Transcriptomic analysis of developing sorghum grains to detect genes related to cell wall biosynthesis and remodelling.

OBJECTIVE: Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important grain produced in the world. Interest for cultivating sorghum is increasing all over the world in the context of climate change, due to its low input and water requirements. Like other cultivated cereals, sorghum has significant nutritional value thanks to its protein, carbohydrate and dietary fiber content, these latter mainly consisting of cell wall polysaccharides. This work describes for the first time a transcriptomic analysis dedicated to identify the genes involved in the biosynthesis and remodelling of cell walls both in the endosperm and outer layers of sorghum grain during its development. Further analysis of these transcriptomic data will improve our understanding of cell wall assembly, which is a key component of grain quality. DATA DESCRIPTION: This research delineates the steps of our analysis, starting with the cultivation conditions and the grain harvest at different stages of development, followed by the laser microdissection applied to separate the endosperm from the outer layers. It also describes the procedures implemented to generate RNA libraries and to obtain a normalized and filtered table of transcript counts, and finally determine the number of putative cell wall-related genes already listed in literature.

A genome-wide association and prediction study in grapevine deciphers the genetic architecture of multiple traits and identifies genes under many new QTLs.

To cope with the challenges facing agriculture, speeding-up breeding programs is a worthy endeavor, especially for perennial species such as grapevine, but requires understanding the genetic architecture of target traits. To go beyond the mapping of quantitative trait loci in bi-parental crosses, we exploited a diversity panel of 279 Vitis vinifera L. cultivars planted in 5 blocks in the vineyard. This panel was phenotyped over several years for 127 traits including yield components, organic acids, aroma precursors, polyphenols, and a water stress indicator. The panel was genotyped for 63k single nucleotide polymorphisms by combining an 18K microarray and genotyping-by-sequencing. The experimental design allowed to reliably assess the genotypic values for most traits. Marker densification via genotyping-by-sequencing markedly increased the proportion of genetic variance explained by single nucleotide polymorphisms, and 2 multi-single nucleotide polymorphism models identified quantitative trait loci not found by a single nucleotide polymorphism-by-single nucleotide polymorphism model. Overall, 489 reliable quantitative trait loci were detected for 41% more response variables than by a single nucleotide polymorphism-by-single nucleotide polymorphism model with microarray-only single nucleotide polymorphisms, many new ones compared with the results from bi-parental crosses. A prediction accuracy higher than 0.42 was obtained for 50% of the response variables. Our overall approach as well as quantitative trait locus and prediction results provide insights into the genetic architecture of target traits. New candidate genes and the application into breeding are discussed.

The Road to Sorghum Domestication: Evidence From Nucleotide Diversity and Gene Expression Patterns.

Native African cereals (sorghum, millets) ensure food security to millions of low-income people from low fertility and drought-prone regions of Africa and Asia. In spite of their agronomic importance, the genetic bases of their phenotype and adaptations are still not well-understood. Here we focus on Sorghum bicolor, which is the fifth cereal worldwide for grain production and constitutes the staple food for around 500 million people. We leverage transcriptomic resources to address the adaptive consequences of the domestication process. Gene expression and nucleotide variability were analyzed in 11 domesticated and nine wild accessions. We documented a downregulation of expression and a reduction of diversity both in nucleotide polymorphism (30%) and gene expression levels (18%) in domesticated sorghum. These findings at the genome-wide level support the occurrence of a global reduction of diversity during the domestication process, although several genes also showed patterns consistent with the action of selection. Nine hundred and forty-nine genes were significantly differentially expressed between wild and domesticated gene pools. Their functional annotation points to metabolic pathways most likely contributing to the sorghum domestication syndrome, such as photosynthesis and auxin metabolism. Coexpression network analyzes revealed 21 clusters of genes sharing similar expression patterns. Four clusters (totaling 2,449 genes) were significantly enriched in differentially expressed genes between the wild and domesticated pools and two were also enriched in domestication and improvement genes previously identified in sorghum. These findings reinforce the evidence that the combined and intricated effects of the domestication and improvement processes do not only affect the behaviors of a few genes but led to a large rewiring of the transcriptome. Overall, these analyzes pave the way toward the identification of key domestication genes valuable for genetic resources characterization and breeding purposes.

Transcriptional Regulation of Sorghum Stem Composition: Key Players Identified Through Co-expression Gene Network and Comparative Genomics Analyses.

