastid genome without a reverse transcriptase gene as an intron. To clarify the phylogenetic relationships of the algae with red-algal derived plastids, phylogenetic analyses of 32 taxa were performed, including three previously sequenced cryptophyte plastid genomes containing 93 protein-coding genes. The stramenopiles were found to have branched out from the Chromista taxa (cryptophytes, haptophytes, and stramenopiles), while the cryptophytes and haptophytes were consistently grouped into sister relationships with high resolution. PMID:26047475

  15. Dynamic composition, shaping and organization of plastid nucleoids

    PubMed Central

    Powikrowska, Marta; Oetke, Svenja; Jensen, Poul E.; Krupinska, Karin

    2014-01-01

    In this article recent progress on the elucidation of the dynamic composition and structure of plastid nucleoids is reviewed from a structural perspective. Plastid nucleoids are compact structures of multiple copies of different forms of ptDNA, RNA, enzymes for replication and gene expression as well as DNA binding proteins. Although early electron microscopy suggested that plastid DNA is almost free of proteins, it is now well established that the DNA in nucleoids similarly as in the nuclear chromatin is associated with basic proteins playing key roles in organization of the DNA architecture and in regulation of DNA associated enzymatic activities involved in transcription, replication, and recombination. This group of DNA binding proteins has been named plastid nucleoid associated proteins (ptNAPs). Plastid nucleoids are unique with respect to their variable number, genome copy content and dynamic distribution within different types of plastids. The mechanisms underlying the shaping and reorganization of plastid nucleoids during chloroplast development and in response to environmental conditions involve posttranslational modifications of ptNAPs, similarly to those changes known for histones in the eukaryotic chromatin, as well as changes in the repertoire of ptNAPs, as known for nucleoids of bacteria. Attachment of plastid nucleoids to membranes is proposed to be important not only for regulation of DNA availability for replication and transcription, but also for the coordination of photosynthesis and plastid gene expression. PMID:25237313

  16. Transport of nuclear-encoded proteins into secondarily evolved plastids.

    PubMed

    Hempel, Franziska; Bozarth, Andrew; Sommer, Maik S; Zauner, Stefan; Przyborski, Jude M; Maier, Uwe-G

    2007-09-01

    Many algal groups evolved by engulfment and intracellular reduction of a eukaryotic phototroph within a heterotrophic cell. Via this process, so-called secondary plastids evolved, surrounded by three or four membranes. In these organisms most of the genetic material encoding plastid functions is localized in the cell nucleus, with the result that many proteins have to pass three, four, or even five membranes to reach their final destination within the plastid. In this article, we review recent models and findings that help to explain important cellular mechanisms involved in the complex process of protein transport into secondary plastids. PMID:17696773

  17. Microtubules restrict plastid sedimentation in protonemata of the moss Ceratodon

    NASA Technical Reports Server (NTRS)

    Schwuchow, J.; Sack, F. D.

    1994-01-01

    Apical cells of protonemata of the moss Ceratodon purpureus are unusual among plant cells with sedimentation in that only some amyloplasts sediment and these do not fall completely to the bottom of vertical cells. To determine whether the cytoskeleton restricts plastid sedimentation, the effects of amiprophos-methyl (APM) and cytochalasin D (CD) on plastid position were quantified. APM treatments of 30-60 min increased the plastid sedimentation that is normally seen along the length of untreated or control cells. Longer APM treatments often resulted in more dramatic plastid sedimentation, and in some cases almost all plastids sedimented to the lowermost point in the cell. In contrast, the microfilament inhibitor CD did not affect longitudinal plastid sedimentation compared to untreated cells, although it did disturb or eliminate plastid zonation in the tip. These data suggest that microtubules restrict the sedimentation of plastids along the length of the cell and that microtubules are load-bearing for all the plastids in the apical cell. This demonstrates the importance of the cytoskeleton in maintaining organelle position and cell organization against the force of gravity.

  18. Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation

    SciTech Connect

    Nawrath, C.; Poirier, Y.; Somerville, C. )

    1994-12-20

    In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plans accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-[mu]m granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand. 20 refs., 6 figs.

  19. Updating algal evolutionary relationships through plastid genome sequencing: did alveolate plastids emerge through endosymbiosis of an ochrophyte?

    PubMed Central

    Ševčíková, Tereza; Horák, Aleš; Klimeš, Vladimír; Zbránková, Veronika; Demir-Hilton, Elif; Sudek, Sebastian; Jenkins, Jerry; Schmutz, Jeremy; Přibyl, Pavel; Fousek, Jan; Vlček, Čestmír; Lang, B. Franz; Oborník, Miroslav; Worden, Alexandra Z.; Eliáš, Marek

    2015-01-01

    Algae with secondary plastids of a red algal origin, such as ochrophytes (photosynthetic stramenopiles), are diverse and ecologically important, yet their evolutionary history remains controversial. We sequenced plastid genomes of two ochrophytes, Ochromonas sp. CCMP1393 (Chrysophyceae) and Trachydiscus minutus (Eustigmatophyceae). A shared split of the clpC gene as well as phylogenomic analyses of concatenated protein sequences demonstrated that chrysophytes and eustigmatophytes form a clade, the Limnista, exhibiting an unexpectedly elevated rate of plastid gene evolution. Our analyses also indicate that the root of the ochrophyte phylogeny falls between the recently redefined Khakista and Phaeista assemblages. Taking advantage of the expanded sampling of plastid genome sequences, we revisited the phylogenetic position of the plastid of Vitrella brassicaformis, a member of Alveolata with the least derived plastid genome known for the whole group. The results varied depending on the dataset and phylogenetic method employed, but suggested that the Vitrella plastids emerged from a deep ochrophyte lineage rather than being derived vertically from a hypothetical plastid-bearing common ancestor of alveolates and stramenopiles. Thus, we hypothesize that the plastid in Vitrella, and potentially in other alveolates, may have been acquired by an endosymbiosis of an early ochrophyte. PMID:26017773

  20. Regulation of Isoprenoid Pheromone Biosynthesis in Bumblebee Males.

    PubMed

    Prchalová, Darina; Buček, Aleš; Brabcová, Jana; Žáček, Petr; Kindl, Jiří; Valterová, Irena; Pichová, Iva

    2016-02-01

    Males of the closely related species Bombus terrestris and Bombus lucorum attract conspecific females by completely different marking pheromones. MP of B. terrestris and B. lucorum pheromones contain mainly isoprenoid (ISP) compounds and fatty acid derivatives, respectively. Here, we studied the regulation of ISP biosynthesis in both bumblebees. RNA-seq and qRT-PCR analyses indicated that acetoacetyl-CoA thiolase (AACT), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), and farnesyl diphosphate synthase (FPPS) transcripts are abundant in the B. terrestris labial gland. Maximal abundance of these transcripts correlated well with AACT enzymatic activity detected in the LG extracts. In contrast, transcript abundances of AACT, HMGR, and FPPS in B. lucorum were low, and AACT activity was not detected in LGs. These results suggest that transcriptional regulation plays a key role in the control of ISP biosynthetic gene expression and ISP pheromone biosynthesis in bumblebee males. PMID:26632352

  1. Enzymatic modification of proteins with a geranylgeranyl isoprenoid.

    PubMed Central

    Casey, P J; Thissen, J A; Moomaw, J F

    1991-01-01

    The prenylation of several proteins involved in oncogenesis and signal transduction plays an essential role in regulating their biological activities. Two distinct isoprenoids are known to be involved in this modification, the 15-carbon farnesyl and 20-carbon geranylgeranyl groups. Thus far, identified farnesylated proteins contain methionine or serine at the COOH terminus, while those modified by geranylgeranyl end in leucine. This report describes the characterization of an enzyme activity that transfers the geranylgeranyl group to candidate proteins. The enzyme, termed a "protein geranylgeranyltransferase," exhibits a marked preference for substrate proteins that contain leucine at the COOH terminus. In fact, the enzyme will efficiently modify a normally farnesylated protein, Ha-ras, if its COOH-terminal amino acid is switched from serine to leucine. Additional studies characterize this enzyme and suggest that it is responsible for the geranylgeranyl modification of a number of GTP-binding proteins (or their subunits) that contain a consensus prenylation sequence ending in leucine. Images PMID:1924324

  2. Mevalonate-suppressive dietary isoprenoids for bone health.

    PubMed

    Mo, Huanbiao; Yeganehjoo, Hoda; Shah, Anureet; Mo, Warren K; Soelaiman, Ima Nirwana; Shen, Chwan-Li

    2012-12-01

    Osteoclastogenesis and osteoblastogenesis, the balancing acts for optimal bone health, are under the regulation of small guanosine triphosphate-binding proteins (GTPases) including Ras, Rac, Rho and Rab. The activities of GTPases require post-translational modification with mevalonate-derived prenyl pyrophosphates. Mevalonate deprivation induced by competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase (e.g., statins) prevents the activation of GTPases, suppresses the expression of the receptor for activation of nuclear factor kappa B (NFκB) ligand (RANKL) and activation of NFκB and, consequently, inhibits osteoclast differentiation and induces osteoclast apoptosis. In contrast, statin-mediated inactivation of GTPases enhances alkaline phosphatase activity and the expression of bone morphogenetic protein-2, vascular epithelial growth factor, and osteocalcin in osteoblasts and induces osteoblast proliferation and differentiation. Animal studies show that statins inhibit bone resorption and increase bone formation. The anabolic effect of statins and other mevalonate pathway-suppressive pharmaceuticals resembles the anti-osteoclastogenic and bone-protective activities conferred by dietary isoprenoids, secondary products of plant mevalonate metabolism. The tocotrienols, vitamin E molecules with HMG CoA reductase-suppressive activity, induce mevalonate deprivation and concomitantly suppress the expression of RANKL and cyclooxygenase-2, the production of prostaglandin E2 and the activation of NFκB. Accordingly, tocotrienols inhibit osteoclast differentiation and induce osteoclast apoptosis, impacts reminiscent of those of statins. In vivo studies confirm the bone protective activity of tocotrienols at nontoxic doses. Blends of tocotrienols, statins and isoprenoids widely found in fruits, vegetables, grains, herbs, spices, and essential oils may synergistically suppress osteoclastogenesis while promoting osteoblastogenesis, offering a novel

  3. Volatile isoprenoids as defense compounds during abiotic stress in tropical plants

    NASA Astrophysics Data System (ADS)

    Jardine, K.

    2015-12-01

    Emissions of volatile isoprenoids from tropical forests play central roles in atmospheric processes by fueling atmospheric chemistry resulting in modified aerosol and cloud lifecycles and their associated feedbacks with the terrestrial biosphere. However, the identities of tropical isoprenoids, their biological and environmental controls, and functions within plants and ecosystems remain highly uncertain. As part of the DOE ARM program's GoAmazon 2014/15 campaign, extensive field and laboratory observations of volatile isoprenoids are being conducted in the central Amazon. Here we report the results of our completed and ongoing activities at the ZF2 forest reserve in the central Amazon. Among the results of the research are the suprisingly high abundance of light-dependent volatile isoprenoid emissions across abundant tree genera in the Amazon in both primary and secondary forests, the discovery of highly reactive monoterpene emissions from Amazon trees, and evidence for the importance of volatile isoprenoids in protecting photosynthesis during oxidative stress under elevated temperatures including energy consumption and direct antioxidant functions and a tight connection betwen volatile isoprenoid emissions, photorespiration, and CO2 recycling within leaves. The results highlight the need to model allocation of carbon to isoprenoids during elevated temperature stress in the tropics.

  4. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  5. Origin and Evolution of Plastids and Photosynthesis in Eukaryotes

    PubMed Central

    McFadden, Geoffrey I.

    2014-01-01

    Recent progress in understanding the origins of plastids from endosymbiotic cyanobacteria is reviewed. Establishing when during geological time the endosymbiosis occurred remains elusive, but progress has been made in defining the cyanobacterial lineage most closely related to plastids, and some mechanistic insight into the possible existence of cryptic endosymbioses perhaps involving Chlamydia-like infections of the host have also been presented. The phylogenetic affinities of the host remain obscure. The existence of a second lineage of primary plastids in euglyphid amoebae has now been confirmed, but the quasipermanent acquisition of plastids by animals has been shown to be more ephemeral than initially suspected. A new understanding of how plastids have been integrated into their hosts by transfer of photosynthate, by endosymbiotic gene transfer and repatriation of gene products back to the endosymbiont, and by regulation of endosymbiont division is presented in context. PMID:24691960

  6. Unequal distribution of plastids during generative cell formation in Impatiens.

    PubMed

    van Went, J L

    1984-07-01

    This paper describes the unequal distribution of plastids in the developing microspores of Impatiens walleriana and Impatiens glandulifera which leads to the exclusion of plastids from the generative cell. During the development from young microspore to the onset of mitosis a change in the organization of the cytoplasm and distribution of organelles is gradually established. This includes the formation of vacuoles at the poles of the elongate-shaped microspores, the movement of the nucleus to a position near the microspore wall in the central part of the cell, and the accumulation of the plastids to a position near the wall at the opposite side of the cell. In Impatiens walleriana, the accumulated plastids are separated from each other by ER cisterns, and some mitochondria are also accumulated. In both Impatiens species, the portion of the microspore in which the generative cell will be formed is completely devoid of plastids at the time mitosis starts. PMID:24257638

  7. Localization of the pre-squalene segment of the isoprenoid biosynthetic pathway in mammalian peroxisomes.

    PubMed

    Kovacs, Werner J; Tape, Khanichi N; Shackelford, Janis E; Duan, Xueying; Kasumov, Takhar; Kelleher, Joanne K; Brunengraber, Henri; Krisans, Skaidrite K

    2007-03-01

    Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal beta-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool. PMID:17180682

  8. The isoprenoid pathway and transcriptional response to its inhibitors in the yeast Saccharomyces cerevisiae.

    PubMed

    Kuranda, Klaudia; François, Jean; Palamarczyk, Grazyna

    2010-02-01

    This review presents new insights into the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae, in particular the short-term transcriptional response to two inhibitors, lovastatin and zaragozic acid (ZA). Whereas lovastatin blocks whole isoprenoid pathway, ZA only blocks the sterol branch. Consequently, their effects on the cellular level of farnesyl diphosphate (FPP) are different. Lovastatin decreases the FPP level, whereas ZA, by inhibiting the main FPP-consuming enzyme, increases FPP availability in the cell. We discuss the role of genes whose expression is affected by both inhibitors and consider possible association of these genes with the regulation of the isoprenoid pathway. PMID:19744247

  9. High isoprenoid flux Escherichia coli as a host for carotenoids production.

    PubMed

    Suh, Wonchul

    2012-01-01

    A noncarotenogenic microbe E. coli was engineered for high production of carotenoids. To increase the isoprenoid flux, the chromosomal native promoters of the rate-controlling steps (dxs, idi and ispDispF) in the isoprenoid pathway were replaced with a strong bacteriophage T5 promoter (P(T5)) by using the λ-Red recombinase system in combination with the Flp/FRT site-specific recombination system for marker excision and P1 transduction for gene trait stacking. The resulting high isoprenoid flux E. coli can be used as a starting strain to produce various carotenoids by introducing heterologous carotenoid genes. In this study, the high isoprenoid flux E. coli was transformed with a plasmid carrying the β-carotene biosynthetic genes from Pantoea stewartii for β-carotene production. PMID:22144352

  10. Complete Plastid Genome Sequence of the Brown Alga Undaria pinnatifida

    PubMed Central

    Liu, Tao; Wang, Guoliang; Chi, Shan; Liu, Cui; Wang, Haiyang

    2015-01-01

    In this study, we fully sequenced the circular plastid genome of a brown alga, Undaria pinnatifida. The genome is 130,383 base pairs (bp) in size; it contains a large single-copy (LSC, 76,598 bp) and a small single-copy region (SSC, 42,977 bp), separated by two inverted repeats (IRa and IRb: 5,404 bp). The genome contains 139 protein-coding, 28 tRNA, and 6 rRNA genes; none of these genes contains introns. Organization and gene contents of the U. pinnatifida plastid genome were similar to those of Saccharina japonica. There is a co-linear relationship between the plastid genome of U. pinnatifida and that of three previously sequenced large brown algal species. Phylogenetic analyses of 43 taxa based on 23 plastid protein-coding genes grouped all plastids into a red or green lineage. In the large brown algae branch, U. pinnatifida and S. japonica formed a sister clade with much closer relationship to Ectocarpus siliculosus than to Fucus vesiculosus. For the first time, the start codon ATT was identified in the plastid genome of large brown algae, in the atpA gene of U. pinnatifida. In addition, we found a gene-length change induced by a 3-bp repetitive DNA in ycf35 and ilvB genes of the U. pinnatifida plastid genome. PMID:26426800

  11. The foundation of extranuclear inheritance: plastid and mitochondrial genetics.

    PubMed

    Hagemann, Rudolf

    2010-03-01

    In 1909 two papers by Correns and by Baur published in volume 1 of Zeitschrift für induktive Abstammungs- und Vererbungslehre (now Molecular Genetics and Genomics) reported on the non-Mendelian inheritance of chlorophyll deficiencies. These papers, reporting the very first cases of extranuclear inheritance, laid the foundation for a new field: non-Mendelian or extranuclear genetics. Correns observed a purely maternal inheritance (in Mirabilis), whereas Baur found a biparental inheritance (in Pelargonium). Correns suspected the non-Mendelian factors in the cytoplasm, while Baur believed that the plastids carry these extranuclear factors. In the following years, Baur's hypothesis was proved to be correct. Baur subsequently developed the theory of plastid inheritance. In many genera the plastids are transmitted only uniparentally by the mother, while in a few genera there is a biparental plastid inheritance. Commonly there is random sorting of plastids during ontogenetic development. Renner and Schwemmle as well as geneticists in other countries added additional details to this theory. Pioneering studies on mitochondrial inheritance in yeast started in 1949 in the group of Ephrussi and Slonimski; respiration-deficient cells (petites in yeast, poky in Neurospora) were demonstrated to be due to mitochondrial mutations. Electron microscopical and biochemical studies (1962-1964) showed that plastids and mitochondria contain organelle-specific DNA molecules. These findings laid the molecular basis for the two branches of extranuclear inheritance: plastid and mitochondrial genetics. PMID:20140454

