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Stefan Martens - One of the best experts on this subject based on the ideXlab platform.

  • metabolomic and genetic analyses of flavonol synthesis in arabidopsis thaliana support the in vivo involvement of Leucoanthocyanidin dioxygenase
    Planta, 2009
    Co-Authors: Ralf Stracke, Stefan Martens, Lutz Bartelniewoehner, Hirofumi Ishihara, Martin Sagasser, Bernd Weisshaar
    Abstract:

    Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a Leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme.

  • characterization of major enzymes and genes involved in flavonoid and proanthocyanidin biosynthesis during fruit development in strawberry fragaria ananassa
    Archives of Biochemistry and Biophysics, 2007
    Co-Authors: Joao R M Almeida, Eleonora Damico, Anja Preuss, Fabrizio Carbone, Bettina Deiml, Fabienne Mourgues, Gaetano Perrotta, Thilo C Fischer, Arnaud Bovy, Stefan Martens
    Abstract:

    The biosynthesis of flavonoids and proanthocyanidins was studied in cultivated strawberry (Fragaria x ananassa) by combining biochemical and molecular approaches. Chemical analyses showed that ripe strawberries accumulate high amounts of pelargonidin-derived anthocyanins, and a larger pool of 3',4'-hydroxylated proanthocyanidins. Activities and properties of major recombinant enzymes were demonstrated by means of in vitro assays, with special emphasis on specificity for the biologically relevant 4'- and 3',4'-hydroxylated compounds. Only Leucoanthocyanidin reductase showed a strict specificity for the 3',4'-hydroxylated leucocyanidin, while other enzymes accepted either hydroxylated substrate with different relative activity rates. The structure of late flavonoid pathway genes, leading to the synthesis of major compounds in ripe fruits, was elucidated. Complex developmental and spatial expression patterns were shown for phenylpropanoid and flavonoid genes in fruits throughout ripening as well as in leaves, petals and roots. Presented results elucidate key steps in the biosynthesis of strawberry flavonoid end products. (c) 2007 Elsevier Inc. All rights reserved.

  • ectopic expression of a basic helix loop helix gene transactivates parallel pathways of proanthocyanidin biosynthesis structure expression analysis and genetic control of Leucoanthocyanidin 4 reductase and anthocyanidin reductase genes in lotus corni
    Plant Physiology, 2006
    Co-Authors: Francesco Paolocci, Stefan Martens, Mark P Robbins, Laura Madeo, Sergio Arcioni, F Damiani
    Abstract:

    Proanthocyanidins (PAs) are plant secondary metabolites and are composed primarily of catechin and epicatechin units in higher plant species. Due to the ability of PAs to bind reversibly with plant proteins to improve digestion and reduce bloat, engineering this pathway in leaves is a major goal for forage breeders. Here, we report the cloning and expression analysis of anthocyanidin reductase (ANR) and Leucoanthocyanidin 4-reductase (LAR), two genes encoding enzymes committed to epicatechin and catechin biosynthesis, respectively, in Lotus corniculatus. We show the presence of two LAR gene families (LAR1 and LAR2) and that the steady-state levels of ANR and LAR1 genes correlate with the levels of PAs in leaves of wild-type and transgenic plants. Interestingly, ANR and LAR1, but not LAR2, genes produced active proteins following heterologous expression in Escherichia coli and are affected by the same basic helix-loop-helix transcription factor that promotes PA accumulation in cells of palisade and spongy mesophyll. This study provides direct evidence that the same subclass of transcription factors can mediate the expression of the structural genes of both branches of PA biosynthesis.

  • Heterologous expression of dihydroflavonol 4‐reductases from various plants
    FEBS Letters, 2002
    Co-Authors: Stefan Martens, Teemu H. Teeri, Gert Forkmann
    Abstract:

    Abstract Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of dihydroflavonols to the respective flavan 3,4-diols (Leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway. Several cDNA clones encoding DFR have been isolated from different plant species. Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far. Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids. Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker’s yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR. These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences. Furthermore, this tool allows the stereospecific synthesis of 14C-labeled Leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.

Simon P. Robinson - One of the best experts on this subject based on the ideXlab platform.

  • proanthocyanidin synthesis and expression of genes encoding Leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves
    Plant Physiology, 2005
    Co-Authors: Jochen Bogs, Mark O Downey, John S Harvey, Anthony R Ashton, Gregory J Tanner, Simon P. Robinson
    Abstract:

    Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and Leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

  • Analysis of the Expression of Anthocyanin Pathway Genes
    Plant physiology, 1996
    Co-Authors: Paul K. Boss, Christopher Davies, Simon P. Robinson
    Abstract:

    Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 1 O weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3Hl, dihydroflavonol 4-reductase [DFR], Leucoanthocyanidin dioxygen- ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFCT]) was determined. In flowers and grape berry skins, expres- sion of all of the genes, except UFCT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFCT then increased 1 O weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFCT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFCT is regulated independently of the other genes, suggest- ing that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.

Andrew C. Allan - One of the best experts on this subject based on the ideXlab platform.

