Focus on putative serine carboxypeptidase-like acyltransferases in grapevine.

Grapevine (Vitis vinifera L.) berry synthesizes and accumulates a large array of phenolic compounds (e.g. flavonoids and hydroxycinnamic acid derivatives), some of which result from acylation mechanisms. In grapevine, the genes encoding enzymes responsible for such acylation are largely unknown. Enzymes classified as serine carboxypeptidases (SCPs), able to transfer acyl moieties from a glucose ester, have previously been characterized in plants, and named serine carboxypeptidase-like acyltransferases (SCL-ATs). We performed genome-wide identification of SCP sequences in V. vinifera. Phylogenetic analysis revealed that only 12 grapevine SCPs, grouped in clade IA with previously characterized SCPL-AT could have an acylation function. Interestingly, seven putative SCP-ATs are grouped in a 400 kb cluster in chromosome 3. The expression level of putative SCPL-ATs has been evaluated at key stages of grape berry development in the main tissues and compared with the content of acylated phenolic compounds in the corresponding samples. The expression levels of VvGAT1 and VvGAT2 and that of VvSCP5 were increased in hairy-roots overexpressing transcription factors inducing the biosynthesis of proanthocyanidins and anthocyanins, respectively. These findings open the way for the functional characterization of the identified putative SCPL-AT from grapevine.

A group of grapevine MYBA transcription factors located in chromosome 14 control anthocyanin synthesis in vegetative organs with different specificities compared with the berry color locus.

Grapevine organs accumulate anthocyanins in a cultivar-specific and environmentally induced manner. The MYBA1-A2 genes within the berry color locus in chromosome 2 represent the major genetic determinants of fruit color. The simultaneous occurrence of transposon insertions and point mutations in these genes is responsible for most white-skinned phenotypes; however, the red pigmentation found in vegetative organs suggests the presence of additional regulators. This work describes a genomic region of chromosome 14 containing three closely related R2R3-MYB genes, named MYBA5, MYBA6 and MYBA7. Ectopic expression of the latter two genes in grapevine hairy roots promoted anthocyanin accumulation without affecting other phenylpropanoids. Transcriptomic profiling of hairy roots expressing MYBA1, MYBA6 and MYBA7 showed that these regulators share the activation of late biosynthetic and modification/transport-related genes, but differ in the activation of the FLAVONOID-3'5'-HYDROXYLASE (F3'5'H) family. An alternatively spliced MYBA6 variant was incapable of activating anthocyanin synthesis, however, because of the lack of an MYC1 interaction domain. MYBA1, MYBA6.1 and MYBA7 activated the promoters of UDP-GLUCOSE:FLAVONOID 3-O-GLUCOSYLTRANSFERASE (UFGT) and ANTHOCYANIN 3-O-GLUCOSIDE-6″-O-ACYLTRANSFERASE (3AT), but only MYBA1 induced F3'5'H in concordance with the low proportion of tri-hydroxylated anthocyanins found in MYBA6-A7 hairy roots. This putative new color locus is related to the red/cyanidic pigmentation of vegetative organs in black- and white-skinned cultivars, and forms part of the UV-B radiation response pathway orchestrated by ELONGATED HYPOCOTYL 5 (HY5). These results demonstrate the involvement of additional anthocyanin regulators in grapevine and suggest an evolutionary divergence between the two grape color loci for controlling additional targets of the flavonoid pathway.

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

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.

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.

ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides.

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.

Selection of candidate genes for grape proanthocyanidin pathway by an integrative approach.

Proanthocyanidins (PA) play a major role in plant protection against biotic and abiotic stresses. Moreover these molecules are known to be beneficial for human health and are responsible for astringency of foods and beverages such as wine and thus have a great impact on the final quality of the product. Genes playing a role in the PA pathway are only partially known. The amount of available transcriptomic and genetic data to select candidate genes without a priori knowledge from orthologous function increases every day. However, the methods used so far generate so many candidate genes that it is impossible to validate all of them. In this study, we used an integrative strategy based on different screening methods to select a reduced list of candidate genes. We have crossed results from different screening methods including QTL mapping and three transcriptomic studies to select 20 candidate genes, located in QTL intervals and fulfilling at least two transcriptomic screenings. This list includes three glucosyltransferases, already suspected to have a role in the PA biosynthetic pathway. Among the 17 remaining genes, we selected three genes to perform further analysis by association genetic studies. For each of these genes, we found a polymorphism linked to PA variation. The three genes (VvMybC2-L1, VvGAT-like and VvCob-like), not previously known to play a role in PA synthesis, are promising candidates for further molecular physiology studies.

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.

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 AM3green 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.

Ectopic expression of VlmybA1 in grapevine activates a narrow set of genes involved in anthocyanin synthesis and transport.

The colour of the red wine is essentially due to the release of anthocyanins from the red skin of grape berries during the process of wine making. Anthocyanins are synthesized during ripening of the berries under the control of VvMYBA1 transcription factor that controls the expression of UFGT. In order to identify the whole set of downstream regulated genes, we targeted constitutive ectopic expression of VlmybA1-2 into grapevine hairy roots and plants. The ectopic expression of VlmybA1-2 triggered de novo production and storage of anthocyanins in all transgenic vegetative organs, leading to a very intense red coloration, and did not interfere with proanthocyanidin (PA) biosynthesis. The ectopic red pigmentation was due to the accumulation of anthocyanins in vacuoles and anthocyanin vacuolar inclusion (AVIs) in all organs but only in specific tissues. A transcriptomic analysis using a 14 K oligoarray revealed that the ectopic expression of VlmybA1-2 activated only few genes, most of which are involved in both PA and anthocyanin biosynthesis, while the expression of BAN and LAR (two specific genes of the PA biosynthesis pathway) was unaffected. Among these, 4 genes emerged given the amplitude of their up-regulation, quantitatively similar to VlmybA1-2 itself. In addition to the previously described UFGT, this set comprised an isogen of GST, an O-methyltransferase, both of which are supposed to play a role in the anthocyanin biosynthesis pathway, as well as a candidate gene putatively involved in the vacuolar anthocyanin transport in grapevine (anthoMATE). Together, these results suggest that MybA1 activates the last steps of anthocyanin synthesis and transport through the regulation of a narrow, specific spectrum of genes regulated as a cluster.

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.

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.