  12. Apoptosis and cell-cycle arrest in human and murine tumor cells are initiated by isoprenoids.

    PubMed

    Mo, H; Elson, C E

    1999-04-01

    Diverse classes of phytochemicals initiate biological responses that effectively lower cancer risk. One class of phytochemicals, broadly defined as pure and mixed isoprenoids, encompasses an estimated 22,000 individual components. A representative mixed isoprenoid, gamma-tocotrienol, suppresses the growth of murine B16(F10) melanoma cells, and with greater potency, the growth of human breast adenocarcinoma (MCF-7) and human leukemic (HL-60) cells. beta-Ionone, a pure isoprenoid, suppresses the growth of B16 cells and with greater potency, the growth of MCF-7, HL-60 and human colon adenocarcinoma (Caco-2) cells. Results obtained with diverse cell lines differing in ras and p53 status showed that the isoprenoid-mediated suppression of growth is independent of mutated ras and p53 functions. beta-Ionone suppressed the growth of human colon fibroblasts (CCD-18Co) but only when present at three-fold the concentration required to suppress the growth of Caco-2 cells. The isoprenoids initiated apoptosis and, concomitantly arrested cells in the G1 phase of the cell cycle. Both suppress 3-hydroxy-3-methylglutaryl CoA reductase activity. beta-Ionone and lovastatin interfered with the posttranslational processing of lamin B, an activity essential to assembly of daughter nuclei. This interference, we postulate, renders neosynthesized DNA available to the endonuclease activities leading to apoptotic cell death. Lovastatin-imposed mevalonate starvation suppressed the glycosylation and translocation of growth factor receptors to the cell surface. As a consequence, cells were arrested in the G1 phase of the cell cycle. This rationale may apply to the isoprenoid-mediated G1-phase arrest of tumor cells. The additive and potentially synergistic actions of these isoprenoids in the suppression of tumor cell proliferation and initiation of apoptosis coupled with the mass action of the diverse isoprenoid constituents of plant products may explain, in part, the impact of fruit, vegetable

  13. Inhibition of the isoprenoid biosynthesis pathway; detection of intermediates by UPLC-MS/MS.

    PubMed

    Henneman, Linda; van Cruchten, Arno G; Kulik, Willem; Waterham, Hans R

    2011-04-01

    The isoprenoid biosynthesis pathway provides the cell with a variety of compounds which are involved in multiple cellular processes. Inhibition of this pathway with statins and bisphosphonates is widely applied in the treatment of hypercholesterolemia and metabolic bone disease, respectively. In addition, since isoprenylation of proteins is an important therapeutic target in cancer research there is interest in interfering with isoprenoid biosynthesis, for which new inhibitors to block farnesylation and geranylgeranylation of small GTPases are being developed. We recently developed a sensitive method using UPLC-MS/MS that allows the direct detection and quantification of all intermediates of the mevalonate pathway from MVA to GGPP which can be used to verify the specificity of inhibitors of the isoprenoid biosynthesis pathway. We here investigated the specificity of several inhibitors of the isoprenoid biosynthesis pathway in HepG2 cells, fibroblasts and lymphoblasts. The nitrogen-containing bisphosphonates pamidronate and zoledronate specifically inhibit farnesyl pyrophosphate synthase indicated by the accumulation of IPP/DMAPP. However, zaragozic acid A, a squalene synthase inhibitor, causes an increase of MVA in addition to the expected increase of FPP. Analysis of isoprenoid intermediate profiles after incubation with 6-fluoromevalonate showed a very nonspecific result with an increase in MVA, MVAP, MVAPP and IPP/DMAPP. These results show that inhibitors of a particular enzyme of the isoprenoid biosynthesis pathway can have additional effects on other enzymes of the pathway either direct or indirect through accumulation of isoprenoid intermediates. Our method can be used to test new inhibitors and their effect on overall isoprenoid biosynthesis. PMID:21237288

  14. Globulixanthone F, a new polyoxygenated xanthone with an isoprenoid group and two antimicrobial biflavonoids from the stem bark of Symphonia globulifera.

    PubMed

    Mkounga, Pierre; Fomum, Zacharias T; Meyer, Michèle; Bodo, Bernard; Nkengfack, Augustin E

    2009-06-01

    Bioassay-guided fractionation of the stem bark of Symphonia globulifera has yielded three known xanthones, ugaxanthone (1), mbarraxanthone (2) and gentisein (3), two biflavonoid derivatives named GB2 (4) and manniflavanone GB3 (5), and one new polyoxygenated xanthone with an isoprenoid group, named globulixanthone F (6). The structures of these compounds were elucidated by means of spectroscopic methods. The spectral data of 1 and 2 are reported here for the first time, as well as the antimicrobial activity of globulixanthone F against a range of microorganisms. We also report the total synthesis of the xanthone skeleton. PMID:19634326

  15. Low frequency paternal transmission of plastid genes in Brassicaceae.

    PubMed

    Schneider, Anja; Stelljes, Christian; Adams, Caroline; Kirchner, Stefan; Burkhard, Gabi; Jarzombski, Sabine; Broer, Inge; Horn, Patricia; Elsayed, Ashraf; Hagl, Peter; Leister, Dario; Koop, Hans-Ulrich

    2015-04-01

    Plastid-encoded genes are maternally inherited in most plant species. Transgenes located on the plastid genome are thus within a natural confinement system, preventing their distribution via pollen. However, a low-frequency leakage of plastids via pollen seems to be universal in plants. Here we report that a very low-level paternal inheritance in Arabidopsis thaliana occurs under field conditions. As pollen donor an Arabidopsis accession (Ler-Ely) was used, which carried a plastid-localized atrazine resistance due to a point mutation in the psbA gene. The frequency of pollen transmission into F1 plants, based on their ability to express the atrazine resistance was 1.9 × 10(-5). We extended our analysis to another cruciferous species, the world-wide cultivated crop Brassica napus. First, we isolated a fertile and stable plastid transformant (T36) in a commercial cultivar of B. napus (cv Drakkar). In T36 the aadA and the bar genes were integrated in the inverted repeat region of the B. napus plastid DNA following particle bombardment of hypocotyl segments. Southern blot analysis confirmed transgene integration and homoplasmy of plastid DNA. Line T36 expressed Basta resistance from the inserted bar gene and this trait was used to estimate the frequency of pollen transmission into F1 plants. A frequency of <2.6 × 10(-5) was determined in the greenhouse. Taken together, our data show a very low rate of paternal plastid transmission in Brassicacea. Moreover, the establishment of plastid transformation in B. napus facilitates a safe use of this important crop plant for plant biotechnology. PMID:25343875

  16. Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle.

    PubMed

    Morris, Wayne L; Ducreux, Laurence J M; Hedden, Peter; Millam, Steve; Taylor, Mark A

    2006-01-01

    Potato tubers were engineered to express a bacterial gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (DXS) in order to investigate the effects of perturbation of isoprenoid biosynthesis. Twenty-four independent transgenic lines out of 38 generated produced tubers with significantly elongated shape that also exhibited an early tuber sprouting phenotype. Expression analysis of nine transgenic lines (four exhibiting the phenotype and five showing a wild-type phenotype) demonstrated that the phenotype was strongly associated with dxs expression. At harvest, apical bud growth had already commenced in dxs-expressing tubers whereas in control lines no bud growth was evident until dormancy was released after 56-70 d of storage. The initial phase of bud growth in dxs tubers was followed by a lag period of approximately 56 d, before further elongation of the developing sprouts could be detected. Thus dxs expression results in the separation of distinct phases in the dormancy and sprouting processes. In order to account for the sprouting phenotype, the levels of plastid-derived isoprenoid growth regulators were measured in transgenic and control tubers. The major difference measured was an increase in the level of trans-zeatin riboside in tubers at harvest expressing dxs. Additionally, compared with controls, in some dxs-expressing lines, tuber carotenoid content increased approximately 2-fold, with most of the increase accounted for by a 6-7-fold increase in phytoene. PMID:16873449

  17. Plastid transformation in sugar beet: Beta vulgaris.

    PubMed

    De Marchis, Francesca; Bellucci, Michele

    2014-01-01

    Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of utmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories; conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species. PMID:24599867

  18. Streptophyte Terrestrialization in Light of Plastid Evolution.

    PubMed

    de Vries, Jan; Stanton, Amanda; Archibald, John M; Gould, Sven B

    2016-06-01

    Key steps in evolution are often singularities. The emergence of land plants is one such case and it is not immediately apparent why. A recent analysis found that the zygnematophycean algae represent the closest relative to embryophytes. Intriguingly, many exaptations thought essential to conquer land are common among various streptophytes, but zygnematophycean algae share with land plants the transfer of a few plastid genes to the nucleus. Considering the contribution of the chloroplast to terrestrialization highlights potentially novel exaptations that currently remain unexplored. We discuss how the streptophyte chloroplast evolved into what we refer to as the embryoplast, and argue this was as important for terrestrialization by freshwater algae as the host cell-associated exaptations that are usually focused upon. PMID:26895731

  19. Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

    PubMed Central

    De Diego, Nuria; Muñoz, Francisco J.; Baroja-Fernández, Edurne; Li, Jun; Ricarte-Bermejo, Adriana; Baslam, Marouane; Aranjuelo, Iker; Almagro, Goizeder; Humplík, Jan F.; Novák, Ondřej; Spíchal, Lukáš; Doležal, Karel; Pozueta-Romero, Javier

    2015-01-01

    and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs. PMID:25811607

  20. [Plastid genome engineering: novel optimization strategies and applications].

    PubMed

    Zhou, Fei; Lu, Shizhan; Gao, Liang; Zhang, Juanjuan; Lin, Yongjun

    2015-08-01

    The plastid genome engineering system allows site-specific modifications via two homologous recombination events. It is much safer, more precise and efficient compared with the nuclear transformation system. This technology can be applied to the basic research to expand plastid genome function analysis, and it also provides an excellent platform for not only high-level production of recombinant proteins but also plant breeding. In this review, we summarize the state of the art and progresses in this field. We focus on novel breeding strategies in transformation system improvement and new tools to enhance plastid transgene expression levels. In addition, we highlight selected applications in resistance engineering and quality improvement via metabolic engineering. We believe that by overcoming current technological limitations in the plastid transformation system can another green revolution for crop breeding beckon. PMID:26266781

  1. The plastid genome of the red alga Laurencia.

    PubMed

    Verbruggen, Heroen; Costa, Joana F

    2015-06-01

    We present the 174,935 nt long plastid genome of the red alga Laurencia sp. JFC0032. It is the third plastid genome characterized for the largest order of red algae (Ceramiales). The circular-mapping plastid genome is small compared to most florideophyte red algae, and our comparisons show a trend toward smaller plastid genome sizes in the family Rhodomelaceae, independent from a similar trend in Cyanidiophyceae. The Laurencia genome is densely packed with 200 annotated protein-coding genes (188 widely conserved, 3 open reading frames shared with other red algae and 9 hypothetical coding regions). It has 29 tRNAs, a single-copy ribosomal RNA cistron, a tmRNA, and the RNase P RNA. PMID:26986672

  2. The complete plastid genome sequence of Bomarea edulis (Alstroemeriaceae: Liliales).

    PubMed

    Kim, Jung Sung; Kim, Hyoung Tae; Yoon, Chang Young; Kim, Joo-Hwan

    2016-05-01

    Bomarea, a member of the family Alstroemeriaceae, is distributed from Chile to Mexico and includes approximately 120 species. Recent molecular phylogenetic studies have clarified the monophyly of the family within the order Liliales and the sister relationship with the family Colchicaceae. At this time, five plastid genomes of Liliales have been analyzed at the familial level. To examine plastid genome variation at the generic level, we sequenced the plastid genome of Bomarea edulis, which is the most widely distributed species in the genus, and compared it with Alstroemeria aurea. The plastid genome sequence of B. edulis was 154,925 bp in length with a similar structure as A. aurea, excluding the IR-LSC junction. Ycf68 and infA were pseudogenes caused by frameshift mutations, and the ycf15 gene was deleted, similar to A. aurea. PMID:25319309

  3. Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control[C][W

    PubMed Central

    Pulido, Pablo; Toledo-Ortiz, Gabriela; Phillips, Michael A.; Wright, Louwrance P.; Rodríguez-Concepción, Manuel

    2013-01-01

    Plastids provide plants with metabolic pathways that are unique among eukaryotes, including the methylerythritol 4-phosphate pathway for the production of isoprenoids essential for photosynthesis and plant growth. Here, we show that the first enzyme of the pathway, deoxyxylulose 5-phosphate synthase (DXS), interacts with the J-protein J20 in Arabidopsis thaliana. J-proteins typically act as adaptors that provide substrate specificity to heat shock protein 70 (Hsp70), a molecular chaperone. Immunoprecipitation experiments showed that J20 and DXS are found together in vivo and confirmed the presence of Hsp70 chaperones in DXS complexes. Mutants defective in J20 activity accumulated significantly increased levels of DXS protein (but no transcripts) and displayed reduced levels of DXS enzyme activity, indicating that loss of J20 function causes posttranscriptional accumulation of DXS in an inactive form. Furthermore, J20 promotes degradation of DXS following a heat shock. Together, our data indicate that J20 might identify unfolded or misfolded (damaged) forms of DXS and target them to the Hsp70 system for proper folding under normal conditions or degradation upon stress. PMID:24104567

  4. Identification of functional lox sites in the plastid genome.

    PubMed

    Corneille, Sylvie; Lutz, Kerry A; Azhagiri, Arun K; Maliga, Pal

    2003-09-01

    Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid. PMID:12969428

  5. Exceptional paternal inheritance of plastids in Arabidopsis suggests that low-frequency leakage of plastids via pollen may be universal in plants.

    PubMed

    Azhagiri, Arun K; Maliga, Pal

    2007-12-01

    Plastid DNA is absent in pollen or sperm cells of Arabidopsis thaliana. Accordingly, plastids and mitochondria, in a standard genetic cross, are transmitted to the seed progeny by the maternal parent only. Our objective was to test whether paternal plastids are transmitted by pollen as an exception. The maternal parent in our cross was a nuclear male sterile (ms1-1/ms1-1), spectinomycin-sensitive Ler plant. It was fertilized with pollen of a male fertile RLD-Spc1 plant carrying a plastid-encoded spectinomycin resistance mutation. Seedlings with paternal plastids were selected by spectinomycin resistance encoded in the paternal plastid DNA. Our data, in general, support maternal inheritance of plastids in A. thaliana. However, we report that paternal plastids are transmitted to the seed progeny in Arabidopsis at a low (3.9 x 10(-5)) frequency. This observation extends previous reports in Antirrhinum majus, Epilobium hirsutum, Nicotiana tabacum, Petunia hybrida, and the cereal crop Setaria italica to a cruciferous species suggesting that low-frequency paternal leakage of plastids via pollen may be universal in plants previously thought to exhibit strict maternal plastid inheritance. The genetic tools employed here will facilitate testing the effect of Arabidopsis nuclear mutations on plastid inheritance and allow for the design of mutant screens to identify nuclear genes controlling plastid inheritance. PMID:17931353

  6. Protein import and the origin of red complex plastids.

    PubMed

    Gould, Sven B; Maier, Uwe-G; Martin, William F

    2015-06-15

    The number and nature of endosymbioses involving red algal endosymbionts are debated. Gene phylogenies have become the most popular tool to untangle this issue, but they deliver conflicting results. As gene and lineage sampling has increased, so have both the number of conflicting trees and the number of suggestions in the literature for multiple tertiary, and even quaternary, symbioses that might reconcile the tree conflicts. Independent lines of evidence that can address the issue are needed. Here we summarize the mechanism and machinery of protein import into complex red plastids. The process involves protein translocation machinery, known as SELMA, that arose once in evolution, that facilitates protein import across the second outermost of the four plastid membranes, and that is always targeted specifically to that membrane, regardless of where it is encoded today. It is widely accepted that the unity of protein import across the two membranes of primary plastids is strong evidence for their single cyanobacterial origin. Similarly, the unity of SELMA-dependent protein import across the second outermost plastid membrane constitutes strong evidence for the existence of a single red secondary endosymbiotic event at the common origin of all red complex plastids. We furthermore propose that the two outer membranes of red complex plastids are derived from host endoplasmic reticulum in the initial red secondary endosymbiotic event. PMID:26079086

  7. The Plastid Genome of Eutreptiella Provides a Window into the Process of Secondary Endosymbiosis of Plastid in Euglenids