  • peach myb7 activates transcription of the proanthocyanidin pathway gene encoding Leucoanthocyanidin reductase but not anthocyanidin reductase
    Frontiers in Plant Science, 2015
    Co-Authors: Hui Zhou, Andrew C. Allan, Kui Linwang, Liao Liao, Chao Gu, Ziqi Lu
    Abstract:

    Proanthocyanidins (PAs) are a group of natural phenolic compounds that have a great effect on both flavour and nutritious value of fruit. It has been shown that PA synthesis is regulated by R2R3-MYB transcription factors (TFs) via activation of PA-specific pathway genes encoding Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Here, we report the isolation and characterization of a MYB gene designated PpMYB7 in peach. The peach PpMYB7 represents a new group of R2R3-MYB genes regulating PA synthesis in plants. It is able to activate transcription of PpLAR1 but not PpANR, and has a broader selection of potential bHLH partners compared with PpMYBPA1. Transcription of PpMYB7 can be activated by the peach basic leucine-zipper 5 TF (PpbZIP5) via response to ABA. Our study suggests a transcriptional network regulating PA synthesis in peach, with the results aiding the understanding of the functional divergence between R2R3-MYB TFs in plants.

  • Transcriptional analysis of apple fruit proanthocyanidin biosynthesis
    Journal of Experimental Botany, 2012
    Co-Authors: Rebecca A. Henry-kirk, Tony K. Mcghie, Christelle M. Andre, Roger P. Hellens, Andrew C. Allan
    Abstract:

    Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple. Abbreviations: ANOVA analysis of variance ANR anthocyanidin reductase DAD diode array detector DAFB days after full bloom DFR dihydroflavonol reductase LAR Leucoanthocyanidin reductase LC-MS liquid chromatography/mass spectrometry PA proanthocyanidin qPCR real-time quantitative PCR

Lijun Wang - One of the best experts on this subject based on the ideXlab platform.

  • Isolation and Characterization of cDNAs Encoding Leucoanthocyanidin Reductase and Anthocyanidin Reductase from Populus trichocarpa
    PLOS ONE, 2013
    Co-Authors: Lijun Wang, Li Yuan, Yuanzhong Jiang, Li Yang, Wanxiang Lu, Abdul Karim
    Abstract:

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (-)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P

  • isolation and characterization of cdnas encoding Leucoanthocyanidin reductase and anthocyanidin reductase from populus trichocarpa
    PLOS ONE, 2013
    Co-Authors: Li Yuan, Lijun Wang, Yuanzhong Jiang, Li Yang, Wanxiang Lu, Abdul Karim
    Abstract:

    Proanthocyanidins (PAs) contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA) and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) are two key enzymes of the PA biosynthesis that produce the main subunits: (+)-catechin and (-)-epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05) in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  • molecular cloning and characterization of ptrlar3 a gene encoding Leucoanthocyanidin reductase from populus trichocarpa and its constitutive expression enhances fungal resistance in transgenic plants
    Journal of Experimental Botany, 2012
    Co-Authors: Li Yuan, Lijun Wang, Yuanzhong Jiang, Lili Zhao, Li Yang
    Abstract:

    The flavonoid-derived proanthocyanidins (PAs) are one class of the major defence phenolics in poplar leaves. Transcriptional activation of PA biosynthetic genes, resulting in PA accumulation in leaves, was detected following infection by the fungal Marssonina brunnea f.sp. multigermtubi using digital gene expression analysis. In order to study PA biosynthesis and its induction by fungi, a putative Leucoanthocyanidin reductase gene, PtrLAR3, was isolated from Populus trichocarpa. Sequence comparison of PtrLAR3 with other known Leucoanthocyanidin reductase proteins revealed high amino acid sequence similarity. Semi-quantitative reverse-transcription (RT) PCR and quantitative real-time PCR analysis demonstrated that PtrLAR3 was expressed in various tissues and the highest level of expression was observed in roots. Overexpression of PtrLAR3 in Chinese white poplar (Populus tomentosa Carr.) led to a significant plant-wide increase in PA levels. In vitro assays showed that crude leaf extracts from 35S:PtrLAR3 transformants were able to inhibit significantly the hyphal growth of M. brunnea f.sp. multigermtubi compared to the extracts from control plants. The transgenic 35S:PtrLAR3 poplar plants displayed a significant (P < 0.05) reduction in their disease symptoms compared with the control. RT-PCR analysis showed that PtrLAR3 expression was up-regulated in all transformants. These results suggested that constitutive expression of endogenous PtrLAR3 could be exploited to improve resistance to fungal pathogens in poplar.

Anthony R Ashton - One of the best experts on this subject based on the ideXlab platform.

  • proanthocyanidin synthesis and expression of genes encoding Leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves
    Plant Physiology, 2005
    Co-Authors: Jochen Bogs, Mark O Downey, John S Harvey, Anthony R Ashton, Gregory J Tanner, Simon P. Robinson
    Abstract:

    Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, Leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and Leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

  • the arabidopsis tds4 gene encodes Leucoanthocyanidin dioxygenase ldox and is essential for proanthocyanidin synthesis and vacuole development
    Plant Journal, 2003
    Co-Authors: Sharon Abrahams, Gregory J Tanner, Amanda R Walker, P J Larkin, Anthony R Ashton
    Abstract:

    Summary The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by Leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme Leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.

  • proanthocyanidin biosynthesis in plants purification of legume Leucoanthocyanidin reductase and molecular cloning of its cdna
    Journal of Biological Chemistry, 2003
    Co-Authors: Gregory J Tanner, Sharon Abrahams, P J Larkin, Kathy T Francki, John M Watson, Anthony R Ashton
    Abstract:

    Abstract Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of ∼10 μmol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known. Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome. This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.