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Peter M. Bramley - One of the best experts on this subject based on the ideXlab platform.
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Proteome changes in tomato lines transformed with Phytoene Synthase-1 in the sense and antisense orientations
Journal of Experimental Botany, 2012Co-Authors: F Robertson, P. Kaisa Koistinen, Christopher Gerrish, John M. Halket, Raj K. P. Patel, Paul D. Fraser, Peter M. BramleyAbstract:The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene Phytoene Synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.
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Increases in cell elongation, plastid compartment size and Phytoene Synthase activity underlie the phenotype of the high pigment-1 mutant of tomato
Planta, 2003Co-Authors: P. J. Cookson, Paul D. Fraser, Peter M. Bramley, Cathie A. Shipton, J. W. Kiano, S. Romer, W. Schuch, Kevin A. PykeAbstract:A characteristic trait of the high pigment-1 (hp-1) mutant phenotype of tomato (Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and Phytoene Synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of Phytoene Synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of Phytoene Synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.
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Evaluation of transgenic tomato plants expressing an additional Phytoene Synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene Synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene Synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene Synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene Synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate Synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene Synthase.
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Phytoene Synthase from tomato (Lycopersicon esculentum) chloroplasts--partial purification and biochemical properties.
Planta, 2000Co-Authors: Paul D. Fraser, Wolfgang Schuch, Peter M. BramleyAbstract:Phytoene Synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, Phytoene Synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although Phytoene Synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) Synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with Km values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of Phytoene Synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences.
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Phytoene Synthase-2 enzyme activity in tomato does not contribute to carotenoid synthesis in ripening fruit.
Plant molecular biology, 1999Co-Authors: Paul D. Fraser, Wolfgang Schuch, Joy W. Kiano, Mark R. Truesdale, Peter M. BramleyAbstract:The characteristic yellow fruit phenotype of the r,r mutant and Psy-1 (Phytoene Synthase-1) antisense tomatoes is due to a mutated or down-regulated Phytoene Synthase protein, respectively, resulting in the virtual absence of carotenoids. Based on detailed carotenoid determinations Psy-1 appeared to barely contribute to the formation of carotenoids in chloroplast-containing tissues. Despite the virtual absence of carotenoids in ripe fruit the formation of Phytoene in vitro was detected in fruit of both mutants. When [14C]isopentenyl pyrophosphate (IPP) was used as the substrate for Phytoene Synthase a reduction (e.g. r,r mutant, 5-fold) in the formation of Phytoene was observed with an accumulation (e.g. r,r mutant, 2-fold) of the immediate precursor geranylgeranyl pyrophosphate (GGPP). Contrastingly, reduced Phytoene Synthase activity was not detected when [3H]GGPP was used as the substrate. The profile of Phytoene formation during ripening was also different in the down-regulated mutants compared to the wild-type. Using specific primers, RT-PCR analysis detected the presence of Psy-2 transcripts in the down-regulated mutants and wild-type throughout fruit development and ripening. These data were supported by the detection of Phytoene Synthase protein on western blots. Both GGPP formation and Phytoene desaturation were elevated in these mutants. Therefore, it appears that despite the absence of carotenoids in ripe fruit, both the mutants have the enzymic capability to synthesize carotenoids in this tissue. Implications of the data with respect to the regulation of carotenoid formation and the channelling of prenyl lipid precursors in tomato (and its potential manipulation) are discussed.
Paul D. Fraser - One of the best experts on this subject based on the ideXlab platform.