    PubMed Central

    Hrdá, Štěpánka; Fousek, Jan; Szabová, Jana; Hampl, Vladimír; Vlček, Čestmír

    2012-01-01

    Euglenids are a group of protists that comprises species with diverse feeding modes. One distinct and diversified clade of euglenids is photoautotrophic, and its members bear green secondary plastids. In this paper we present the plastid genome of the euglenid Eutreptiella, which we assembled from 454 sequencing of Eutreptiella gDNA. Comparison of this genome and the only other available plastid genomes of photosynthetic euglenid, Euglena gracilis, revealed that they contain a virtually identical set of 57 protein coding genes, 24 genes fewer than the genome of Pyramimonas parkeae, the closest extant algal relative of the euglenid plastid. Searching within the transcriptomes of Euglena and Eutreptiella showed that 6 of the missing genes were transferred to the nucleus of the euglenid host while 18 have been probably lost completely. Euglena and Eutreptiella represent the deepest bifurcation in the photosynthetic clade, and therefore all these gene transfers and losses must have happened before the last common ancestor of all known photosynthetic euglenids. After the split of Euglena and Eutreptiella only one additional gene loss took place. The conservation of gene content in the two lineages of euglenids is in contrast to the variability of gene order and intron counts, which diversified dramatically. Our results show that the early secondary plastid of euglenids was much more susceptible to gene losses and endosymbiotic gene transfers than the established plastid, which is surprisingly resistant to changes in gene content. PMID:22448269

  8. Block of the Mevalonate Pathway Triggers Oxidative and Inflammatory Molecular Mechanisms Modulated by Exogenous Isoprenoid Compounds

    PubMed Central

    Tricarico, Paola Maura; Kleiner, Giulio; Valencic, Erica; Campisciano, Giuseppina; Girardelli, Martina; Crovella, Sergio; Knowles, Alessandra; Marcuzzi, Annalisa

    2014-01-01

    Deregulation of the mevalonate pathway is known to be involved in a number of diseases that exhibit a systemic inflammatory phenotype and often neurological involvements, as seen in patients suffering from a rare disease called mevalonate kinase deficiency (MKD). One of the molecular mechanisms underlying this pathology could depend on the shortage of isoprenoid compounds and the subsequent mitochondrial damage, leading to oxidative stress and pro-inflammatory cytokines’ release. Moreover, it has been demonstrated that cellular death results from the balance between apoptosis and pyroptosis, both driven by mitochondrial damage and the molecular platform inflammasome. In order to rescue the deregulated pathway and decrease inflammatory markers, exogenous isoprenoid compounds were administered to a biochemical model of MKD obtained treating a murine monocytic cell line with a compound able to block the mevalonate pathway, plus an inflammatory stimulus. Our results show that isoprenoids acted in different ways, mainly increasing the expression of the evaluated markers [apoptosis, mitochondrial dysfunction, nucleotide-binding oligomerization-domain protein-like receptors 3 (NALP3), cytokines and nitric oxide (NO)]. Our findings confirm the hypothesis that inflammation is triggered, at least partially, by the shortage of isoprenoids. Moreover, although further studies are necessary, the achieved results suggest a possible role for exogenous isoprenoids in the treatment of MKD. PMID:24758928

  9. A small portion of plastid transcripts is polyadenylated in the flagellate Euglena gracilis.

    PubMed

    Záhonová, Kristína; Hadariová, Lucia; Vacula, Rostislav; Yurchenko, Vyacheslav; Eliáš, Marek; Krajčovič, Juraj; Vesteg, Matej

    2014-03-01

    Euglena gracilis possesses secondary plastids of green algal origin. In this study, E. gracilis expressed sequence tags (ESTs) derived from polyA-selected mRNA were searched and several ESTs corresponding to plastid genes were found. PCR experiments failed to detect SL sequence at the 5'-end of any of these transcripts, suggesting plastid origin of these polyadenylated molecules. Quantitative PCR experiments confirmed that polyadenylation of transcripts occurs in the Euglena plastids. Such transcripts have been previously observed in primary plastids of plants and algae as low-abundance intermediates of transcript degradation. Our results suggest that a similar mechanism exists in secondary plastids. PMID:24492004

  10. Role of horizontal gene transfer in the evolution of photosynthetic eukaryotes and their plastids.

    PubMed

    Keeling, Patrick J

    2009-01-01

    Plastids are the organelles derived from a cyanobacterium through endosymbiosis. Unlike mitochondria, plastids are not found in all eukaryotes, but their evolution has an added layer of complexity since plastids have moved between eukaryotic lineages by secondary and tertiary endosymbiotic events. This complex history, together with the genetic integration between plastids and their host, has led to many opportunities for gene flow between phylogenetically distinct lineages. Some intracellular transfers do not lead to a protein functioning in a new environment, but many others do and the protein makeup of many plastids appears to have been influenced by exogenous sources as well. Here, different evolutionary sources and cellular destinations of gene flow that has affected the plastid lineage are reviewed. Most horizontal gene transfer (HGT) affecting the modern plastid has taken place via the host nucleus, in the form of genes for plastid-targeted proteins. The impact of this varies greatly from lineage to lineage, but in some cases such transfers can be as high as one fifth of analyzed genes. More rarely, genes have also been transferred to the plastid genome itself, and plastid genes have also been transferred to other non-plant, non-algal lineages. Overall, the proteome of many plastids has emerged as a mosaic of proteins from many sources, some from within the same cell (e.g., cytosolic genes or genes left over from the replacement of an earlier plastid), some from the plastid of other algal lineages, and some from completely unrelated sources. PMID:19271204

  11. Function of isoprenoid quinones and chromanols during oxidative stress in plants.

    PubMed

    Kruk, Jerzy; Szymańska, Renata; Nowicka, Beatrycze; Dłużewska, Jolanta

    2016-09-25

    Isoprenoid quinones and chromanols in plants fulfill both signaling and antioxidant functions under oxidative stress. The redox state of the plastoquinol pool (PQ-pool), which is modulated by interaction with reactive oxygen species (ROS) during oxidative stress, has a major regulatory function in both short- and long-term acclimatory responses. By contrast, the scavenging of ROS by prenyllipids affects signaling pathways where ROS play a role as signaling molecules. As the primary antioxidants, isoprenoid quinones and chromanols are synthesized under high-light stress in response to any increased production of ROS. During photo-oxidative stress, these prenyllipids are continuously synthesized and oxidized to other compounds. In turn, their oxidation products (hydroxy-plastochromanol, plastoquinol-C, plastoquinone-B) can still have an antioxidant function. The oxidation products of isoprenoid quinones and chromanols formed specifically in the face of singlet oxygen, can be indicators of singlet oxygen stress. PMID:26970272

  12. A Genetic and Pharmacological Analysis of Isoprenoid Pathway by LC-MS/MS in Fission Yeast

    PubMed Central

    Takami, Tomonori; Fang, Yue; Zhou, Xin; Jaiseng, Wurentuya; Ma, Yan; Kuno, Takayoshi

    2012-01-01

    Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In hmg1-1, a mutant of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), ergosterol (a final sterol product), and squalene (an intermediate pathway product), were decreased to approximately 80% and 10%, respectively, compared with that of wild-type cells. Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level. PMID:23145048

  13. rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

    PubMed

    Kinsella, B T; Maltese, W A

    1992-02-25

    p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic. PMID:1740442

  14. Plastid ultrastructure and photosynthesis in greening petaloid hypsophylls.

    PubMed

    Weidner, M; Franz, A; Napp-Zinn, K

    1985-02-01

    The ultrastructural and biochemicalphysiological aspects of postfloral greening have been studied in hypsophylls of Heliconia aurantiaca Ghiesbr., Guzmania cf. x magnifica Richter and Spathiphyllum wallisii Regel. In all three species the greening of the hypsophylls is due to plastid transformation, chloroplast formation proceeding from the initially different types of plastids. The degradation process of the original plastid structures and the mode of thylakoid formation are distinct in each case. In none of the species do the transformed plastids look identical to the chloroplasts of the corresponding foliage leaves. On a chlorophyll basis, the rate of photosynthesis of the greened hypsophylls surpasses the rate of the leaves considerably in Spathiphyllum, but is much lower in Heliconia (no data for Guzmania). In all species, anatomy, plastid structure, pigments, 77° K-fluorescence emission, ribulose-1,5-bis-phosphate carboxylase activities and short-term photosynthesis (14)CO2-assimilation patterns prove the greened hypsophylls to be capable of providing additional carbon to the developing fruits, thus supplementing the import of organic matter from the foliage leaves. PMID:24249334

  15. Overexpression of Plastid Transketolase in Tobacco Results in a Thiamine Auxotrophic Phenotype[OPEN

    PubMed Central

    Khozaei, Mahdi; Fisk, Stuart; Lawson, Tracy; Gibon, Yves; Sulpice, Ronan; Stitt, Mark; Lefebvre, Stephane C.; Raines, Christine A.

    2015-01-01

    To investigate the effect of increased plastid transketolase on photosynthetic capacity and growth, tobacco (Nicotiana tabacum) plants with increased levels of transketolase protein were produced. This was achieved using a cassette composed of a full-length Arabidopsis thaliana transketolase cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed a major and unexpected effect of plastid transketolase overexpression as the transgenic tobacco plants exhibited a slow-growth phenotype and chlorotic phenotype. These phenotypes were complemented by germinating the seeds of transketolase-overexpressing lines in media containing either thiamine pyrophosphate or thiamine. Thiamine levels in the seeds and cotyledons were lower in transketolase-overexpressing lines than in wild-type plants. When transketolase-overexpressing plants were supplemented with thiamine or thiamine pyrophosphate throughout the life cycle, they grew normally and the seed produced from these plants generated plants that did not have a growth or chlorotic phenotype. Our results reveal the crucial importance of the level of transketolase activity to provide the precursor for synthesis of intermediates and to enable plants to produce thiamine and thiamine pyrophosphate for growth and development. The mechanism determining transketolase protein levels remains to be elucidated, but the data presented provide evidence that this may contribute to the complex regulatory mechanisms maintaining thiamine homeostasis in plants. PMID:25670766

  16. Evidence for the Retention of Two Evolutionary Distinct Plastids in Dinoflagellates with Diatom Endosymbionts

    PubMed Central

    Hehenberger, Elisabeth; Imanian, Behzad; Burki, Fabien; Keeling, Patrick J.

    2014-01-01

    Dinoflagellates harboring diatom endosymbionts (termed “dinotoms”) have undergone a process often referred to as “tertiary endosymbiosis”—the uptake of algae containing secondary plastids and integration of those plastids into the new host. In contrast to other tertiary plastids, and most secondary plastids, the endosymbiont of dinotoms is distinctly less reduced, retaining a number of cellular features, such as their nucleus and mitochondria and others, in addition to their plastid. This has resulted in redundancy between host and endosymbiont, at least between some mitochondrial and cytosolic metabolism, where this has been investigated. The question of plastidial redundancy is particularly interesting as the fate of the host dinoflagellate plastid is unclear. The host cytosol possesses an eyespot that has been postulated to be a remnant of the ancestral peridinin plastid, but this has not been tested, nor has its possible retention of plastid functions. To investigate this possibility, we searched for plastid-associated pathways and functions in transcriptomic data sets from three dinotom species. We show that the dinoflagellate host has indeed retained genes for plastid-associated pathways and that these genes encode targeting peptides similar to those of other dinoflagellate plastid-targeted proteins. Moreover, we also identified one gene encoding an essential component of the dinoflagellate plastid protein import machinery, altogether suggesting the presence of a functioning plastid import system in the host, and by extension a relict plastid. The presence of the same plastid-associated pathways in the endosymbiont also extends the known functional redundancy in dinotoms, further confirming the unusual state of plastid integration in this group of dinoflagellates. PMID:25172904

  17. The Recovery of Plastid Function Is Required for Optimal Response to Low Temperatures in Arabidopsis

    PubMed Central

    Kindgren, Peter; Dubreuil, Carole; Strand, Åsa

    2015-01-01

    Cold acclimation is an essential response in higher plants to survive freezing temperatures. Here, we report that two independent mutant alleles of the H-subunit of Mg-chelatase, CHLH, gun5-1 and cch in Arabidopsis are sensitive to low temperatures. Plants were grown in photoperiodic conditions and exposed to low temperatures for short- and long-term periods. Tetrapyrrole biosynthesis was initially significantly inhibited in response to low temperature but recovered in wild type (Col-0), although the tetrapyrrole levels were lower in cold compared to control conditions. The gun5-1 and cch alleles showed an inability to recover chlorophyll biosynthesis in addition to a significant decrease in freezing tolerance. We found that the impaired plastid function in the CHLH mutant plants resulted in compromised de novo protein synthesis at low temperatures. The expression of the transcription factors CBF1-3 was super-induced in gun5-1 and cch mutant alleles but expression levels of their target genes, COR15a, COR47 and COR78 were similar or even lower compared to Col-0. In addition, the protein levels of COR15a were lower in gun5-1 and cch and a general defect in protein synthesis could be seen in the gun5-1 mutant following a 35S labelling experiment performed at low temperature. Taken together, our results demonstrate the importance of a functional chloroplast for the cold acclimation process and further suggest that impaired plastid function could result in inhibition of protein synthesis at low temperature. PMID:26366569

  18. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  19. Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis1[OPEN

    PubMed Central

    Lange, Iris; Poirier, Brenton C.; Herron, Blake K.; Lange, Bernd Markus

    2015-01-01

    In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants. PMID:26282236

  20. Composition and content of normal and isoprenoid hydrocarbons in the Pavel meteorite

    NASA Astrophysics Data System (ADS)

    Ivanov, Kh. P.; Stoianova, R. Zh.; Stoev, G.

    The composition and content of alkanes in extracts from the Pavel meteorite are examined and the content of individual hydrocarbons in the entire meteorite is determined. Preliminary evidence is found for the existence of isoprenoids, steranes, and triterpanes in the meteorite, substances that have not previously been found in meteorites.

  1. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  2. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  3. Protein synthesis during the initial phase of the temperature-induced bleaching response in Euglena gracilis

    SciTech Connect

    Ortiz, W. )

    1990-05-01

    Growing cultures of photoheterotrophic Euglena gracilis experience an increase in chlorophyll accumulation during the initial phase of the temperature-induced bleaching response suggesting an increase in the synthesis of plastid components at the bleaching temperature of 33{degree}C. A primary goal of this work was to establish whether an increase in the synthesis of plastid proteins accompanies the observed increase in chlorophyll accumulation. In vivo pulse-labeling experiments with ({sup 35}S)sodium sulfate were carried out with cells grown at room temperature or at 33{degree}C. The synthesis of a number of plastid polypeptides of nucleocytoplasmic origin, including some presumably novel polypeptides, increased in cultures treated for 15 hours at 33{degree}C. In contrast, while synthesis of thylakoid proteins by the plastid protein synthesis machinery decreased modestly, synthesis of the large subunit of the enzyme ribulosebisphosphate carboxylase was strongly affected at the elevated temperature. Synthesis of novel plastid-encoded polypeptides was not induced at the bleaching temperature. It is concluded that protein synthesis in plastids declines during the initial phase of the temperature response in Euglena despite an overall increase in cellular protein synthesis and an increase in chlorophyll accumulation per cell.

  4. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes.

    PubMed

    Wheeler, Glen; Ishikawa, Takahiro; Pornsaksit, Varissa; Smirnoff, Nicholas

    2015-01-01

    Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, L-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, L-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant. PMID:25768426

  5. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis.

    PubMed

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-03-29

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the "recycling" of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  6. Is there a plastid in Perkinsus atlanticus (Phylum Perkinsozoa)?

    PubMed

    Teles-Grilo, M Leonor; Tato-Costa, Joana; Duarte, Sérgio M; Maia, Alexandre; Casal, Graça; Azevedo, Carlos

    2007-06-01

    Perkinsus atlanticus is a pathogenic protist that infects the clam Ruditapes decussatus. The recent proposal for the inclusion of the genus Perkinsus in a new phylum, Perkinsozoa, in the infra-kingdom Alveolata, gave rise to controversies whether this genus should form a phylum on its own. Molecular analysis of some conserved nuclear genes shows a closer proximity of the genus Perkinsus to the dinoflagellates than to the apicomplexans. Studies on extranuclear genomes, however, could also be very helpful for a more precise definition of those phyla. In Perkinsozoa, there have been until now no reports about the isolation of mitochondria as well as no conclusive results about the presence of any plastids, therefore a comparison with the data already obtained in Apicomplexa and Dinoflagellata has not yet been possible. In this work, we identify a plastid in Perkinsus atlanticus, using ultrastructural techniques and inhibition growth tests. It will be important to analyze the plastid genome at a molecular level, in order to confirm if the plastid in Perkinsus is more similar to those of Dinoflagellata or Apicomplexa. Such information will doubtless contribute to a more precise determination of the phylogenetic position of the genus Perkinsus. PMID:17498932

  7. Plastid transformation for abiotic stress tolerance in plants.

    PubMed

    Bansal, K C; Singh, A K; Wani, S H

    2012-01-01

    Abiotic stresses such as drought, salinity, and extreme temperatures are major limiting factors in plant growth and development and pose serious threat to global agricultural production. Here we describe a procedure, using a tobacco plastid transformation vector, to generate transplastomic plants with an enhanced ability to tolerate abiotic stresses such as salinity, drought, or cold stress. The procedure involves biolistic delivery of a plastid transformation vector into explants, antibiotic selection procedures, and -identification of transplastomic lines. The plastid transformation vector contains an aadA gene that encodes resistance to spectinomycin as a selectable marker along with the gene of interest for developing transplastomic plants that are tolerant to abiotic stresses. Shoot buds appear over the surface of bombarded explants following spectinomycin selection. Transplastomic shoots are multiplied following several rounds of -spectinomycin selection. Homoplasmic transplastomic lines are confirmed by spectinomycin and streptomycin double selection over a period of 4-5 weeks. The available reports suggest that transplastomic technology is a useful tool for expressing genes in plastids or chloroplasts for enhancing abiotic stress tolerance in plants. PMID:22895771

  8. Production Of Cellulase In Plastids Of Transgenic Plants

    DOEpatents

    Lamppa, Gayle

    2002-08-06

    A genetic construct encoding a fusion protein including endogluconase E1 and a transit peptide is used to transform plants. The plants produce cellulase by expressing the genetic construct. The cellulase is targeted to plastids and can be collected and purified.