Most sorghum biomass accumulates in stem secondary cell walls (SCW). As sorghum stems are used as raw materials for various purposes such as feed, energy and fiber reinforced polymers, identifying the genes responsible for SCW establishment is highly important. Taking advantage of studies performed in model species, most of the structural genes contributing at the molecular level to the SCW biosynthesis in sorghum have been proposed while their regulatory factors have mostly not been determined. Validation of the role of several MYB and NAC transcription factors in SCW regulation in Arabidopsis and a few other species has been provided. In this study, we contributed to the recent efforts made in grasses to uncover the mechanisms underlying SCW establishment. We reported updated phylogenies of NAC and MYB in 9 different species and exploited findings from other species to highlight candidate regulators of SCW in sorghum. We acquired expression data during sorghum internode development and used co-expression analyses to determine groups of co-expressed genes that are likely to be involved in SCW establishment. We were able to identify two groups of co-expressed genes presenting multiple evidences of involvement in SCW building. Gene enrichment analysis of MYB and NAC genes provided evidence that while NAC SECONDARY WALL THICKENING PROMOTING FACTOR NST genes and SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN gene functions appear to be conserved in sorghum, NAC master regulators of SCW in sorghum may not be as tissue compartmentalized as in Arabidopsis. We showed that for every homolog of the key SCW MYB in Arabidopsis, a similar role is expected for sorghum. In addition, we unveiled sorghum MYB and NAC that have not been identified to date as being involved in cell wall regulation. Although specific validation of the MYB and NAC genes uncovered in this study is needed, we provide a network of sorghum genes involved in SCW both at the structural and regulatory levels.

New flavanol O-glycosides in grape and wine.

The presence of monomeric and dimeric flavan-3-ol monohexosides was investigated in grapes and wines. Polyphenol extracts were prepared from grape seeds and skins (Syrah, Merlot, and Cabernet-Sauvignon) sampled at three different developmental stages. Different wines (Tannat, Alicante, Syrah, Merlot, and Grenache) were also studied. The different samples obtained were analyzed by UPLC-ESI-IT-MS. Specific molecular ions corresponding to flavan-3-ol hexosides were detected by using targeted molecular ions with specific m/z values: 451 for (epi)catechin hexosides, and 739 for procyanidin dimer hexosides. 4'-O-ß-glucosyl-(+)-catechin and 7-O-ß-glucosyl-(+)-catechin were obtained by using a glucosyl transferase from apple, UGT71A15, and their structures determined by NMR. These glucosylated monomers and the dimers were identified in all analyzed grape seed and several skin extracts at the different stages and in wines made from different varieties.

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.

Cultivar Diversity of Grape Skin Polyphenol Composition and Changes in Response to Drought Investigated by LC-MS Based Metabolomics.

Phenolic compounds represent a large family of plant secondary metabolites, essential for the quality of grape and wine and playing a major role in plant defense against biotic and abiotic stresses. Phenolic composition is genetically driven and greatly affected by environmental factors, including water stress. A major challenge for breeding of grapevine cultivars adapted to climate change and with high potential for wine-making is to dissect the complex plant metabolic response involved in adaptation mechanisms. A targeted metabolomics approach based on ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode has been developed for high throughput profiling of the phenolic composition of grape skins. This method enables rapid, selective, and sensitive quantification of 96 phenolic compounds (anthocyanins, phenolic acids, stilbenoids, flavonols, dihydroflavonols, flavan-3-ol monomers, and oligomers…), and of the constitutive units of proanthocyanidins (i.e., condensed tannins), giving access to detailed polyphenol composition. It was applied on the skins of mature grape berries from a core-collection of 279 Vitis vinifera cultivars grown with or without watering to assess the genetic variation for polyphenol composition and its modulation by irrigation, in two successive vintages (2014-2015). Distribution of berry weights and δ13C values showed that non irrigated vines were subjected to a marked water stress in 2014 and to a very limited one in 2015. Metabolomics analysis of the polyphenol composition and chemometrics analysis of this data demonstrated an influence of water stress on the biosynthesis of different polyphenol classes and cultivar differences in metabolic response to water deficit. Correlation networks gave insight on the relationships between the different polyphenol metabolites and related biosynthetic pathways. They also established patterns of polyphenol response to drought, with different molecular families affected either positively or negatively in the different cultivars, with potential impact on grape and wine quality.

The composition of cell walls from grape skin in Vitis vinifera intraspecific hybrids.