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Proteome changes in tomato lines transformed with Phytoene Synthase-1 in the sense and antisense orientations
Journal of Experimental Botany, 2012Co-Authors: F Robertson, P. Kaisa Koistinen, Christopher Gerrish, John M. Halket, Raj K. P. Patel, Paul D. Fraser, Peter M. BramleyAbstract:The commercial cultivation of genetically engineered (GE) crops in Europe has met with considerable consumer resistance, which has led to vigorous safety assessments including the measurement of substantial equivalence between the GE and parent lines. This necessitates the identification and quantification of significant changes to the metabolome and proteome in the GE crop. In this study, the quantitative proteomic analysis of tomato fruit from lines that have been transformed with the carotenogenic gene Phytoene Synthase-1 (Psy-1), in the sense and antisense orientations, in comparison with a non-transformed, parental line is described. Multidimensional protein identification technology (MudPIT), with tandem mass spectrometry, has been used to identify proteins, while quantification has been carried out with isobaric tags for relative and absolute quantification (iTRAQ). Fruit from the GE plants showed significant alterations to their proteomes compared with the parental line, especially those from the Psy-1 sense transformants. These results demonstrate that MudPIT and iTRAQ are suitable techniques for the verification of substantial equivalence of the proteome in GE crops.
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carotenoid synthesis and Phytoene Synthase activity during mating of blakeslea trispora
Phytochemistry, 2012Co-Authors: Jurgen Breitenbach, Paul D. Fraser, Gerhard SandmannAbstract:Carotenoid formation was investigated in wild type and carotenogenic mutants of Blakeslea trispora after mating (-) and (+) strains. The highest yields of carotenoids, especially β-carotene was observed following mating. In vitro incorporation of geranylgeranyl pyrophosphate into Phytoene and β-carotene corresponded to increased carotenogenesis in the mated strains. Immuno determination of Phytoene Synthase protein levels revealed that the amounts of this enzyme is concurrent with the increases in carotenoid content. In fungi, Phytoene Synthase together with lycopene cyclase are encoded by a fusion gene crtYB or carRA with two individual domains. These domains were both heterologously expressed in an independent manner and antisera raised against both. These antisera were used, to assess protein levels in mated and non-mated B. trispora. The Phytoene Synthase domain was detected as an individual soluble protein with a molecular weight of 40 kDa and the lycopene cyclase an individual protein of mass about 30 kDa present in the membrane fraction following sub-cellular fractionation. This result demonstrates a post-translational cleavage of the protein transcribed from a single mRNA into independent functional Phytoene Synthase and lycopene cyclase.
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Increases in cell elongation, plastid compartment size and Phytoene Synthase activity underlie the phenotype of the high pigment-1 mutant of tomato
Planta, 2003Co-Authors: P. J. Cookson, Paul D. Fraser, Peter M. Bramley, Cathie A. Shipton, J. W. Kiano, S. Romer, W. Schuch, Kevin A. PykeAbstract:A characteristic trait of the high pigment-1 (hp-1) mutant phenotype of tomato (Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and Phytoene Synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of Phytoene Synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of Phytoene Synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.
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Evaluation of transgenic tomato plants expressing an additional Phytoene Synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene Synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene Synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene Synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene Synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate Synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene Synthase.
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Phytoene Synthase from tomato (Lycopersicon esculentum) chloroplasts--partial purification and biochemical properties.
Planta, 2000Co-Authors: Paul D. Fraser, Wolfgang Schuch, Peter M. BramleyAbstract:Phytoene Synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, Phytoene Synthase has been purified 600-fold from tomato (Lycopersicon esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa, with an isoelectric point of 4.6. Although Phytoene Synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) Synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with Km values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 μM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including β-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of Phytoene Synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences.
Norihiko Misawa - One of the best experts on this subject based on the ideXlab platform.
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Enrichment of carotenoids in flaxseed by introducing a bacterial Phytoene Synthase gene.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Masaki Fujisawa, Norihiko MisawaAbstract:Carotenoids are well-known natural pigments, typically ranging from yellow to red. Carotenoids are industrially utilized as functional materials due to their strong antioxidant properties. Phytoene synthesis is known to be a rate-determining step in the entire carotenoid biosynthetic pathway in plants. We show methods of pathway engineering for the enrichment of carotenoids in flaxseed (linseed; Linum usitatissimum L.), which is an industrially important oleaginous crop. A Phytoene Synthase gene (crtB) derived from a soil bacterium Pantoea ananatis (formerly called Erwinia uredovora) strain 20D3 was introduced into L. usitatissimum WARD cultivar. The resulting transgenic flax plants formed orange seeds, which contained Phytoene, alpha-carotene, beta-carotene, and lutein. The total carotenoid amount in the transgenic seeds was 156 microg/g fresh weight at the maximum, corresponding to 18.6-fold increase compared with that of untransformed controls.