  9. Protein networks identify novel symbiogenetic genes resulting from plastid endosymbiosis

    PubMed Central

    Méheust, Raphaël; Zelzion, Ehud; Bhattacharya, Debashish; Lopez, Philippe; Bapteste, Eric

    2016-01-01

    The integration of foreign genetic information is central to the evolution of eukaryotes, as has been demonstrated for the origin of the Calvin cycle and of the heme and carotenoid biosynthesis pathways in algae and plants. For photosynthetic lineages, this coordination involved three genomes of divergent phylogenetic origins (the nucleus, plastid, and mitochondrion). Major hurdles overcome by the ancestor of these lineages were harnessing the oxygen-evolving organelle, optimizing the use of light, and stabilizing the partnership between the plastid endosymbiont and host through retargeting of proteins to the nascent organelle. Here we used protein similarity networks that can disentangle reticulate gene histories to explore how these significant challenges were met. We discovered a previously hidden component of algal and plant nuclear genomes that originated from the plastid endosymbiont: symbiogenetic genes (S genes). These composite proteins, exclusive to photosynthetic eukaryotes, encode a cyanobacterium-derived domain fused to one of cyanobacterial or another prokaryotic origin and have emerged multiple, independent times during evolution. Transcriptome data demonstrate the existence and expression of S genes across a wide swath of algae and plants, and functional data indicate their involvement in tolerance to oxidative stress, phototropism, and adaptation to nitrogen limitation. Our research demonstrates the “recycling” of genetic information by photosynthetic eukaryotes to generate novel composite genes, many of which function in plastid maintenance. PMID:26976593

  10. Multifunctionality of plastid nucleoids as revealed by proteome analyses.

    PubMed

    Melonek, Joanna; Oetke, Svenja; Krupinska, Karin

    2016-08-01

    Protocols aimed at the isolation of nucleoids and transcriptionally active chromosomes (TACs) from plastids of higher plants have been established already decades ago, but only recent improvements in the mass spectrometry methods enabled detailed proteomic characterization of their components. Here we present a comprehensive analysis of the protein compositions obtained from two proteomic studies of TAC fractions isolated from Arabidopsis/mustard and spinach chloroplasts, respectively, as well as nucleoid fractions from Arabidopsis, maize and pea. Interestingly, different approaches as well as the use of diverse starting materials resulted in the detection of varying protein catalogues with a number of shared proteins. Possible reasons for the discrepancies between the protein repertoires and for missing out some of the nucleoid proteins that have been identified previously by other means than mass spectrometry as well as the repeated identification of "unexpected" proteins indicating potential links between DNA/RNA-associated nucleoid core functions and energy metabolism as well as biosynthetic activities of plastids will be discussed. In accordance with the nucleoid association of proteins involved in key functions of plastids including photosynthesis, the phenotypes of mutants lacking one or the other plastid nucleoid-associated protein (ptNAP) show the importance of nucleoid proteins for overall plant development and growth. This article is part of a Special Issue entitled: Plant Proteomics--a bridge between fundamental processes and crop production, edited by Dr. Hans-Peter Mock. PMID:26987276

  11. Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

    PubMed Central

    Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P

    1999-01-01

    Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829

  12. Excision of plastid marker genes using directly repeated DNA sequences.

    PubMed

    Mudd, Elisabeth A; Madesis, Panagiotis; Avila, Elena Martin; Day, Anil

    2014-01-01

    Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. PMID:24599849

  13. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    PubMed

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions. PMID:24310436

  14. Agrobacterium tumefaciens Tumor Morphology Root Plastid Localization and Preferential Usage of Hydroxylated Prenyl Donor Is Important for Efficient Gall Formation1[C][W][OA

    PubMed Central

    Ueda, Nanae; Kojima, Mikiko; Suzuki, Katsunori; Sakakibara, Hitoshi

    2012-01-01

    Upon Agrobacterium tumefaciens infection of a host plant, Tumor morphology root (Tmr) a bacterial adenosine phosphate-isopentenyltransferase (IPT), creates a metabolic bypass in the plastid for direct synthesis of trans-zeatin (tZ) with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate as the prenyl donor. To understand the biological importance of Tmr function for gall formation, we compared Tmr and Trans-zeatin secretion (Tzs) another agrobacterial IPT that functions within the bacterial cell. Although there is no significant difference in their substrate specificities in vitro, ectopic overexpression of Tzs in Arabidopsis (Arabidopsis thaliana) resulted in the accumulation of comparable amounts of tZ- and N6-(∆2-isopentenyl)adenine (iP)-type cytokinins, whereas overexpression of Tmr resulted exclusively in the accumulation of tZ-type cytokinins. Ectopic expression of Tzs in plant cells yields only small amounts of the polypeptide in plastid-enriched fractions. Obligatory localization of Tzs into Arabidopsis plastid stroma by translational fusions with ferredoxin transit peptide (TP-Tzs) increased the accumulation of both tZ- and iP-type cytokinins. Replacement of tmr on the Ti plasmid with tzs, TP-tzs, or an Arabidopsis plastidic IPT induced the formation of smaller galls than wild-type A. tumefaciens, and they were accompanied by the accumulation of iP-type cytokinins. Tmr is thus specialized for plastid localization and preferential usage of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate in vivo and is important for efficient gall formation. PMID:22589470

  15. Bioactive Isoprenoid-Derived Natural Products from a Dongsha Atoll Soft Coral Sinularia erecta.

    PubMed

    Huang, Chiung-Yao; Tseng, Yen-Ju; Chokkalingam, Uvarani; Hwang, Tsong-Long; Hsu, Chi-Hsin; Dai, Chang-Feng; Sung, Ping-Jyun; Sheu, Jyh-Horng

    2016-05-27

    Four new isoprenoids, including two norcembranoids sinulerectols A and B (1 and 2), a cembranoid sinulerectol C (3), and a degraded cembranoid sinulerectadione (4), along with three known isoprenoids, an unnamed norcembrene (5), sinularectin (6), and ineleganolide (7), and a known nitrogen-containing compound (Z)-N-[2-(4-hydroxyphenyl)ethyl]-3-methyldodec-2-enamide (8), were isolated from an extract of the marine soft coral Sinularia erecta. The structure of sinularectin (6) was revised, too. Compounds 3, 4, and 8 exhibited inhibitory activity against the proliferation of a limited panel of cancer cell lines, whereas 1, 2, and 8 displayed potent anti-inflammatory activity in fMLP/CB-stimulated human neutrophils. PMID:27142697

  16. Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs.

    PubMed

    Jomaa, H; Wiesner, J; Sanderbrand, S; Altincicek, B; Weidemeyer, C; Hintz, M; Türbachova, I; Eberl, M; Zeidler, J; Lichtenthaler, H K; Soldati, D; Beck, E

    1999-09-01

    A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured. PMID:10477522

  17. Closed pyrolyses of the isoprenoid algaenan of Botryococcus braunii, L race: geochemical implications for derived kerogens

    NASA Astrophysics Data System (ADS)

    Behar, F.; Derenne, S.; Largeau, C.

    1995-07-01

    Algaenans, i.e., highly aliphatic, nonhydrolysable, insoluble macromolecular constituents, have been identified in a number of microalga cell walls and their selective preservation shown to play a major role in the formation of numerous kerogens. All the algaenans so far examined comprise a network of long polymethylenic chains, except for the L race of Botryococcus braunii. The resistant macromolecular material isolated from the latter, termed PRB L, is based on C 40 isoprenoid chains with a lycopane-type skeleton. Recent comparative studies of PRB L and of Botryococcus-derived sediments provided the first example of kerogen formation via the selective preservation of an "isoprenoid" algaenan. The present study is concerned with PRB L pyrolyses in sealed gold tubes under various temperature/time conditions (260-350°C, 0.5-69 h). For the conversion rates thus obtained, ranging from 30 to 100%, a complete mass balance of the different families of pyrolysis products was established; most of the C 1 to C 40 pyrolysate constituents were identified and the abundances of the above compounds and their variations with conversion progress were determined. This study thus allowed us (1) to derive further information about PRB L chemical structure (location of the ether bridges, contribution of linear chains and their relationships with the C 40 isoprenoid ones), (2) to determine the behaviour of this isoprenoid algaenan to thermal stress (timing of the formation of the different groups of products then released, nature of the primary cleavages, origin and mode of formation of the secondary products, and further degradations), and (3) to show, in connection with previous studies, that PRB L-derived kerogens should exhibit pronounced differences relative to standard type I kerogens, the latter being based on polymethylenic chains, regarding not only the structure of the generated products but also the timing of oil generation (upward shift of the catagenesis zone).

  18. Influence of Manganese Deficiency and Toxicity on Isoprenoid Syntheses 1

    PubMed Central

    Wilkinson, Robert E.; Ohki, Kenneth

    1988-01-01

    Twenty-eight day old wheat (Triticum aestivum L. cv Stacy) response to varying Mn concentration (10.1-10,000 micromolar) in nutrient solution was measured. Manganese concentrations in the most recently matured leaves (blade 1) were 0.21 to 19.03 mmol Mn per kilogram dry weight, respectively. Fresh and dry weights increased to a maximum at the 5 micromolar Mn nutritional level (0.37 millimole Mn per kilogram dry weight) and were decreased at Mn above and below this concentration. Blade 1 chloroplast pigment concentrations increased up to the 20 micromolar Mn nutritional level (1.98 millimole Mn per kilogram dry weight) and decreased at higher Mn concentrations. Thylakoid Mn content was above 1 mole Mn/100 mole chloroplast at Mn nutrition levels which resulted in greatly decreased plant growth. Total phytoene biosynthesis was decreased by Mn deficiency and toxicity. In vitro ent- kaurene synthesis was greatly influenced by Mn concentration with a maximal biosynthesis at 1 micromolar Mn and decreases at Mn levels above and below this concentration. In vivo blade 1 gibberellic acid equivalent concentrations were maximal at 20 parts per million Mn nutrition solution levels (1.98 millimole Mn per kilogram dry weight) and decreased at Mn tissue concentrations above and below this value; additionally, gibberellic acid concentrations were reciprocal to extracted C20 alcohol concentrations. Mn influence on gibberellin and chloroplast pigment biosyntheses exactly matched the measured changes in growth. PMID:16666235

  19. Production of wax esters during aerobic growth of marine bacteria on isoprenoid compounds

    PubMed

    Rontani; Bonin; Volkman

    1999-01-01

    This paper describes the production of isoprenoid wax esters during the aerobic degradation of 6,10,14-trimethylpentadecan-2-one and phytol by four bacteria (Acinetobacter sp. strain PHY9, Pseudomonas nautica [IP85/617], Marinobacter sp. strain CAB [DSMZ 11874], and Marinobacter hydrocarbonoclasticus [ATCC 49840]) isolated from the marine environment. Different pathways are proposed to explain the formation of these compounds. In the case of 6,10, 14-trimethylpentadecan-2-one, these esters result from the condensation of some acidic and alcoholic metabolites produced during the biodegradation, while phytol constitutes the alcohol moiety of most of the esters produced during growth on this isoprenoid alcohol. The amount of these esters formed increased considerably in N-limited cultures, in which the ammonium concentration corresponds to conditions often found in marine sediments. This suggests that the bacterial formation of isoprenoid wax esters might be favored in such environments. Although conflicting evidence exists regarding the stability of these esters in sediments, it seems likely that, under some conditions, bacterial esterification can enhance the preservation potential of labile compounds such as phytol. PMID:9872783

  20. Squalenes, phytanes and other isoprenoids as major neutral lipids of methanogenic and thermoacidophilic 'archaebacteria'

    NASA Technical Reports Server (NTRS)

    Tornabene, T. G.; Langworthy, T. A.; Holzer, G.; Oro, J.

    1979-01-01

    The neutral lipids from nine species of methanogenic bacteria (five methanobacilli, two methanococci, a methanospirillum and a methanosarcina) and two thermoacidophilic bacteria (Thermo-plasma and Sulfolobus) have been analyzed. The neutral lipids were found to comprise a wide range (C14 to C30) of polyisoprenyl hydrocarbons with varying degrees of saturation. The principal components represented the three major isoprenoid series (C20 phytanyl, C25 pentaisoprenyl, and C30 squalenyl), in contrast with the neutral lipids of extreme halophiles, which consist predominantly of C2O (phytanyl, geranylgeraniol), C30 (squalenes), C40 (carotenes) and C50 (bacterioruberins compounds), as reported by Kates (1978). These results, which indicate strong general similarities between genetically diverse organisms, support the classification of these organisms in a separate phylogenetic group. The occurrence of similar isoprenoid compounds in petroleum and ancient sediments and the fact that the methanogens, halophiles and thermoacidophiles live in conditions presumed to have prevailed in archaen times suggest that the isoprenoid compounds in petroleum compounds and sediment may have been directly synthesized by organisms of this type

  1. Proteolysis in Plastids of Arabidopsis Thaliana: Functional Analysis of ClpS1,2,T and their Physical and Genetic Interactions with the ClpPR Protease Core Complex and Clp Chaperones

    SciTech Connect

    van Wijk, Klaas

    2009-01-12

    Chloroplasts are essential organelles required for plant growth and biomass production. They synthesize many essential secondary metabolites (e.g. hormones, isoprenoids, amino acids, etc.) and house the photosynthetic apparatus needed for conversion of light energy and CO2 into chemical energy [in the form of reduced carbohydrates, ATP and NADPH]. Thus chloroplasts are essential for life on earth and essential for production of bioenergy. Formation and maintenance of a functional chloroplast requires an extensive investment in the biogenesis and homeostasis apparatus. Protease and proteolysis play a critical role in these processes, with the Clp gene family being particularly central. Proteolysis of proteins and protein complexes in plastids is poorly understood, and is not only critical for biogenesis, adaptation and maintenance but is also important for plant development. Several years ago, the vanWijk lab identified a large and relatively abundant ClpP/R/S complex, along with ClpC1,C2 and ClpD chaperones and a putative Clp affinity modulator in plastids. So far, no substrate recognition mechanism has been determined for any Clp complex in plants. The purpose of this grant was to initiate functional analysis of three members of the Clp family.

  2. Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes

    PubMed Central

    Reyes-Prieto, Adrian; Moustafa, Ahmed

    2012-01-01

    Studies of photosynthetic eukaryotes have revealed that the evolution of plastids from cyanobacteria involved the recruitment of non-cyanobacterial proteins. Our phylogenetic survey of >100 Arabidopsis nuclear-encoded plastid enzymes involved in amino acid biosynthesis identified only 21 unambiguous cyanobacterial-derived proteins. Some of the several non-cyanobacterial plastid enzymes have a shared phylogenetic origin in the three Plantae lineages. We hypothesize that during the evolution of plastids some enzymes encoded in the host nuclear genome were mistargeted into the plastid. Then, the activity of those foreign enzymes was sustained by both the plastid metabolites and interactions with the native cyanobacterial enzymes. Some of the novel enzymatic activities were favored by selective compartmentation of additional complementary enzymes. The mosaic phylogenetic composition of the plastid amino acid biosynthetic pathways and the reduced number of plastid-encoded proteins of non-cyanobacterial origin suggest that enzyme recruitment underlies the recompartmentation of metabolic routes during the evolution of plastids. PMID:23233874

  3. Is Remusatia (Araceae) Monophyletic? Evidence from Three Plastid Regions

    PubMed Central

    Li, Rong; Yi, Tingshuang; Li, Heng

    2012-01-01

    The genus Remusatia (Araceae) includes four species distributed in the tropical and subtropical Old World. The phylogeny of Remusatia was constructed using parsimony and Bayesian analyses of sequence data from three plastid regions (the rbcL gene, the trnL-trnF intergenic spacer, and the rps16 intron). Phylogenetic analyses of the concatenated plastid data suggested that the monophyly of Remusatia was not supported because R. hookeriana did not form a clade with the other three species R. vivipara, R. yunnanensis, and R. pumila. Nevertheless, the topology of the analysis constraining Remusatia to monophyly was congruent with the topology of the unconstrained analysis. The results confirmed the inclusion of the previously separate genus Gonatanthus within Remusatia and disagreed with the current infrageneric classification of the genus. PMID:22312239

  4. Plastid-nucleus communication involves calcium-modulated MAPK signalling.

    PubMed

    Guo, Hailong; Feng, Peiqiang; Chi, Wei; Sun, Xuwu; Xu, Xiumei; Li, Yuan; Ren, Dongtao; Lu, Congming; David Rochaix, Jean; Leister, Dario; Zhang, Lixin

    2016-01-01

    Chloroplast retrograde signals play important roles in coordinating the plastid and nuclear gene expression and are critical for proper chloroplast biogenesis and for maintaining optimal chloroplast functions in response to environmental changes in plants. Until now, the signals and the mechanisms for retrograde signalling remain poorly understood. Here we identify factors that allow the nucleus to perceive stress conditions in the chloroplast and to respond accordingly by inducing or repressing specific nuclear genes encoding plastid proteins. We show that ABI4, which is known to repress the LHCB genes during retrograde signalling, is activated through phosphorylation by the MAP kinases MPK3/MPK6 and the activity of these kinases is regulated through 14-3-3ω-mediated Ca(2+)-dependent scaffolding depending on the chloroplast calcium sensor protein CAS. These findings uncover an additional mechanism in which chloroplast-modulated Ca(2+) signalling controls the MAPK pathway for the activation of critical components of the retrograde signalling chain. PMID:27399341

  5. Plastid establishment did not require a chlamydial partner

    PubMed Central

    Domman, Daryl; Horn, Matthias; Embley, T. Martin; Williams, Tom A.

