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1.
J Exp Bot ; 67(11): 3537-50, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27241494

RESUMEN

In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, ß-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.


Asunto(s)
Oxidorreductasas de Alcohol/genética , Ácido Gálico/metabolismo , Proteínas de Plantas/genética , Vitis/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Escherichia coli/genética , Organismos Modificados Genéticamente/crecimiento & desarrollo , Filogenia , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ADN , Vitis/enzimología , Vitis/metabolismo
2.
New Phytol ; 201(3): 795-809, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24147899

RESUMEN

Flavonoids are secondary metabolites with multiple functions. In grape (Vitis vinifera), the most abundant flavonoids are proanthocyanidins (PAs), major quality determinants for fruit and wine. However, knowledge about the regulation of PA composition is sparse. Thus, we aimed to identify novel genomic regions involved in this mechanism. Expression quantitative trait locus (eQTL) mapping was performed on the transcript abundance of five downstream PA synthesis genes (dihydroflavonol reductase (VvDFR), leucoanthocyanidin dioxygenase (VvLDOX), leucoanthocyanidin reductase (VvLAR1), VvLAR2 and anthocyanidin reductase (VvANR)) measured by real-time quantitative PCR on a pseudo F1 population in two growing seasons. Twenty-one eQTLs were identified; 17 of them did not overlap with known candidate transcription factors or cis-regulatory sequences. These novel loci and the presence of digenic epistasis support the previous hypothesis of a polygenic regulatory mechanism for PA biosynthesis. In a genomic region co-locating eQTLs for VvDFR, VvLDOX and VvLAR1, gene annotation and a transcriptomic survey suggested that VvMYBC2-L1, a gene coding for an R2R3-MYB protein, is involved in regulating PA synthesis. Phylogenetic analysis showed its high similarity to characterized negative MYB factors. Its spatiotemporal expression profile in grape coincided with PA synthesis. Its functional characterization via overexpression in grapevine hairy roots demonstrated its ability to reduce the amount of PA and to down-regulate expression of PA genes.


Asunto(s)
Mapeo Cromosómico , Frutas/genética , Proteínas de Plantas/metabolismo , Proantocianidinas/metabolismo , Sitios de Carácter Cuantitativo/genética , Factores de Transcripción/metabolismo , Vitis/genética , Vías Biosintéticas/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Estudios de Asociación Genética , Genotipo , Filogenia , Proteínas de Plantas/genética , Raíces de Plantas/genética , Proantocianidinas/biosíntesis , Proantocianidinas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo
3.
Plant Sci ; 207: 18-24, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23602095

RESUMEN

Expression quantitative locus (eQTL) mapping was proposed as a valuable approach to dissect the genetic basis of transcript variation, one of the prime causes of natural phenotypic variation. Few eQTL studies have been performed on woody species due to the difficulty in sample homogenisation. Based on previous knowledge on berry colour formation, we performed eQTL mapping in field experimentation of grapevine with appropriate sampling criteria. The transcript level of VvUFGT, a key enzyme for anthocyanin synthesis was measured by real-time qRT-PCR in grape berry on a 191-individual pseudo-F1 progeny, derived from a cross between Syrah and Grenache cultivars. Two eQTLs were identified: one, explaining 20%, of genotypic variance and co-locating with VvUFGT itself (cis-eQTL), was principally due to the contrast between Grenache alleles; the other, explaining 35% of genotypic variance, was a trans-eQTL due to Syrah allelic contrast and co-located with VvMYBAs, transcription factors known to activate the expression of VvUFGT. This study assessed and validated the feasibility of eQTL mapping approach in grapevine and offered insights and new hypotheses on grape skin colour formation.


Asunto(s)
Antocianinas/genética , Pigmentación , Proteínas de Plantas/genética , Sitios de Carácter Cuantitativo , Vitis/genética , Antocianinas/metabolismo , Mapeo Cromosómico , Frutas/genética , Frutas/metabolismo , Genotipo , Proteínas de Plantas/metabolismo , Reacción en Cadena de la Polimerasa , Vitis/metabolismo
4.
J Agric Food Chem ; 56(14): 5896-904, 2008 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-18582087

RESUMEN

An extraction method on grape berry was optimized for the total flavan-3-ol content measurement with regard to the nature of the sample and the duration of its extraction. This extraction was performed for the first time on the whole pericarp. Flavan-3-ol extractions were achieved on Shiraz ripe samples of pericarp versus skin within different durations: the best results were obtained for the whole pericarp and 1 h duration. Therefore, this more convenient protocol was used to investigate the flavan-3-ol content at different stages through berry development, in parallel with the abundance of transcripts involved in their biosynthesis. Furthermore, flavan-3-ol extractions on pericarp analysis confirmed their presence in both pulp and skin. For the first time, the flavan-3-ol biosynthesis in pulp was demonstrated with both biochemical and transcriptomic analyses since the presence of leucoanthocyanidin reductase (LAR2) and anthocyanin reductase (ANR) transcripts was revealed by real-time PCR. In addition, the percentage of epigallocatechin was different in pulp and skin.


Asunto(s)
Flavonoides/biosíntesis , Frutas/química , Frutas/crecimiento & desarrollo , Vitis/química , Antocianinas/metabolismo , Catequina/análogos & derivados , Catequina/análisis , Flavonoides/análisis , Frutas/metabolismo , Oxidorreductasas/genética , ARN Mensajero/análisis
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