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Production of Phytoene, a carotenoid, and induction of connexin 26 in transgenic mice carrying the Phytoene Synthase gene crtB.
Biochemical and biophysical research communications, 2004Co-Authors: Yoshiko Satomi, Norihiko Misawa, Takashi Maoka, Hoyoku NishinoAbstract:Carotenoids have been recognized as chemopreventive agents against human diseases, such as cancer and cardiovascular disease. Mammalians utilize carotenoids supplied from their food since they are unable to perform the de novo synthesis of carotenoids. We previously created mammalian cultured cells producing Phytoene, a type of carotenoid, and showed that these cells acquired resistance against oxidative stress and oncogenic transformation. In the present study, we established a transgenic mouse line, carrying the crtB gene encoding Phytoene Synthase, which could produce Phytoene endogenously. It was found that connexin 26 was induced in these Phytoene-producing mice. Since it is known that carotenoids enhance gap junctional communication by inducing the expression of connexin genes, the present data suggest that the induction of connexin 26 in Phytoene-producing mice may play a role in controlling cell-to-cell communication. Phytoene-producing mice provide a useful system in which to investigate the in vivo function of the carotenoid Phytoene.
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Evaluation of transgenic tomato plants expressing an additional Phytoene Synthase in a fruit-specific manner.
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Paul D. Fraser, Norihiko Misawa, Wolfgang Schuch, Susanne Römer, Cathie A. Shipton, Philippa B. Mills, Joy W. Kiano, Rachel Drake, Peter M. BramleyAbstract:Phytoene Synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato Phytoene Synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T0) were 2–4-fold higher than the controls, whereas Phytoene, lycopene, β-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T1 and T2 generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5–10-fold elevations in total Phytoene Synthase activity. Metabolic control analysis suggests that the presence of an additional Phytoene Synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate Synthase, and incorporation of isopentenyl diphosphate into Phytoene) were not significantly altered by the presence of the bacterial Phytoene Synthase.
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Production and characterisation of monoclonal antibodies to Phytoene Synthase of lycopersicon esculentum
Phytochemistry, 1998Co-Authors: Paul D. Fraser, Gerhard Sandmann, Norihiko Misawa, Julie Johnson, Wolfgang Schuch, Peter M. BramleyAbstract:Abstract Monoclonal antibodies have been prepared against the tomato ( Lycopersicon esculentum Mill.) fruit ripening-enhanced Phytoene Synthase (PSY1). The antigen was prepared as a fn2 -galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli . The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against β -galactosidase- Phytoene Synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature Phytoene Synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to Phytoene Synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG\1a 2 isotypes) inhibited Phytoene Synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit Phytoene Synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial Phytoene Synthases, nor Phytoene Synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of Phytoene Synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.
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Production of Phytoene, an Oxidative Stress Protective Carotenoid, in Mammalian Cells by Introduction of Phytoene Synthase Gene crtB Isolated from a Bacterium Erwinia uredovora
Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences, 1995Co-Authors: Yoshiko Satomi, Norihiko Misawa, Teruhiko Yoshida, Kazunori Aoki, Mitsuharu Masuda, Michiaki Murakoshi, Nobuo Takasuka, Takashi Sugimura, Hoyoku NishinoAbstract:Carotenoids have been known to be effective in preventing a variety of diseases such as cancers and cardiovascular disorders. Mammalian cells cannot synthesize carotenoids and rely on a dietary source for their intake. Here we report the establishment of mammalian cells synthesizing Phytoene, a kind of carotenoid, by introduction of thecrtB gene, Phytoene Synthase gene, which was previously cloned from a bacterium, Erwiuia uredovora. The Phytoene-producing cells acquired a resistance against oxidative stress induced by Fe3+/adenosine 5'-diphosphate. The data suggest the feasibility of a genetic chemoprevention for oxidative stress-related disorders by endogenous synthesis of Phytoene in mammalian cells.