    2015-01-01

    Primary plastids descend from the cyanobacterial endosymbiont of an ancient eukaryotic host, but the initial selective drivers that stabilized the association between these two cells are still unclear. One hypothesis that has achieved recent prominence suggests that the first role of the cyanobiont was in energy provision for a host cell whose reserves were being depleted by an intracellular chlamydial pathogen. A pivotal claim is that it was chlamydial proteins themselves that converted otherwise unusable cyanobacterial metabolites into host energy stores. We test this hypothesis by investigating the origins of the key enzymes using sophisticated phylogenetics. Here we show a mosaic origin for the relevant pathway combining genes with host, cyanobacterial or bacterial ancestry, but we detect no strong case for Chlamydiae to host transfer under the best-fitting models. Our conclusion is that there is no compelling evidence from gene trees that Chlamydiae played any role in establishing the primary plastid endosymbiosis. PMID:25758953

  6. Production of high levels of poly-3-hydroxybutyrate in plastids of Camelina sativa seeds.

    PubMed

    Malik, Meghna R; Yang, Wenyu; Patterson, Nii; Tang, Jihong; Wellinghoff, Rachel L; Preuss, Mary L; Burkitt, Claire; Sharma, Nirmala; Ji, Yuanyuan; Jez, Joseph M; Peoples, Oliver P; Jaworski, Jan G; Cahoon, Edgar B; Snell, Kristi D

    2015-06-01

    Poly-3-hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed-specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N-terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T1 seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T2 seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight-averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T4 seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed-based platform for PHB production. PMID:25418911

  7. Editing of plastid RNA in Arabidopsis thaliana ecotypes.

    PubMed

    Tillich, Michael; Funk, Helena T; Schmitz-Linneweber, Christian; Poltnigg, Peter; Sabater, Bartolomé; Martin, Mercedes; Maier, Rainer M

    2005-09-01

    Post-transcriptional maturation of plastid-encoded mRNAs from land plants includes editing by making cytidine to uridine alterations at highly specific positions; this usually restores codon identities for conserved amino acids that are important for the proper function of the affected proteins. In contrast to the rather constant number of editing sites their location varies greatly, even between closely related taxa. Here, we experimentally determined the specific pattern of editing sites (the editotype) of the plastid genome of Arabidopsis thaliana ecotype Columbia (Col-0). Based on phylogenetic analyses of plastid open reading frames, we identified 28 editing sites. Two editing events in the genes matK and ndhB seem to have evolved late during the evolution of flowering plants. Strikingly, they are embedded in almost identical sequence elements and seem to be phylogenetically co-processed. This suggests that the two sites are recognized by the same trans-factor, which could help to explain the hitherto enigmatic gain of editing sites in evolution. In order to trace variations in editotype at the subspecies level we examined two other A. thaliana accessions, Cape Verde Islands (Cvi-0) and Wassilewskija (Ws-2), for the Col-0 editing sites. Both Cvi-0 and Ws-2 possess and process the whole set of editing sites as determined in Col-0, but the consequences of RNA editing differ at one position between the ecotypes. PMID:16115067

  8. Plastid DNA polymerases from higher plants, Arabidopsis thaliana

    SciTech Connect

    Mori, Yoko; Kimura, Seisuke; Saotome, Ai; Kasai, Nobuyuki; Sakaguchi, Norihiro; Uchiyama, Yukinobu; Ishibashi, Toyotaka; Yamamoto, Taichi; Chiku, Hiroyuki; Sakaguchi, Kengo . E-mail: kengo@rs.noda.sut.ac.jp

    2005-08-19

    Previously, we described a novel DNA polymerase, designated as OsPolI-like, from rice. The OsPolI-like showed a high degree of sequence homology with the DNA polymerase I of cyanobacteria and was localized in the plastid. Here, we describe two PolI-like polymerases, designated as AtPolI-like A and AtPolI-like B, from Arabidopsis thaliana. In situ hybridization analysis demonstrated expression of both mRNAs in proliferating tissues such as the shoot apical meristem. Analysis of the localizations of GFP fusion proteins showed that AtPolI-like A and AtPolI-like B were localized to plastids. AtPolI-like B expression could be induced by exposure to the mutagen H{sub 2}O{sub 2}. These results suggested that AtPolI-like B has a role in the repair of oxidation-induced DNA damage. Our data indicate that higher plants possess two plastid DNA polymerases that are not found in animals and yeasts.

  9. Seasonality of isoprenoid emissions from a primary rainforest in central Amazonia

    NASA Astrophysics Data System (ADS)

    Alves, E. G.; Jardine, K.; Tota, J.; Jardine, A.; Yáñez-Serrano, A. M.; Karl, T.; Tavares, J.; Nelson, B.; Gu, D.; Stavrakou, T.; Martin, S.; Manzi, A.; Guenther, A.

    2015-10-01

    Tropical rainforests are an important source of isoprenoid and other Volatile Organic Compound (VOC) emissions to the atmosphere. The seasonal variation of these compounds is however still poorly understood. In this study, profiles were collected of the vertical profile of mixing ratios of isoprene, total monoterpenes and total sesquiterpenes, within and above the canopy, in a primary rainforest in central Amazonia, using a Proton Transfer Reaction-Mass Spectrometer (PTR-MS). Fluxes of these compounds from the canopy into the atmosphere were estimated from PTR-MS measurements by using an inverse Lagrangian transport model. Measurements were carried out continuously from September 2010 to January 2011, encompassing the dry and wet seasons. Mixing ratios were higher during the dry (isoprene - 2.68 ± 0.9 ppbv, total monoterpenes - 0.67 ± 0.3 ppbv; total sesquiterpenes - 0.09 ± 0.07 ppbv) than the wet season (isoprene - 1.66 ± 0.9 ppbv, total monoterpenes - 0.47 ± 0.2 ppbv; total sesquiterpenes - 0.03 ± 0.02 ppbv) for all compounds. Ambient air temperature and photosynthetically active radiation (PAR) behaved similarly. Daytime isoprene and total monoterpene mixing ratios were highest within the canopy, rather than near the ground or above the canopy. By comparison, daytime total sesquiterpene mixing ratios were highest near the ground. Daytime fluxes varied significantly between seasons for all compounds. The maximums for isoprene (2.53 ± 0.5 μmol m-2 h-1) and total monoterpenes (1.77 ± 0.05 μmol m-2 h-1) were observed in the late dry season, whereas the maximum for total sesquiterpenes was found during the dry-to-wet transition season (0.77 ± 0.1 μmol m-2 h-1). These flux estimates suggest that the canopy is the main source of isoprenoids to the atmosphere for all seasons. However, uncertainties in turbulence parameterization near the ground could affect estimates of fluxes that come from the ground. Leaf phenology seemed to be an important driver of

  10. Seasonality of isoprenoid emissions from a primary rainforest in central Amazonia

    NASA Astrophysics Data System (ADS)

    Alves, Eliane G.; Jardine, Kolby; Tota, Julio; Jardine, Angela; Yãnez-Serrano, Ana Maria; Karl, Thomas; Tavares, Julia; Nelson, Bruce; Gu, Dasa; Stavrakou, Trissevgeni; Martin, Scot; Artaxo, Paulo; Manzi, Antonio; Guenther, Alex

    2016-03-01

    Tropical rainforests are an important source of isoprenoid and other volatile organic compound (VOC) emissions to the atmosphere. The seasonal variation of these compounds is however still poorly understood. In this study, vertical profiles of mixing ratios of isoprene, total monoterpenes and total sesquiterpenes, were measured within and above the canopy, in a primary rainforest in central Amazonia, using a proton transfer reaction - mass spectrometer (PTR-MS). Fluxes of these compounds from the canopy into the atmosphere were estimated from PTR-MS measurements by using an inverse Lagrangian transport model. Measurements were carried out continuously from September 2010 to January 2011, encompassing the dry and wet seasons. Mixing ratios were higher during the dry (isoprene - 2.68 ± 0.9 ppbv, total monoterpenes - 0.67 ± 0.3 ppbv; total sesquiterpenes - 0.09 ± 0.07 ppbv) than the wet season (isoprene - 1.66 ± 0.9 ppbv, total monoterpenes - 0.47 ± 0.2 ppbv; total sesquiterpenes - 0.03 ± 0.02 ppbv) for all compounds. Ambient air temperature and photosynthetically active radiation (PAR) behaved similarly. Daytime isoprene and total monoterpene mixing ratios were highest within the canopy, rather than near the ground or above the canopy. By comparison, daytime total sesquiterpene mixing ratios were highest near the ground. Daytime fluxes varied significantly between seasons for all compounds. The maximums for isoprene (2.53 ± 0.5 µmol m-2 h-1) and total monoterpenes (1.77 ± 0.05 µmol m-2 h-1) were observed in the late dry season, whereas the maximum for total sesquiterpenes was found during the dry-to-wet transition season (0.77 ± 0.1 µmol m-2 h-1). These flux estimates suggest that the canopy is the main source of isoprenoids emitted into the atmosphere for all seasons. However, uncertainties in turbulence parameterization near the ground could affect estimates of fluxes that come from the ground. Leaf phenology seemed to be an important driver of seasonal

  11. The Sink-Specific Plastidic Phosphate Transporter PHT4;2 Influences Starch Accumulation and Leaf Size in Arabidopsis1[W][OA

    PubMed Central

    Irigoyen, Sonia; Karlsson, Patrik M.; Kuruvilla, Jacob; Spetea, Cornelia; Versaw, Wayne K.

    2011-01-01

    Nonphotosynthetic plastids are important sites for the biosynthesis of starch, fatty acids, and amino acids. The uptake and subsequent use of cytosolic ATP to fuel these and other anabolic processes would lead to the accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. However, the identity of the transporter(s) responsible for Pi export is unclear. The plastid-localized Pi transporter PHT4;2 of Arabidopsis (Arabidopsis thaliana) is expressed in multiple sink organs but is nearly restricted to roots during vegetative growth. We identified and used pht4;2 null mutants to confirm that PHT4;2 contributes to Pi transport in isolated root plastids. Starch accumulation was limited in pht4;2 roots, which is consistent with the inhibition of starch synthesis by excess Pi as a result of a defect in Pi export. Reduced starch accumulation in leaves and altered expression patterns for starch synthesis genes and other plastid transporter genes suggest metabolic adaptation to the defect in roots. Moreover, pht4;2 rosettes, but not roots, were significantly larger than those of the wild type, with 40% greater leaf area and twice the biomass when plants were grown with a short (8-h) photoperiod. Increased cell proliferation accounted for the larger leaf size and biomass, as no changes were detected in mature cell size, specific leaf area, or relative photosynthetic electron transport activity. These data suggest novel signaling between roots and leaves that contributes to the regulation of leaf size. PMID:21960139

  12. High-Frequency Transformation of Undeveloped Plastids in Tobacco Suspension Cells

    PubMed Central

    Langbecker, Camri L.; Ye, Guang-Ning; Broyles, Debra L.; Duggan, Lisa L.; Xu, Charles W.; Hajdukiewicz, Peter T.J.; Armstrong, Charles L.; Staub, Jeffrey M.

    2004-01-01

    Although leaf chloroplast transformation technology was developed more than a decade ago, no reports exist of stable transformation of undeveloped plastids or other specialized plastid types, such as proplastids, etioplasts, or amyloplasts. In this work we report development of a dark-grown tobacco suspension cell model system to investigate the transformation potential of undeveloped plastids. Electron microscope analysis confirmed that the suspension cells carry plastids that are significantly smaller (approximately 50-fold less in volume) and have a very different subcellular localization and developmental state than leaf cell chloroplasts. Using antibiotic selection in the light, we demonstrated that both plastid and nuclear transformation of these cell suspensions is efficient and reproducible, with plastid transformation frequency at least equal to that of leaf chloroplast transformation. Homoplasmic plastid transformants are readily obtained in cell colonies, or in regenerated plants, providing a more consistent and versatile model than the leaf transformation system. Because of the uniformity of the cell suspension model, we could further show that growth rate, selection scheme, particle size, and DNA amount influence the frequency of transformation. Our results indicate that the rate-limiting steps for nuclear and plastid transformation are different, and each must be optimized separately. The suspension cell system will be useful as a model for understanding transformation in those plant species that utilize dark-grown embryogenic cultures and for characterizing the steps that lead to homoplasmic plastid transformation. PMID:15141065

  13. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. PMID:27241217

  14. Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

    PubMed

    Dorrell, Richard G; Hinksman, George A; Howe, Christopher J

    2016-02-01

    Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes. PMID:26768263

  15. Erwin Baur or Carl Correns: who really created the theory of plastid inheritance?

    PubMed

    Hagemann, R

    2000-01-01

    Historical reviews of the field of non-Mendelian genetics and many other publications credit Erwin Baur and Carl Correns equally for the development of the theory of plastid inheritance. However, a study of the original literature indicates that this conclusion is not correct. Analysis of the relevant articles leads to the conclusion that Baur alone deserves credit for the theory of plastid inheritance. In his classic article on the inheritance properties of white-margined Pelargonium plants, Baur (1909) stated: (1) The plastids are carriers of hereditary factors which are able to mutate. (2) In variegated plants, random sorting-out of plastids is taking place. (3) The genetic results indicate a biparental inheritance of plastids by egg cells and sperm cells in Pelargonium. By contrast, Correns held the view that in variegated plants there is a maternally transmitted labile state of the cytoplasm which switches either to a permanently "healthy" state (allowing the "indifferent" plastids to become green chloroplasts) or to a permanently "diseased, ill" cytoplasmic state (causing white plastids and cells). Otto Renner supported Baur's theory and worked out important characteristics of plastid inheritance in the genus Oenothera. In the 1930s Renner reported many more observations, which established plastid inheritance as a widely accepted genetic theory. PMID:11218080

  16. Plastid uridine salvage activity is required for photoassimilate allocation and partitioning in Arabidopsis.

    PubMed

    Chen, Mingjie; Thelen, Jay J

    2011-08-01

    Nucleotides are synthesized from de novo and salvage pathways. To characterize the uridine salvage pathway, two genes, UKL1 and UKL2, that tentatively encode uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) bifunctional enzymes were studied in Arabidopsis thaliana. T-DNA insertions in UKL1 and UKL2 reduced transcript expression and increased plant tolerance to toxic analogs 5-fluorouridine and 5-fluorouracil. Enzyme activity assays using purified recombinant proteins indicated that UKL1 and UKL2 have UK but not UPRT activity. Subcellular localization using a C-terminal enhanced yellow fluorescent protein fusion indicated that UKL1 and UKL2 localize to plastids. The ukl2 mutant shows reduced transient leaf starch during the day. External application of orotate rescued this phenotype in ukl2, indicating pyrimidine pools are limiting for starch synthesis in ukl2. Intermediates for lignin synthesis were upregulated, and there was increased lignin and reduced cellulose content in the ukl2 mutant. Levels of ATP, ADP, ADP-glucose, UTP, UDP, and UDP-glucose were altered in a light-dependent manner. Seed composition of the ukl1 and ukl2 mutants included lower oil and higher protein compared with the wild type. Unlike single gene mutants, the ukl1 ukl2 double mutant has severe developmental defects and reduced biomass accumulation, indicating these enzymes catalyze redundant reactions. These findings point to crucial roles played by uridine salvage for photoassimilate allocation and partitioning. PMID:21828290

  17. Plastid–Nuclear Interaction and Accelerated Coevolution in Plastid Ribosomal Genes in Geraniaceae

    PubMed Central

    Weng, Mao-Lun; Ruhlman, Tracey A.; Jansen, Robert K.

