Sandrine Vialet

Ectopic Expression of VvMybPA2 Promotes Proanthocyanidin Biosynthesis in Grapevine and Suggests Additional Targets in the Pathway

Grapevine (Vitis vinifera) proanthocyanidins contribute to plant defense mechanisms against biotic stress and also play a critical role in organoleptic properties of wine. In grapevine berry, these compounds are mainly accumulated in exocarps and seeds in the very early stages of development. A previous study has already identified VvMybPA1 as the first transcription factor involved in the regulation of the proanthocyanidin pathway during seed development in grapevine. A novel Myb factor, VvMybPA2, which is described in this study, is in contrast mainly expressed in the exocarp of young berries and in the leaves. This transcription factor shows very high protein sequence homology with other plant Myb factors, which regulate flavonoid biosynthesis. Ectopic expression of either VvMybPA1 or VvMybPA2 in grapevine hairy roots induced qualitative and quantitative changes of the proanthocyanidin profiles. High-throughput transcriptomic analyses of transformed grapevine organs identified a large set of putative targets of the VvMybPA1 and VvMybPA2 transcription factors. Both genes significantly activated enzymes of the flavonoid pathway, including anthocyanidin reductase and leucoanthocyanidin reductase 1, the specific terminal steps in the biosynthesis of epicatechin and catechin, respectively, but not leucoanthocyanidin reductase 2. The functional annotation of the genes whose expression was modified revealed putative new actors of the proanthocyanidin pathway, such as glucosyltransferases and transporters.

Grapevine MATE-Type Proteins Act as Vacuolar H+-Dependent Acylated Anthocyanin Transporters

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1∷ or AM3∷green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H+-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.

ABCC1, an ATP Binding Cassette Protein from Grape Berry, Transports Anthocyanidin 3-O-Glucosides

This work provides biochemical evidence that ABCC transporters are directly involved in anthocyanin transport into plant vacuoles. The presence of reduced glutathione is a prerequisite for the transport. Our data support that anthocyanins and glutathione are cotransported but that no glutathione anthocyanin conjugate is formed. Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate.

Grapevine ( Vitis vinifera L . )

Grapevine (Vitis) is considered to be one of the major fruit crops in the world based on hectares cultivated and economic value. Grapes are used not only for wine but also for fresh fruit, dried fruit, and juice production. Wine is by far the major product of grapes, and the focus of this chapter is on wine grape cultivars. Grapevine cultivars of Vitis vinifera L. have a reputation for producing premium quality wines. These premium quality wines are produced from a small number of cultivars that enjoy a high level of consumer acceptance and are firmly entrenched in the market place because of varietal name branding and the association of certain wine styles and regions with specific cultivars. In light of this situation, grapevine improvement by a transgenic approach is attractive when compared to a classical breeding approach. The transfer of individual traits as single genes with a minimum disruption to the original genome would leave the traditional characteristics of the cultivar intact. However, a reliable transformation system is required for a successful transgenic approach to grapevine improvement. There are three criteria for achieving an efficient Agrobacterium-mediated transformation system: (1) the production of highly regenerative transformable tissue, (2) optimal cocultivation conditions for both grapevine tissue and Agrobacterium, and (3) an efficient selection regime for transgenic plant regeneration. In this chapter, we describe a grapevine transformation system that meets these criteria. We also describe a protocol for the production of transformed roots suitable for functional gene studies and for the production of semi-transgenic grafted plants.

A negative MYB regulator of proanthocyanidin accumulation, identified through expression quantitative locus mapping in the grape berry.

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.

Identification and functional characterization of cDNAs coding for hydroxybenzoate/hydroxycinnamate glucosyltransferases co-expressed with genes related to proanthocyanidin biosynthesis

Grape proanthocyanidins (PAs) play a major role in the organoleptic properties of wine. They are accumulated mainly in grape skin and seeds during the early stages of berry development. Despite the recent progress in the identification of genes involved in PA biosynthesis, the mechanisms involved in subunit condensation, galloylation, or fine regulation of the spatio-temporal composition of grape berries in PAs are still not elucidated. Two Myb transcription factors, VvMybPA1 and VvMybPA2, controlling the PA pathway have recently been identified and ectopically over-expressed in an homologous system. In addition to already known PA genes, three genes coding for glucosyltransferases were significantly differentially expressed between hairy roots over-expressing VvMybPA1 or VvMybPA2 and control lines. The involvement of these genes in PA biosynthesis metabolism is unclear. The three glucosyltransferases display high sequence similarities with other plant glucosyltransferases able to catalyse the formation of glucose esters, which are important intermediate actors for the synthesis of different phenolic compounds. Studies of the in vitro properties of these three enzymes (Km, Vmax, substrate specificity, pH sensitivity) were performed through production of recombinant proteins in E. coli and demonstrated that they are able to catalyse the formation of 1-O-acyl-Glc esters of phenolic acids but are not active on flavonoids and stilbenes. The transcripts are expressed in the early stages of grape berry development, mainly in the berry skins and seeds. The results presented here suggest that these enzymes could be involved in vivo in PA galloylation or in the synthesis of hydroxycinnamic esters.

Validation of an Extraction Method on Whole Pericarp of Grape Berry (Vitis vinifera L. cv. Shiraz) to Study Biochemical and Molecular Aspects of Flavan-3-ol Synthesis during Berry Development

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.

Expression QTL mapping in grapevine--revisiting the genetic determinism of grape skin colour.

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.

Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine

Highlight Functional analysis of the four grapevine shikimate dehydrogenases (VvSDH1–4) reveals that two of them are involved in gallic acid biosynthesis.