Ralf Welsch - One of the best experts on this subject based on the ideXlab platform.
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Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro.
Methods in molecular biology (Clifton N.J.), 2019Co-Authors: Maurizio Camagna, Ralf WelschAbstract:Phytoene Synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) Synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires 14C-labeled substrate and allows to determine its incorporation into Phytoene as well as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP Synthase homologs from various plant species.
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Carotenogenesis Is Regulated by 5′UTR-Mediated Translation of Phytoene Synthase Splice Variants
Plant physiology, 2016Co-Authors: Daniel Álvarez, Peter Beyer, Florian Wüst, Björn Voß, Dirk Maass, Patrick Schaub, Ralf WelschAbstract:Phytoene Synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of the first biosynthetically formed carotene. While PSY constitutes a small gene family in most plant taxa, the Brassicaceae, including Arabidopsis (Arabidopsis thaliana), predominantly possess a single PSY gene. This monogenic situation is compensated by the differential expression of two alternative splice variants (ASV), which differ in length and in the exon/intron retention of their 5′UTRs. ASV1 contains a long 5′UTR (untranslated region) and is involved in developmentally regulated carotenoid formation, such as during deetiolation. ASV2 contains a short 5′UTR and is preferentially induced when an immediate increase in the carotenoid pathway flux is required, such as under salt stress or upon sudden light intensity changes. We show that the long 5′UTR of ASV1 is capable of attenuating the translational activity in response to high carotenoid pathway fluxes. This function resides in a defined 5′UTR stretch with two predicted interconvertible RNA conformations, as known from riboswitches, which might act as a flux sensor. The translation-inhibitory structure is absent from the short 5′UTR of ASV2 allowing to bypass translational inhibition under conditions requiring rapidly increased pathway fluxes. The mechanism is not found in the rice (Oryza sativa) PSY1 5′UTR, consistent with the prevalence of transcriptional control mechanisms in taxa with multiple PSY genes. The translational control mechanism identified is interpreted in terms of flux adjustments needed in response to retrograde signals stemming from intermediates of the plastid-localized carotenoid biosynthesis pathway.
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Isolation and functional characterisation of banana Phytoene Synthase genes as potential cisgenes
Planta, 2012Co-Authors: Bulukani Mlalazi, Ralf Welsch, Priver Namanya, Harjeet Khanna, R. Jason Geijskes, Mark D. Harrison, Rob Harding, James L. Dale, Marion BatesonAbstract:Carotenoids occur in all photosynthetic organisms where they protect photosystems from auto-oxidation, participate in photosynthetic energy transfer and are secondary metabolites. Of the more than 600 known plant carotenoids, few can be converted into vitamin A by humans and so these pro-vitamin A carotenoids (pVAC) are important in human nutrition. Phytoene Synthase (PSY) is a key enzyme in the biosynthetic pathway of pVACs and plays a central role in regulating pVAC accumulation in the edible portion of crop plants. Banana is a major commercial crop and serves as a staple crop for more than 30 million people. There is natural variation in fruit pVAC content across different banana cultivars, but this is not well understood. Therefore, we isolated PSY genes from banana cultivars with relatively high (cv. Asupina) and low (cv. Cavendish) pVAC content. We provide evidence that PSY in banana is encoded by two paralogs ( PSY1 and PSY2 ), each with a similar gene structure to homologous genes in other monocots. Further, we demonstrate that PSY2 is more highly expressed in fruit pulp compared to leaf. Functional analysis of PSY1 and PSY2 in rice callus and E. coli demonstrates that both genes encode functional enzymes, and that Asupina PSYs have approximately twice the enzymatic activity of the corresponding Cavendish PSYs. These results suggest that differences in PSY enzyme activity contribute significantly to the differences in Asupina and Cavendish fruit pVAC content. Importantly, Asupina PSY genes could potentially be used to generate new cisgenic or intragenic banana cultivars with enhanced pVAC content.