    2016-01-01

    Plastids and mitochondria have many protein complexes that include subunits encoded by organelle and nuclear genomes. In animal cells, compensatory evolution between mitochondrial and nuclear-encoded subunits was identified and the high mitochondrial mutation rates were hypothesized to drive compensatory evolution in nuclear genomes. In plant cells, compensatory evolution between plastid and nucleus has rarely been investigated in a phylogenetic framework. To investigate plastid–nuclear coevolution, we focused on plastid ribosomal protein genes that are encoded by plastid and nuclear genomes from 27 Geraniales species. Substitution rates were compared for five sets of genes representing plastid- and nuclear-encoded ribosomal subunit proteins targeted to the cytosol or the plastid as well as nonribosomal protein controls. We found that nonsynonymous substitution rates (dN) and the ratios of nonsynonymous to synonymous substitution rates (ω) were accelerated in both plastid- (CpRP) and nuclear-encoded subunits (NuCpRP) of the plastid ribosome relative to control sequences. Our analyses revealed strong signals of cytonuclear coevolution between plastid- and nuclear-encoded subunits, in which nonsynonymous substitutions in CpRP and NuCpRP tend to occur along the same branches in the Geraniaceae phylogeny. This coevolution pattern cannot be explained by physical interaction between amino acid residues. The forces driving accelerated coevolution varied with cellular compartment of the sequence. Increased ω in CpRP was mainly due to intensified positive selection whereas increased ω in NuCpRP was caused by relaxed purifying selection. In addition, the many indels identified in plastid rRNA genes in Geraniaceae may have contributed to changes in plastid subunits. PMID:27190001

  18. The YTA7 gene is involved in the regulation of the isoprenoid pathway in the yeast Saccharomyces cerevisiae.

    PubMed

    Kuranda, Klaudia; Grabinska, Kariona; Berges, Thierry; Karst, Francis; Leberre, Veronique; Sokol, Serguei; François, Jean; Palamarczyk, Grazyna

    2009-05-01

    The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of nonsterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, we searched for its protein partners using a two-hybrid screen, and identified five interacting proteins, among them Yta7p. Subsequently, we showed that Yta7p was a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of YTA7 affected the enzymatic activity of cis-prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7Delta cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase. Altogether, these results provide substantial evidence of Yta7p involvement in the regulation of isoprenoid biosynthesis. PMID:19416104

  19. Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

    PubMed Central

    Wallrapp, Frank H.; Pan, Jian-Jung; Ramamoorthy, Gurusankar; Almonacid, Daniel E.; Hillerich, Brandan S.; Seidel, Ronald; Patskovsky, Yury; Babbitt, Patricia C.; Almo, Steven C.; Jacobson, Matthew P.; Poulter, C. Dale

    2013-01-01

    The number of available protein sequences has increased exponentially with the advent of high-throughput genomic sequencing, creating a significant challenge for functional annotation. Here, we describe a large-scale study on assigning function to unknown members of the trans-polyprenyl transferase (E-PTS) subgroup in the isoprenoid synthase superfamily, which provides substrates for the biosynthesis of the more than 55,000 isoprenoid metabolites. Although the mechanism for determining the product chain length for these enzymes is known, there is no simple relationship between function and primary sequence, so that assigning function is challenging. We addressed this challenge through large-scale bioinformatics analysis of >5,000 putative polyprenyl transferases; experimental characterization of the chain-length specificity of 79 diverse members of this group; determination of 27 structures of 19 of these enzymes, including seven cocrystallized with substrate analogs or products; and the development and successful application of a computational approach to predict function that leverages available structural data through homology modeling and docking of possible products into the active site. The crystallographic structures and computational structural models of the enzyme–ligand complexes elucidate the structural basis of specificity. As a result of this study, the percentage of E-PTS sequences similar to functionally annotated ones (BLAST e-value ≤ 1e−70) increased from 40.6 to 68.8%, and the percentage of sequences similar to available crystal structures increased from 28.9 to 47.4%. The high accuracy of our blind prediction of newly characterized enzymes indicates the potential to predict function to the complete polyprenyl transferase subgroup of the isoprenoid synthase superfamily computationally. PMID:23493556

  20. Identification of long-chain isoprenoid alkylbenzenes in sediments and crude oils

    SciTech Connect

    Sinninghe Damste, J.S.; Kock-Van Dalen, A.C.; De Leeuw, J.W. )

    1988-11-01

    A series of novel methylated phytanylbenzenes have been identified in sediment extracts and oils ranging in age from Miocene to Permian. Identifications were based on comparison of mass spectra and chromatographic data of synthetic methylated phytanylbenzenes with those of geologically occurring methylated phytanylbenzenes and by coinjections with the standards. Although methylated phytanylbenzenes are structurally related to the methylated 2-methyl-2-(4,8,12-trimethyltridecyl)chromans, components also present in the samples studied, the former do not appear to be the diagenetic derivatives of the latter. The methylated phytanylbenzenes are thought to be derived diagenetically from isoprenoid quinones or may represent a direct biosynthetic origin from specific archaebacteria.

  1. Isoprenoids suppress the growth of murine B16 melanomas in vitro and in vivo.

    PubMed

    He, L; Mo, H; Hadisusilo, S; Qureshi, A A; Elson, C E

    1997-05-01

    Sundry mevalonate-derived constituents (isoprenoids) of fruits, vegetables and cereal grains suppress the growth of tumors. This study estimated the concentrations of structurally diverse isoprenoids required to inhibit the increase in a population of murine B16(F10) melanoma cells during a 48-h incubation by 50% (IC50 value). The IC50 values for d-limonene and perillyl alcohol, the monoterpenes in Phase I trials, were 450 and 250 micromol/L, respectively; related cyclic monoterpenes (perillaldehyde, carvacrol and thymol), an acyclic monoterpene (geraniol) and the end ring analog of beta-carotene (beta-ionone) had IC50 values in the range of 120-150 micromol/L. The IC50 value estimated for farnesol, the side-chain analog of the tocotrienols (50 micromol/L) fell midway between that of alpha-tocotrienol (110 micromol/L) and those estimated for gamma- (20 micromol/L) and delta- (10 micromol/L) tocotrienol. A novel tocotrienol lacking methyl groups on the tocol ring proved to be extremely potent (IC50, 0.9 micromol/L). In the first of two diet studies, experimental diets were fed to weanling C57BL female mice for 10 d prior to and 28 d following the implantation of the aggressively growing and highly metastatic B16(F10) melanoma. The isomolar (116 micromol/kg diet) and the Vitamin E-equivalent (928 micromol/kg diet) substitution of d-gamma-tocotrienol for dl-alpha-tocopherol in the AIN-76A diet produced 36 and 50% retardations, respectively, in tumor growth (P < 0.05). In the second study, melanomas were established before mice were fed experimental diets formulated with 2 mmol/kg d-gamma-tocotrienol, beta-ionone individually and in combination. Each treatment increased (P < 0.03) the duration of host survival. Our finding that the effects of individual isoprenoids were additive suggests the possibility that one component of the anticarcinogenic action of plant-based diets is the tumor growth-suppressive action of the diverse isoprenoid constituents of fruits

  2. Isoprenoid hydrocarbons produced by thermal alteration of Nostoc muscorum and Rhodopseudomonas spheroides

    NASA Technical Reports Server (NTRS)

    Philp, R. P.; Brown, S.; Calvin, M.

    1978-01-01

    The potential of algae and photosynthetic bacteria to serve as precursors of kerogen was studied to determine what factors affect the relative rates of formation of precursor hydrocarbons. Cells of Nostoc muscorum and Rhodopseudomonas spheroides were subjected to thermal alteration (by heating samples in glass tubes sealed under nitrogen) for two, four, and twelve weeks. Both unextracted and extracted cells in the absence and presence of montmorillonite were investigated, and the isoprenoid hydrocarbons produced in these experiments were determined. Phytane and five isomeric phytenes were the main hydrocarbons observed; their relative rates of formation in the different experimental conditions are described. No phytadienes, pristane, or pristenes were detected.

  3. Remote sensing of plant emissions of volatile isoprenoids with PRI. Prospects for upscaling (Invited)

    NASA Astrophysics Data System (ADS)

    Penuelas, J.

    2013-12-01

    Josep Peñuelas*1,2, Giovanni Marino1,2,3,4, Joan LLusia1,2, Catherine Morfopoulos1,2,5, Gerard Farre-Armengol1,2, Shawn Kefauver, Alex Guenther6 , Francesca Rapparini7 , Roger Seco1,2,6, Marc Estiarte1,2, Mónica Mejia-Chang1,2, Romà Ogaya1,2, Jordi Sardans1,2 , Andrew Turnipseed6, Peter Harley6, Osvaldo Facini7, Rita Baraldi7, Jim Greenberg6 , Iolanda Filella1,2 1 CSIC, Global Ecology Unit CREAF-CEAB-UAB, Cerdanyola del Vallés 08193, Catalonia, Spain 2 CREAF, Cerdanyola del Vallés 08193, Catalonia, Spain 3 Dipartimento di Bioscienze e Territorio, Università degli Studi del Molise, Contrada Fonte Lappone, 86090 Pesche (IS), Italy 4 Institute for Plant Protection, National Research Council, Via Madonna del Piano 10, 50019 Sesto Fiorentino (FI), Italy 5 Division of Ecology and Evolution, Imperial College, Silwood Park, Ascot, SL5 7PY, UK 6 Atmospheric Chemistry Division, National Center for Atmospheric Research, P.O. Box 3000, Boulder, CO 80307-3000, USA 7 Biometeorology Institute, IBIMET-CNR, Via P. Gobetti 101, Bologna, Italy Abstract Terrestrial plants re-emit around 1-2% of the carbon they fix as isoprene and monoterpenes. These emissions play major roles in the ecological relationships among living organisms and in atmospheric chemistry and climate, and yet their actual quantification at the ecosystem level in different regions is far from being resolved. Phenomenological models are used to estimate the emission rates, but the limited understanding of the function and regulation of these emissions leads to large uncertainties in such estimations. Many measurements have been made at the foliar but few at the ecosystem level, and those that do exist are limited in space and time. We here provide evidence that a simple remote sensing index, the photochemical reflectance index (PRI), which is indicative of light use efficiency (LUE), is a good indirect estimator of foliar isoprenoid emissions and therefore can be used to sense them remotely. These results open

  4. Identification of the Maize Amyloplast Stromal 112-kD Protein as a Plastidic Starch Phosphorylase12

    PubMed Central

    Yu, Ying; Mu, Helen He; Wasserman, Bruce P.; Carman, George M.

    2001-01-01

    Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences showed high identity to plastidic forms of starch phosphorylase (SP) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme. PMID:11154342

  5. The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis

    PubMed Central

    Fujiwara, Makoto T.; Kojo, Kei H.; Kazama, Yusuke; Sasaki, Shun; Abe, Tomoko; Itoh, Ryuuichi D.

    2015-01-01

    Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids. PMID:26500667

  6. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate

    PubMed Central

    Gornik, Sebastian G.; Febrimarsa; Cassin, Andrew M.; MacRae, James I.; Ramaprasad, Abhinay; Rchiad, Zineb; McConville, Malcolm J.; Bacic, Antony; McFadden, Geoffrey I.; Pain, Arnab; Waller, Ross F.

    2015-01-01

    Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes—notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium—highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite’s host. Hematodinium sp. thus represents a further dimension of endosymbiosis—life after the organelle. PMID:25902514

  7. Complete Plastid Genome Sequencing of Four Tilia Species (Malvaceae): A Comparative Analysis and Phylogenetic Implications

    PubMed Central

    Cai, Jie; Ma, Peng-Fei; Li, Hong-Tao; Li, De-Zhu

    2015-01-01

    Tilia is an ecologically and economically important genus in the family Malvaceae. However, there is no complete plastid genome of Tilia sequenced to date, and the taxonomy of Tilia is difficult owing to frequent hybridization and polyploidization. A well-supported interspecific relationships of this genus is not available due to limited informative sites from the commonly used molecular markers. We report here the complete plastid genome sequences of four Tilia species determined by the Illumina technology. The Tilia plastid genome is 162,653 bp to 162,796 bp in length, encoding 113 unique genes and a total number of 130 genes. The gene order and organization of the Tilia plastid genome exhibits the general structure of angiosperms and is very similar to other published plastid genomes of Malvaceae. As other long-lived tree genera, the sequence divergence among the four Tilia plastid genomes is very low. And we analyzed the nucleotide substitution patterns and the evolution of insertions and deletions in the Tilia plastid genomes. Finally, we build a phylogeny of the four sampled Tilia species with high supports using plastid phylogenomics, suggesting that it is an efficient way to resolve the phylogenetic relationships of this genus. PMID:26566230

  8. The Endosomal Protein CHARGED MULTIVESICULAR BODY PROTEIN1 Regulates the Autophagic Turnover of Plastids in Arabidopsis

    PubMed Central

    Spitzer, Christoph; Li, Faqiang; Buono, Rafael; Roschzttardtz, Hannetz; Chung, Taijoon; Zhang, Min

    2015-01-01

    Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents. PMID:25649438

  9. Differential coloring reveals that plastids do not form networks for exchanging macromolecules.

    PubMed

    Schattat, Martin H; Griffiths, Sarah; Mathur, Neeta; Barton, Kiah; Wozny, Michael R; Dunn, Natalie; Greenwood, John S; Mathur, Jaideep

    2012-04-01

    Stroma-filled tubules named stromules are sporadic extensions of plastids. Earlier, photobleaching was used to demonstrate fluorescent protein diffusion between already interconnected plastids and formed the basis for suggesting that all plastids are able to form networks for exchanging macromolecules. However, a critical appraisal of literature shows that this conjecture is not supported by unequivocal experimental evidence. Here, using photoconvertible mEosFP, we created color differences between similar organelles that enabled us to distinguish clearly between organelle fusion and nonfusion events. Individual plastids, despite conveying a strong impression of interactivity and fusion, maintained well-defined boundaries and did not exchange fluorescent proteins. Moreover, the high pleomorphy of etioplasts from dark-grown seedlings, leucoplasts from roots, and assorted plastids in the accumulation and replication of chloroplasts5 (arc5), arc6, and phosphoglucomutase1 mutants of Arabidopsis thaliana suggested that a single plastid unit might be easily mistaken for interconnected plastids. Our observations provide succinct evidence to refute the long-standing dogma of interplastid connectivity. The ability to create and maintain a large number of unique biochemical factories in the form of singular plastids might be a key feature underlying the versatility of green plants as it provides increased internal diversity for them to combat a wide range of environmental fluctuations and stresses. PMID:22474180

  10. Fluorescent Protein Aided Insights on Plastids and their Extensions: A Critical Appraisal

    PubMed Central

    Delfosse, Kathleen; Wozny, Michael R.; Jaipargas, Erica-Ashley; Barton, Kiah A.; Anderson, Cole; Mathur, Jaideep

    2016-01-01

    Multi-colored fluorescent proteins targeted to plastids have provided new insights on the dynamic behavior of these organelles and their interactions with other cytoplasmic components and compartments. Sub-plastidic components such as thylakoids, stroma, the inner and outer membranes of the plastid envelope, nucleoids, plastoglobuli, and starch grains have been efficiently highlighted in living plant cells. In addition, stroma filled membrane extensions called stromules have drawn attention to the dynamic nature of the plastid and its interactions with the rest of the cell. Use of dual and triple fluorescent protein combinations has begun to reveal plastid interactions with mitochondria, the nucleus, the endoplasmic reticulum and F-actin and suggests integral roles of plastids in retrograde signaling, cell to cell communication as well as plant-pathogen interactions. While the rapid advances and insights achieved through fluorescent protein based research on plastids are commendable it is necessary to endorse meaningful observations but subject others to closer scrutiny. Here, in order to develop a better and more comprehensive understanding of plastids and their extensions we provide a critical appraisal of recent information that has been acquired using targeted fluorescent protein probes. PMID:26834765

  11. Rampant Gene Loss in the Underground Orchid Rhizanthella gardneri Highlights Evolutionary Constraints on Plastid Genomes

    PubMed Central

    Delannoy, Etienne; Fujii, Sota; Colas des Francs-Small, Catherine; Brundrett, Mark; Small, Ian

    2011-01-01

    Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer. PMID:21289370

  12. Evidence for horizontal transfer of mitochondrial DNA to the plastid genome in a bamboo genus

    PubMed Central

    Ma, Peng-Fei; Zhang, Yu-Xiao; Guo, Zhen-Hua; Li, De-Zhu

    2015-01-01

    In flowering plants, three genomes (nuclear, mitochondrial, and plastid) coexist and intracellular horizontal transfer of DNA is prevalent, especially from the plastid to the mitochondrion genome. However, the plastid genomes are generally conserved in evolution and have long been considered immune to foreign DNA. Recently, the opposite direction of DNA transfer from the mitochondrial to the plastid genome has been reported in two eudicot lineages. Here we sequenced 6 plastid genomes of bamboos, three of which are neotropical woody species and three are herbaceous ones. Several unusual features were found, including the duplication of trnT-GGU and loss of one copy of rps19 due to contraction of inverted repeats (IRs). The most intriguing was the ~2.7 kb insertion in the plastid IR regions in the three herbaceous bamboos. Furthermore, the insertion was documented to be horizontally transferred from the mitochondrial to the plastid genome. Our study provided evidence of the mitochondrial-to-plastid DNA transfer in the monocots, demonstrating again that this rare event does occur in other angiosperm lineages. However, the mechanism underlying the transfer remains obscure, and more studies in other plants may elucidate it in the future. PMID:26100509

  13. Precise excision of plastid DNA by the large serine recombinase Bxb1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To elucidate the precise excision of transgene, tobacco plastid genome was transformed with a vector (pTCH-BxbPB) that contains a stuffer DNA fragment flanked by directly oriented attB and attP recognition sites for the Bxb1 recombinase. The transformed plastid genomes containing the recognition si...

  14. Combined heat shock protein 90 and ribosomal RNA sequence phylogeny supports multiple replacements of dinoflagellate plastids.

    PubMed

    Shalchian-Tabrizi, Kamran; Minge, Marianne A; Cavalier-Smith, Tom; Nedreklepp, Joachim M; Klaveness, Dag; Jakobsen, Kjetill S

    2006-01-01

    Dinoflagellates harbour diverse plastids obtained from several algal groups, including haptophytes, diatoms, cryptophytes, and prasinophytes. Their major plastid type with the accessory pigment peridinin is found in the vast majority of photosynthetic species. Some species of dinoflagellates have other aberrantly pigmented plastids. We sequenced the nuclear small subunit (SSU) ribosomal RNA (rRNA) gene of the "green" dinoflagellate Gymnodinium chlorophorum and show that it is sister to Lepidodinium viride, indicating that their common ancestor obtained the prasinophyte (or other green alga) plastid in one event. As the placement of dinoflagellate species that acquired green algal or haptophyte plastids is unclear from small and large subunit (LSU) rRNA trees, we tested the usefulness of the heat shock protein (Hsp) 90 gene for dinoflagellate phylogeny by sequencing it from four species with aberrant plastids (G. chlorophorum, Karlodinium micrum, Karenia brevis, and Karenia mikimotoi) plus Alexandrium tamarense, and constructing phylogenetic trees for Hsp90 and rRNAs, separately and together. Analyses of the Hsp90 and concatenated data suggest an ancestral origin of the peridinin-containing plastid, and two independent replacements of the peridinin plastid soon after the early radiation of the dinoflagellates. Thus, the Hsp90 gene seems to be a promising phylogenetic marker for dinoflagellate phylogeny. PMID:16677346

  15. In silico analysis of 454 data yields the complete American cranberry (Vaccinium macrocarpon Ait.) plastid genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The complete plastid genome sequence of the cranberry cultivar “HyRed” was reconstructed using next generation sequencing (NGS) data by in silico procedures. We used previously generated “HyRed” 454 shotgun sequence data to isolate plastid data via homology comparisons with complete sequences from s...

  16. Endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate.

    PubMed

    Gornik, Sebastian G; Febrimarsa; Cassin, Andrew M; MacRae, James I; Ramaprasad, Abhinay; Rchiad, Zineb; McConville, Malcolm J; Bacic, Antony; McFadden, Geoffrey I; Pain, Arnab; Waller, Ross F

    2015-05-01

    Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes--notably, the apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium--highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite's host. Hematodinium sp. thus represents a further dimension of endosymbiosis--life after the organelle. PMID:25902514

  17. Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis

    NASA Technical Reports Server (NTRS)

    MacCleery, S. A.; Kiss, J. Z.

    1999-01-01

    Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

  18. Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

    PubMed Central

    Siddell, S G; Ellis, R J

    1975-01-01

    The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts. Images PLATE 1 PMID:1147911

  19. RNase P RNA from the Recently Evolved Plastid of Paulinella and from Algae

    PubMed Central

    Bernal-Bayard, Pilar; Puerto-Galán, Leonor; Vioque, Agustín

    2014-01-01

    The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore) that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro. PMID:25402646

  20. RNase P RNA from the recently evolved plastid of Paulinella and from algae.

    PubMed

    Bernal-Bayard, Pilar; Puerto-Galán, Leonor; Vioque, Agustín

    2014-01-01

    The RNase P RNA catalytic subunit (RPR) encoded in some plastids has been found to be functionally defective. The amoeba Paulinella chromatophora contains an organelle (chromatophore) that is derived from the recent endosymbiotic acquisition of a cyanobacterium, and therefore represents a model of the early steps in the acquisition of plastids. In contrast with plastid RPRs the chromatophore RPR retains functionality similar to the cyanobacterial enzyme. The chromatophore RPR sequence deviates from consensus at some positions but those changes allow optimal activity compared with mutated chromatophore RPR with the consensus sequence. We have analyzed additional RPR sequences identifiable in plastids and have found that it is present in all red algae and in several prasinophyte green algae. We have assayed in vitro a subset of the plastid RPRs not previously analyzed and confirm that these organelle RPRs lack RNase P activity in vitro. PMID:25402646

  1. Inducible gene expression from the plastid genome by a synthetic riboswitch

    PubMed Central

    Verhounig, Andreas; Karcher, Daniel; Bock, Ralph

    2010-01-01

    Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology. PMID:20308585

  2. Plastid transformation of sporelings and suspension-cultured cells from the liverwort Marchantia polymorpha L.

    PubMed

    Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki

    2014-01-01

    We describe simple and efficient plastid transformation methods for suspension-cultured cells and sporelings of the liverwort, Marchantia polymorpha L. Use of rapidly proliferating cells such as suspension-cultured cells and sporelings, which are immature thalli developing from spores, as targets made plastid transformation by particle bombardment efficient. Selection on a sucrose-free medium and linearization of the transformation vector significantly improved the recovery rate of plastid transformants. With the methods described here, a few plastid transformants are obtained from a single bombardment of sporelings, while more efficient plastid transformation is expected in suspension-cultured cells, ~60 transformants from a single bombardment. Homoplasmic transformants of thalli are obtained immediately after primary selection, whereas homoplasmic transformants from suspension-cultured cells are obtained after 12-16 weeks of repeated subculture. PMID:24599873

  3. Inducible gene expression from the plastid genome by a synthetic riboswitch.

    PubMed

    Verhounig, Andreas; Karcher, Daniel; Bock, Ralph

    2010-04-01

    Riboswitches are natural RNA sensors that regulate gene expression in response to ligand binding. Riboswitches have been identified in prokaryotes and eukaryotes but are unknown in organelles (mitochondria and plastids). Here we have tested the possibility to engineer riboswitches for plastids (chloroplasts), a genetic system that largely relies on translational control of gene expression. To this end, we have used bacterial riboswitches and modified them in silico to meet the requirements of translational regulation in plastids. These engineered switches were then tested for functionality in vivo by stable transformation of the tobacco chloroplast genome. We report the identification of a synthetic riboswitch that functions as an efficient translational regulator of gene expression in plastids in response to its exogenously applied ligand theophylline. This riboswitch provides a novel tool for plastid genome engineering that facilitates the tightly regulated inducible expression of chloroplast genes and transgenes and thus has wide applications in functional genomics and biotechnology. PMID:20308585

  4. Whole Mitochondrial and Plastid Genome SNP Analysis of Nine Date Palm Cultivars Reveals Plastid Heteroplasmy and Close Phylogenetic Relationships among Cultivars

    PubMed Central

    Sabir, Jamal S. M.; Arasappan, Dhivya; Bahieldin, Ahmed; Abo-Aba, Salah; Bafeel, Sameera; Zari, Talal A.; Edris, Sherif; Shokry, Ahmed M.; Gadalla, Nour O.; Ramadan, Ahmed M.; Atef, Ahmed; Al-Kordy, Magdy A.; El-Domyati, Fotoh M.; Jansen, Robert K.

    2014-01-01

    Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular

  5. Conversion of isoprenoid oil by catalytic cracking and hydrocracking over nanoporous hybrid catalysts.

    PubMed

    Kimura, Toshiyuki; Liu, Chen; Li, Xiaohong; Maekawa, Takaaki; Asaoka, Sachio

    2012-01-01

    In order to produce petroleum alternatives from biomass, a significant amount of research has been focused on oils from microalgae due to their origin, which would not affect food availability. Nanoporous hybrid catalysts composed of ns Al₂O₃ and zeolites have been proven to be very useful compared to traditional catalysts in hydrotreating (HT), hydrocracking (HC), and catalytic cracking (CC) of large molecules. To evaluate the reaction scheme and products from model isoprenoid compounds of microalgae oil, nanoporous hybrid catalyst technologies (CC: ns Al₂O₃/H-USY and ns Al₂O₃/H-GaAlMFI; HC: [Ni-Mo/γ-Al₂O₃]/ns Al₂O₃/H-beta) were studied. The major product from CC on ns Al₂O₃/H-USY was highly aromatic gasoline, while the product from HC was half-isoparaffinic/olefinic kerosene. Although more than 50 wt% of the products from HT/CC on the USY catalyst was liquefied petroleum gas due to overcracking, the product from HT/CC on the MFI catalyst was high-octane-number gasoline. Delightfully, the product from HT/HC was kerosene and its average number was 11, with more than 80 wt% being isoparaffinic. As a result, it was demonstrated that hydrotreating may convert isoprenoid oil from microalgae over nanoporous hybrid catalysts into a variety of products. PMID:22791962

  6. Isoprenoid emissions of trees in a tropical rainforest in Xishuangbanna, SW China

    NASA Astrophysics Data System (ADS)

    Wilske, B.; Cao, K.-F.; Schebeske, G.; Chen, J.-W.; Wang, A.; Kesselmeier, J.

    Isoprenoid emissions of eight tropical tree species of SE Asia were investigated using dynamic Teflon bag branch enclosures. Emission potentials of four species were considerably deviating from a previous report. Two species, Garcinia cowa and Celtis philippensis, emitted isoprene with standard emission factors, given as carbon on dry weight basis of 20.7 and 0.2μgg-1h-1, respectively, before the peak of the rainy season. After the peak of the rainy reason the standard emission changed to 17.5 and 0.7μgg-1h-1, respectively. The other six species emitted monoterpenes with low standard emission factors between <0.1 and 0.5μgg-1h-1. Four out of five species investigated at two different times of the year showed seasonal differences in emission rates and composition. Total isoprenoid emissions were generally higher with new leaf flush than with aged leaves. Overall, the results suggest that better understanding of volatile organic compounds (VOC) emission from tropical species of SE Asia requires investigations that cover different seasons.

  7. Voltammetric detection and profiling of isoprenoid quinones hydrophobically transferred from bacterial cells.

    PubMed

    Le, Dung Quynh; Morishita, Aya; Tokonami, Shiho; Nishino, Tomoaki; Shiigi, Hiroshi; Miyake, Masami; Nagaoka, Tsutomu

    2015-08-18

    We have developed a novel bacterial detection technique by desiccating a bacterial suspension deposited on an electrode. It was also found that the use of an indium-tin-oxide (ITO) electrode dramatically improved the resolution of the voltammogram, allowing us to observe two pairs of redox peaks, each assigned to the adsorption of isoprenoid ubiquinone (UQn) and menaquinone (MKn), which were present in the bacterial cell envelopes, giving midpeak potentials of -0.015 and -0.25 V versus Ag|AgCl|saturated KCl| at pH 7.0, respectively. Most of the microorganisms classified in both the Gram-negative and -positive bacteria gave well-defined redox peaks, demonstrating that this procedure made the detection of the quinones possible without solvent extraction. It has been demonstrated that the present technique can be used not only for the detection of bacteria, but also for profiling of the isoprenoid quinones, which play important roles in electron and proton transfer in microorganisms. In this respect, the present technique provides a much more straightforward way than the solvent extraction in that one sample can be prepared in 1 min by heat evaporation of a suspension containing the targeted bacteria, which has been applied on the ITO electrode. PMID:26218886

  8. Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

    PubMed Central

    Dykstra, Kaitlyn M.; Allen, Cheryl; Born, Ella J.; Tong, Huaxiang; Holstein, Sarah A.

    2015-01-01

    Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents. PMID:26595805

  9. Stereochemical studies of acyclic isoprenoids-XII. Lipids of methanogenic bacteria and possible contributions to sediments

    USGS Publications Warehouse

    Risatti, J.B.; Rowland, S.J.; Yon, D.A.; Maxwell, J.R.

    1984-01-01

    Abundant volatile lipids of Methanobacterium thermoautotrophicum and Methanosarcina barkeri include isoprenoid hydrocarbons (??? C30), and C15, C20 and C25 isoprenoid alcohols. M. barkeri contains 2,6,10,15,19-pentamethyleicosane, whose relative stereochemistry is the same as found in marine sediments, indicating that it is a marker of methanogenic activity. The C20, C30 and C25 alkenes in M. thermoautotrophicum also have a preferred sterochemistry; the latter have the 2,6,10,14,18-pentamethyleicosanyl skeleton, suggesting that the alkane in marine sediments may derive from methanogens. The stereochemistry of squalane in a marine sediment is also compatible with an origin in methanogens; in contrast, the stereochemistry of pristane in M. thermoautotrophicum indicates a fossil fuel contaminant origin, suggesting that this and certain other alkanes reported in archaebacteria might also be of contaminant origin. There is, therefore, little evidence at present that the pristane in immature marine sediments originates in methanogens. The C15 and C20 saturated alcohols in M. thermoautotrophicum have mainly the all-R configuration. If this is generally true for methanogens, the C20 alcohol in the Messel shale may originate mainly from methanogens, whereas that in the Green River shale may originate mainly from photosynthetic organisms. ?? 1984.

  10. Process-based modelling of isoprenoid emissions from evergreen leaves of Quercus ilex (L.)

    NASA Astrophysics Data System (ADS)

    Grote, R.; Mayrhofer, S.; Fischbach, R. J.; Steinbrecher, R.; Staudt, M.; Schnitzler, J.-P.

    Monoterpenes play an important role in regulating the trace gas composition of the lower troposphere. Therefore, realistic estimates of the daily as well as seasonal variations of monoterpene emission source strength on the Earth surface are required. Monoterpenes are emitted by Holm oak ( Quercus ilex L.) and other species lacking specific foliar terpene storage structures and their development is dependent on light and temperature. In the present work we describe a process-based emission model taking into account the physiological/phenological state of Holm oak leaves and biochemical processes leading to the formation of monoterpenes. The model 'seasonal isoprenoid synthase model-biochemical isoprenoid biosynthesis model' (SIM-BIM2) is developed based on a previous version which was used to simulate isoprene emissions from deciduous oaks. The current model considers additional enzymatic reactions in Holm oak chloroplasts that lead to the formation of monoterpenes. The comparison of simulated and measured biochemical properties as well as emission rates displayed that the ability of the model to dynamically adjust monoterpene biosynthesis capacity by modulating the amount of monoterpene synthase activities in dependence of the weather pattern led to realistic simulations of light-dependent monoterpene emission rates. Differences to simulation results obtained by a widely used alternative model [Guenther, A.B., Zimmerman, P.R., Harley, P.C., Monson, R.K., Fall, R., 1993. Isoprene and monoterpene emission rate variability—model evaluations and sensitivity analyses. Journal of Geophysical Research 98, 12609-12617] are discussed.

  11. Conversion of Isoprenoid Oil by Catalytic Cracking and Hydrocracking over Nanoporous Hybrid Catalysts

    PubMed Central

    Kimura, Toshiyuki; Liu, Chen; Li, Xiaohong; Maekawa, Takaaki; Asaoka, Sachio

    2012-01-01

    In order to produce petroleum alternatives from biomass, a significant amount of research has been focused on oils from microalgae due to their origin, which would not affect food availability. Nanoporous hybrid catalysts composed of ns Al2O3 and zeolites have been proven to be very useful compared to traditional catalysts in hydrotreating (HT), hydrocracking (HC), and catalytic cracking (CC) of large molecules. To evaluate the reaction scheme and products from model isoprenoid compounds of microalgae oil, nanoporous hybrid catalyst technologies (CC: ns Al2O3/H-USY and ns Al2O3/H-GaAlMFI; HC: [Ni-Mo/γ-Al2O3]/ns Al2O3/H-beta) were studied. The major product from CC on ns Al2O3/H-USY was highly aromatic gasoline, while the product from HC was half-isoparaffinic/olefinic kerosene. Although more than 50 wt% of the products from HT/CC on the USY catalyst was liquefied petroleum gas due to overcracking, the product from HT/CC on the MFI catalyst was high-octane-number gasoline. Delightfully, the product from HT/HC was kerosene and its average number was 11, with more than 80 wt% being isoparaffinic. As a result, it was demonstrated that hydrotreating may convert isoprenoid oil from microalgae over nanoporous hybrid catalysts into a variety of products. PMID:22791962

  12. Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation

    PubMed Central

    DeGraw, Amanda J.; Palsuledesai, Charuta; Ochocki, Joshua D.; Dozier, Jonathan K.; Lenevich, Stepan; Rashidian, Mohammad; Distefano, Mark D.

    2010-01-01

    Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here we investigate the utility of alkyne-containing isoprenoid analogues for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed Click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and DFT calculations suggest that the substrate specificity of PFTase may vary depending on whether azide- or alkyne-based isoprenoid analogues are employed. These results demonstrate the utility of alkyne-containing analogues for chemical proteomic applications. PMID:21040496

  13. Improvement of Pharmaceutical Properties of Isoprenoid Compounds through the Formation of Cyclodextrin Pseudorotaxane-Like Supramolecules.