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A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes
Plant physiology, 2008Co-Authors: Ralf Welsch, Salim Al-babili, Florian Wüst, Cornelia Bär, Peter BeyerAbstract:We here report on the characterization of a novel third Phytoene Synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5' untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.
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Structural and functional characterization of the Phytoene Synthase promoter from Arabidopsis thaliana
Planta, 2002Co-Authors: Ralf Welsch, Peter Beyer, Joaquin Medina, Giovanni Giuliano, Johannes Von LintigAbstract:The expression of the gene coding for the carotenogenic enzyme Phytoene Synthase is highly regulated. To study this, its promoter and truncated versions thereof were translationally fused to the luciferase gene as a reporter and these constructs were used to transform Arabidopsis thaliana. The full-length promoter was shown to be active in the dark, but mediated positive responses towards different light qualities (far-red, red, blue and white light). Among the herbicides tested, norflurazon and gabaculine showed no notable effects, while CPTA abolished light induction completely. Response towards different light qualities was mediated by a TATA box-proximal promoter region up to position –300, containing G-box-like elements involved in the distinction of different monochromatic light qualities applied. This is detected in electrophoretic mobility shift assays (EMSAs), which reveal differential complex formation. A TATA box distal region of the promoter was shown to be responsible for a high basal promoter activity that was not modulated by different light qualities. Using EMSAs, a novel cis-acting element ATCTA occurring in tandem between positions –854 and –841 proved to be decisive in this respect. The motif was found in several other promoter regions involved in carotenoid and tocopherol biosynthesis, as well as in the promoter regions mediating the expression of photosynthesis-related genes. The functional equivalence of the motifs was shown by successfully using the respective regions in EMSAs. We conclude that the ATCTA motif represents an element capable of mediating a coordinated regulation of these pathways at the transcriptional level.
Eleanore T. Wurtzel - One of the best experts on this subject based on the ideXlab platform.
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The Phytoene Synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content
BMC plant biology, 2015Co-Authors: Charles Ampomah-dwamena, Eleanore T. Wurtzel, Nicky Driedonks, David H. Lewis, Maria Shumskaya, Xiuyin Chen, Richard V. Espley, Andrew C. AllanAbstract:Background Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme Phytoene Synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation.
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plastid localization of the key carotenoid enzyme Phytoene Synthase is altered by isozyme allelic variation and activity
The Plant Cell, 2012Co-Authors: Maria Shumskaya, Louis M T Bradbury, Regina Monaco, Eleanore T. WurtzelAbstract:Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene Synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zea mays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis.
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the Phytoene Synthase gene family in the grasses subfunctionalization provides tissue specific control of carotenogenesis
Plant Signaling & Behavior, 2009Co-Authors: Faqiang Li, Oren Tsfadia, Eleanore T. WurtzelAbstract:Carotenoids are a complex class of isoprenoid pigments playing diverse roles in plants and providing nutritional value. Metabolic engineering of the biosynthetic pathway has been of interest to specifically address global vitamin A deficiency by breeding cereal crop staples in the Poaceae (Grass family) for elevated levels of provitamin A carotenoids. However, there remain open questions about the rate-controlling steps that limit predictability of metabolic engineering in plants, whether by transgenic or nontransgenic means. We decided to focus on the first committed biosynthetic step which is mediated by Phytoene Synthase. Our studies revealed that in the Grasses, PSY is encoded by three genes. Maize transcript profiling, together with carotenoid and ABA analysis, revealed that the three PSY copies have subfunctionalized and provide the Grasses with a fine tine control of carotenogenesis in response to various developmental and external cues. Promoter analysis supports subfunctionalization; cis-element analysis of maize PSY1 alleles and comparison with Grass orthologs suggests that man’s selection of yellow maize endosperm has occurred at the expense of a change of gene regulation in photosynthetic tissue as compared to the progenitor white endosperm PSY1 allele.