    PubMed

    Higashi, Taishi; Tanaka, Haruki; Yoshimatsu, Ayumi; Ikeda, Haruna; Arima, Kanako; Honjo, Masatoshi; Iwamoto, Chihiro; Motoyama, Keiichi; Arima, Hidetoshi

    2016-01-01

    The purpose of this study was to design cyclodextrin (CyD)-based pseudorotaxane-like supramolecular complexes with various isoprenoid compounds, such as reduced coenzyme Q10 (R-CoQ10), squalene, tocotrienol, and teprenone, and to evaluate their pharmaceutical properties. Squalene, tocotrienol, and teprenone formed precipitates with β-CyD and γ-CyD in aqueous solution, whereas R-CoQ10 formed precipitates with γ-CyD aqueous solution. The results of powder X-ray diffraction and (1)H-NMR analyses indicated that these precipitates are derived from pseudorotaxane-like supramolecular complexes. The photostability of teprenone was markedly improved by complexation with CyDs, especially in the γ-CyD system. In addition, the dispersion rates of teprenone in the γ-CyD system were higher than those in the β-CyD system, compared with the corresponding physical mixtures. In conclusion, pharmaceutical properties such as photostability and dispersion rates of isoprenoid compounds were improved by the formation of pseudorotaxane-like supramolecular complexes with β-CyD and/or γ-CyD. PMID:26852798

  14. Plastid biotechnology for crop production: present status and future perspectives

    PubMed Central

    Daniell, Henry

    2012-01-01

    The world population is expected to reach an estimated 9.2 billion by 2050. Therefore, food production globally has to increase by 70% in order to feed the world, while total arable land, which has reached its maximal utilization, may even decrease. Moreover, climate change adds yet another challenge to global food security. In order to feed the world in 2050, biotechnological advances in modern agriculture are essential. Plant genetic engineering, which has created a new wave of global crop production after the first green revolution, will continue to play an important role in modern agriculture to meet these challenges. Plastid genetic engineering, with several unique advantages including transgene containment, has made significant progress in the last two decades in various biotechnology applications including development of crops with high levels of resistance to insects, bacterial, fungal and viral diseases, different types of herbicides, drought, salt and cold tolerance, cytoplasmic male sterility, metabolic engineering, phytoremediation of toxic metals and production of many vaccine antigens, biopharmaceuticals and biofuels. However, useful traits should be engineered via chloroplast genomes of several major crops. This review provides insight into the current state of the art of plastid engineering in relation to agricultural production, especially for engineering agronomic traits. Understanding the bottleneck of this technology and challenges for improvement of major crops in a changing climate are discussed. PMID:21437683

  15. Chloroplast unfolded protein response, a new plastid stress signaling pathway?

    PubMed

    Ramundo, Silvia; Rochaix, Jean-David

    2014-01-01

    A unique feature of the ATP-dependent ClpP protease of eukaryotic photosynthetic organisms is that its catalytic subunit ClpP1 is encoded by the chloroplast genome. Attempts to inactivate this subunit through chloroplast transformation have failed because it is essential for cell survival. To study the function of ClpP we have developed a repressible chloroplast gene expression system in Chlamydomonas reinhardtii. This system is based on the use of a chimeric nuclear gene in which the vitamin-repressible MetE promoter and Thi4 riboswitch have been fused to the coding sequence of Nac2. Upon entry into the chloroplast the Nac2 protein specifically interacts with the psbD 5'UTR and is required for the proper processing/translation of the psbD mRNA. This property can be conveyed to any chloroplast mRNA by replacing its 5'UTR with that of psbD. In this study we have chosen clpP1 as plastid target gene and examined the cellular events induced upon depletion of ClpP through transcriptomic, proteomic, biochemical and electron microscope analysis. Among the most striking features, a massive increase in protein abundance occurs for plastid chaperones, proteases and proteins involved in membrane assembly/disassembly strongly suggesting the existence of a chloroplast unfolded protein response. PMID:25482768

  16. Rice Debranching Enzyme Isoamylase3 Facilitates Starch Metabolism and Affects Plastid Morphogenesis

    PubMed Central

    Yun, Min-Soo; Umemoto, Takayuki; Kawagoe, Yasushi

    2011-01-01

    Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3–green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice. PMID:21551159

  17. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes

    PubMed Central

    Wheeler, Glen; Ishikawa, Takahiro; Pornsaksit, Varissa; Smirnoff, Nicholas

    2015-01-01

    Ascorbic acid (vitamin C) is an enzyme co-factor in eukaryotes that also plays a critical role in protecting photosynthetic eukaryotes against damaging reactive oxygen species derived from the chloroplast. Many animal lineages, including primates, have become ascorbate auxotrophs due to the loss of the terminal enzyme in their biosynthetic pathway, l-gulonolactone oxidase (GULO). The alternative pathways found in land plants and Euglena use a different terminal enzyme, l-galactonolactone dehydrogenase (GLDH). The evolutionary processes leading to these differing pathways and their contribution to the cellular roles of ascorbate remain unclear. Here we present molecular and biochemical evidence demonstrating that GULO was functionally replaced with GLDH in photosynthetic eukaryote lineages following plastid acquisition. GULO has therefore been lost repeatedly throughout eukaryote evolution. The formation of the alternative biosynthetic pathways in photosynthetic eukaryotes uncoupled ascorbate synthesis from hydrogen peroxide production and likely contributed to the rise of ascorbate as a major photoprotective antioxidant. DOI: http://dx.doi.org/10.7554/eLife.06369.001 PMID:25768426

  18. Complete Plastid Genome Sequence of the Basal Asterid Ardisia polysticta Miq. and Comparative Analyses of Asterid Plastid Genomes

    PubMed Central

    Ku, Chuan; Hu, Jer-Ming; Kuo, Chih-Horng

    2013-01-01

    Ardisia is a basal asterid genus well known for its medicinal values and has the potential for development of novel phytopharmaceuticals. In this genus of nearly 500 species, many ornamental species are commonly grown worldwide and some have become invasive species that caused ecological problems. As there is no completed plastid genome (plastome) sequence in related taxa, we sequenced and characterized the plastome of Ardisia polysticta to find plastid markers of potential utility for phylogenetic analyses at low taxonomic levels. The complete A. polysticta plastome is 156,506 bp in length and has gene content and organization typical of most asterids and other angiosperms. We identified seven intergenic regions as potentially informative markers with resolution for interspecific relationships. Additionally, we characterized the diversity of asterid plastomes with respect to GC content, plastome organization, gene content, and repetitive sequences through comparative analyses. The results demonstrated that the genome organizations near the boundaries between inverted repeats (IRs) and single-copy regions (SCs) are polymorphic. The boundary organization found in Ardisia appears to be the most common type among asterids, while six other types are also found in various asterid lineages. In general, the repetitive sequences in genic regions tend to be more conserved, whereas those in noncoding regions are usually lineage-specific. Finally, we inferred the whole-plastome phylogeny with the available asterid sequences. With the improvement in taxon sampling of asterid orders and families, our result highlights the uncertainty of the position of Gentianales within euasterids I. PMID:23638113

  19. A Plastid without a Genome: Evidence from the Nonphotosynthetic Green Algal Genus Polytomella1[W][OPEN

    PubMed Central

    Smith, David Roy; Lee, Robert W.

    2014-01-01

    Polytomella spp. are free-living, nonphotosynthetic green algae closely related to the model organism Chlamydomonas reinhardtii. Although colorless, Polytomella spp. have a plastid, but it is still unknown whether they harbor a plastid genome. We took a next generation sequencing approach, along with transcriptome sequencing, to search for a plastid genome and an associated gene expression system in Polytomella spp. Illumina sequencing of total DNA from four Polytomella spp. did not produce any recognizable plastid-derived reads but did generate a large number of mitochondrial DNA sequences. Transcriptomic analysis of Polytomella parva uncovered hundreds of putative nuclear-encoded, plastid-targeted proteins, which support the presence of plastid-based metabolic functions, similar to those observed in the plastids of other nonphotosynthetic algae. Conspicuously absent, however, were any plastid-targeted proteins involved in the expression, replication, or repair of plastid DNA. Based on these findings and earlier findings, we argue that the Polytomella genus represents the first well-supported example, to our knowledge, of a primary plastid-bearing lineage without a plastid genome. PMID:24563281

  20. Direct Estimate of the Spontaneous Mutation Rate Uncovers the Effects of Drift and Recombination in the Chlamydomonas reinhardtii Plastid Genome.

    PubMed

    Ness, Rob W; Kraemer, Susanne A; Colegrave, Nick; Keightley, Peter D

    2016-03-01

    Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input of de novo mutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation in Chlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic data set of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies in C. reinhardtii and demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome. PMID:26615203

  1. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

    PubMed Central

    Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  2. Is ftsH the Key to Plastid Longevity in Sacoglossan Slugs?

    PubMed Central

    de Vries, Jan; Habicht, Jörn; Woehle, Christian; Huang, Changjie; Christa, Gregor; Wägele, Heike; Nickelsen, Jörg; Martin, William F.; Gould, Sven B.

    2013-01-01

    Plastids sequestered by sacoglossan sea slugs have long been a puzzle. Some sacoglossans feed on siphonaceous algae and can retain the plastids in the cytosol of their digestive gland cells. There, the stolen plastids (kleptoplasts) can remain photosynthetically active in some cases for months. Kleptoplast longevity itself challenges current paradigms concerning photosystem turnover, because kleptoplast photosystems remain active in the absence of nuclear algal genes. In higher plants, nuclear genes are essential for plastid maintenance, in particular, for the constant repair of the D1 protein of photosystem II. Lateral gene transfer was long suspected to underpin slug kleptoplast longevity, but recent transcriptomic and genomic analyses show that no algal nuclear genes are expressed from the slug nucleus. Kleptoplast genomes themselves, however, appear expressed in the sequestered state. Here we present sequence data for the chloroplast genome of Acetabularia acetabulum, the food source of the sacoglossan Elysia timida, which can maintain Acetabularia kleptoplasts in an active state for months. The data reveal what might be the key to sacoglossan kleptoplast longevity: plastids that remain photosynthetically active within slugs for periods of months share the property of encoding ftsH, a D1 quality control protease that is essential for photosystem II repair. In land plants, ftsH is always nuclear encoded, it was transferred to the nucleus from the plastid genome when Charophyta and Embryophyta split. A replenishable supply of ftsH could, in principle, rescue kleptoplasts from D1 photodamage, thereby influencing plastid longevity in sacoglossan slugs. PMID:24336424

  3. Does complete plastid genome sequencing improve species discrimination and phylogenetic resolution in Araucaria?

    PubMed

    Ruhsam, Markus; Rai, Hardeep S; Mathews, Sarah; Ross, T Gregory; Graham, Sean W; Raubeson, Linda A; Mei, Wenbin; Thomas, Philip I; Gardner, Martin F; Ennos, Richard A; Hollingsworth, Peter M

    2015-09-01

    Obtaining accurate phylogenies and effective species discrimination using a small standardized set of plastid genes is challenging in evolutionarily young lineages. Complete plastid genome sequencing offers an increasingly easy-to-access source of characters that helps address this. The usefulness of this approach, however, depends on the extent to which plastid haplotypes track morphological species boundaries. We have tested the power of complete plastid genomes to discriminate among multiple accessions of 11 of 13 New Caledonian Araucaria species, an evolutionarily young lineage where the standard DNA barcoding approach has so far failed and phylogenetic relationships have remained elusive. Additionally, 11 nuclear gene regions were Sanger sequenced for all accessions to ascertain the success of species discrimination using a moderate number of nuclear genes. Overall, fewer than half of the New Caledonian Araucaria species with multiple accessions were monophyletic in the plastid or nuclear trees. However, the plastid data retrieved a phylogeny with a higher resolution compared to any previously published tree of this clade and supported the monophyly of about twice as many species and nodes compared to the nuclear data set. Modest gains in discrimination thus are possible, but using complete plastid genomes or a small number of nuclear genes in DNA barcoding may not substantially raise species discriminatory power in many evolutionarily young lineages. The big challenge therefore remains to develop techniques that allow routine access to large numbers of nuclear markers scaleable to thousands of individuals from phylogenetically disparate sample sets. PMID:25611173

  4. Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

    PubMed

    Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

    2016-01-01

    Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

  5. ATP-dependent molecular chaperones in plastids--More complex than expected.

    PubMed

    Trösch, Raphael; Mühlhaus, Timo; Schroda, Michael; Willmund, Felix

    2015-09-01

    Plastids are a class of essential plant cell organelles comprising photosynthetic chloroplasts of green tissues, starch-storing amyloplasts of roots and tubers or the colorful pigment-storing chromoplasts of petals and fruits. They express a few genes encoded on their organellar genome, called plastome, but import most of their proteins from the cytosol. The import into plastids, the folding of freshly-translated or imported proteins, the degradation or renaturation of denatured and entangled proteins, and the quality-control of newly folded proteins all require the action of molecular chaperones. Members of all four major families of ATP-dependent molecular chaperones (chaperonin/Cpn60, Hsp70, Hsp90 and Hsp100 families) have been identified in plastids from unicellular algae to higher plants. This review aims not only at giving an overview of the most current insights into the general and conserved functions of these plastid chaperones, but also into their specific plastid functions. Given that chloroplasts harbor an extreme environment that cycles between reduced and oxidized states, that has to deal with reactive oxygen species and is highly reactive to environmental and developmental signals, it can be presumed that plastid chaperones have evolved a plethora of specific functions some of which are just about to be discovered. Here, the most urgent questions that remain unsolved are discussed, and guidance for future research on plastid chaperones is given. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25596449

  6. Engineering plastid genomes: methods, tools, and applications in basic research and biotechnology.

    PubMed

    Bock, Ralph

    2015-01-01

    The small bacterial-type genome of the plastid (chloroplast) can be engineered by genetic transformation, generating cells and plants with transgenic plastid genomes, also referred to as transplastomic plants. The transformation process relies on homologous recombination, thereby facilitating the site-specific alteration of endogenous plastid genes as well as the precisely targeted insertion of foreign genes into the plastid DNA. The technology has been used extensively to analyze chloroplast gene functions and study plastid gene expression at all levels in vivo. Over the years, a large toolbox has been assembled that is now nearly comparable to the techniques available for plant nuclear transformation and that has enabled new applications of transplastomic technology in basic and applied research. This review describes the state of the art in engineering the plastid genomes of algae and land plants (Embryophyta). It provides an overview of the existing tools for plastid genome engineering, discusses current technological limitations, and highlights selected applications that demonstrate the immense potential of chloroplast transformation in several key areas of plant biotechnology. PMID:25494465

  7. Transgenic maize lines with cell-type specific expression of fluorescent proteins in plastids.

    PubMed

    Sattarzadeh, Amir; Fuller, Jonathan; Moguel, Salvador; Wostrikoff, Katia; Sato, Shirley; Covshoff, Sarah; Clemente, Tom; Hanson, Maureen; Stern, David B

    2010-02-01

    Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated. PMID:20051034

  8. Is ftsH the key to plastid longevity in sacoglossan slugs?

    PubMed

    de Vries, Jan; Habicht, Jörn; Woehle, Christian; Huang, Changjie; Christa, Gregor; Wägele, Heike; Nickelsen, Jörg; Martin, William F; Gould, Sven B

    2013-01-01

    Plastids sequestered by sacoglossan sea slugs have long been a puzzle. Some sacoglossans feed on siphonaceous algae and can retain the plastids in the cytosol of their digestive gland cells. There, the stolen plastids (kleptoplasts) can remain photosynthetically active in some cases for months. Kleptoplast longevity itself challenges current paradigms concerning photosystem turnover, because kleptoplast photosystems remain active in the absence of nuclear algal genes. In higher plants, nuclear genes are essential for plastid maintenance, in particular, for the constant repair of the D1 protein of photosystem II. Lateral gene transfer was long suspected to underpin slug kleptoplast longevity, but recent transcriptomic and genomic analyses show that no algal nuclear genes are expressed from the slug nucleus. Kleptoplast genomes themselves, however, appear expressed in the sequestered state. Here we present sequence data for the chloroplast genome of Acetabularia acetabulum, the food source of the sacoglossan Elysia timida, which can maintain Acetabularia kleptoplasts in an active state for months. The data reveal what might be the key to sacoglossan kleptoplast longevity: plastids that remain photosynthetically active within slugs for periods of months share the property of encoding ftsH, a D1 quality control protease that is essential for photosystem II repair. In land plants, ftsH is always nuclear encoded, it was transferred to the nucleus from the plastid genome when Charophyta and Embryophyta split. A replenishable supply of ftsH could, in principle, rescue kleptoplasts from D1 photodamage, thereby influencing plastid longevity in sacoglossan slugs. PMID:24336424

  9. Genome-wide gene expression profiles in response to plastid division perturbations.

    PubMed

    Maple, Jodi; Winge, Per; Tveitaskog, Astrid Elisabeth; Gargano, Daniela; Bones, Atle M; Møller, Simon Geir

    2011-11-01

    Plastids are vital organelles involved in important metabolic functions that directly affect plant growth and development. Plastids divide by binary fission involving the coordination of numerous protein components. A tight control of the plastid division process ensures that: there is a full plastid complement during and after cell division, specialized cell types have optimal plastid numbers; the division rate is modulated in response to stress, metabolic fluxes and developmental status. However, how this control is exerted by the host nucleus is unclear. Here, we report a genome-wide microarray analysis of three accumulation and replication of chloroplasts (arc) mutants that show a spectrum of altered plastid division characteristics. To ensure a comprehensive data set, we selected arc3, arc5 and arc11 because they harbour mutations in protein components of both the stromal and cytosolic division machinery, are of different evolutionary origin and display different phenotypic severities in terms of chloroplast number, size and volume. We show that a surprisingly low number of genes are affected by altered plastid division status, but that the affected genes encode proteins important for a variety of fundamental plant processes. PMID:21713643

  10. Massive difference in synonymous substitution rates among mitochondrial, plastid, and nuclear genes of Phaeocystis algae.

    PubMed

    Smith, David Roy; Arrigo, Kevin R; Alderkamp, Anne-Carlijn; Allen, Andrew E

    2014-02-01

    We are just beginning to understand how mutation rates differ among mitochondrial, plastid, and nuclear genomes. In most seed plants the mitochondrial mutation rate is estimated to be lower than those of the plastid and nucleus, whereas in the red alga Porphyra the opposite is true, and in certain green algae all three genomes appear to have similar rates of mutation. Relative rate statistics of organelle vs nuclear genes, however, are lacking for lineages that acquired their plastids through secondary endosymbiosis, but recent organelle DNA analyses suggest that they may differ drastically from what is observed in lineages with primary plastids, such as green plants and red algae. Here, by measuring synonymous nucleotide substitutions, we approximate the relative mutation rates within the haptophyte genus Phaeocystis, which has a red-algal-derived, secondary plastid. Synonymous-site divergence data indicate that for Phaeocystis antarctica and P. globosa the mitochondrial mutation rate is 10 and 3 times that of the plastid and nucleus, respectively. This differs drastically from relative rate estimates for primary-plastid-bearing lineages and presents a much more dynamic view of organelle vs nuclear mutation rates across the eukaryotic domain. PMID:24216019