BACKGROUND: Monastrell is a red grape cultivar adapted to the dry environmental conditions of Murcia, SE Spain. Its berries seem to be characterized by a rigid cell wall structure, which could make difficult the winemaking process. Cabernet Sauvignon cultivar is used to complement Monastrell wines in this region owing to its high phenolic content with high extractability. This study explores the skin cell wall composition of grapes from plants resulting from intraspecific crosses of Vitis vinifera cultivars Monastrell × Cabernet Sauvignon. Moreover, the morphology of the cell wall material (CWM) from some representative samples was visualized by transmission optical microscopy. RESULTS: The total sugar content of CWM from nine out of ten genotypes of the progeny was lower than that from Monastrell. Seven out of ten genotypes showed lower phenolic content than Cabernet Sauvignon. The CWM from nine out of ten hybrids presented lower protein content than that from Monastrell. CONCLUSION: This study confirms that skin cell walls from Monastrell × Cabernet Sauvignon hybrid grapes presented major differences in composition compared with their parents. These data could help in the development of new cultivars adapted to the dry conditions of SE Spain and with a cell wall composition favouring extractability. © 2017 Society of Chemical Industry.

A Fast and Robust UHPLC-MRM-MS Method to Characterize and Quantify Grape Skin Tannins after Chemical Depolymerization.

A rapid, sensitive, and selective analysis method using ultra high performance liquid chromatography coupled with triple-quadrupole mass spectrometry (UHPLC-QqQ-MS) has been developed for the characterization and quantification of grape skin flavan-3-ols after acid-catalysed depolymerization in the presence of phloroglucinol (phloroglucinolysis). The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, this fast gradient robust method allows analysis of constitutive units of grape skin proanthocyanidins, including some present in trace amounts, in a single injection, with a throughput of 6 samples per hour. This method was applied to a set of 214 grape skin samples from 107 different red and white grape cultivars grown under two conditions in the vineyard, irrigated or non-irrigated. The results of triplicate analyses confirmed the robustness of the method, which was thus proven to be suitable for high-throughput and large-scale metabolomics studies. Moreover, these preliminary results suggest that analysis of tannin composition is relevant to investigate the genetic bases of grape response to drought.

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.

BAHD or SCPL acyltransferase? What a dilemma for acylation in the world of plant phenolic compounds.

Phenolic compounds are secondary metabolites involved in several plant growth and development processes, including resistance to biotic and abiotic stresses. The biosynthetic pathways leading to the vast diversity of plant phenolic products often include an acylation step, with phenolic compounds being the donor or acceptor molecules. To date, two acyltransferase families using phenolic compounds as acceptor or donor molecules have been described, with each using a different 'energy-rich' acyl donor. BAHD-acyltransferases, named after the first four biochemically characterized enzymes of the group, use acyl-CoA thioesters as donor molecules, whereas SCPL (Serine CarboxyPeptidase Like)-acyltransferases use 1-O-ß-glucose esters. Here, common and divergent specifications found in the literature for both enzyme families were analyzed to answer the following questions. Are both acyltransferases involved in the synthesis of the same molecule (or same group of molecules)? Are both acyltransferases recruited in the same plant? How does the subcellular localization of these enzymes impact metabolite trafficking in plant cells?

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.

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.

Dissecting genetic architecture of grape proanthocyanidin composition through quantitative trait locus mapping.

BACKGROUND: Proanthocyanidins (PAs), or condensed tannins, are flavonoid polymers, widespread throughout the plant kingdom, which provide protection against herbivores while conferring organoleptic and nutritive values to plant-derived foods, such as wine. However, the genetic basis of qualitative and quantitative PA composition variation is still poorly understood. To elucidate the genetic architecture of the complex grape PA composition, we first carried out quantitative trait locus (QTL) analysis on a 191-individual pseudo-F1 progeny. Three categories of PA variables were assessed: total content, percentages of constitutive subunits and composite ratio variables. For nine functional candidate genes, among which eight co-located with QTLs, we performed association analyses using a diversity panel of 141 grapevine cultivars in order to identify causal SNPs. RESULTS: Multiple QTL analysis revealed a total of 103 and 43 QTLs, respectively for seed and skin PA variables. Loci were mainly of additive effect while some loci were primarily of dominant effect. Results also showed a large involvement of pairwise epistatic interactions in shaping PA composition. QTLs for PA variables in skin and seeds differed in number, position, involvement of epistatic interaction and allelic effect, thus revealing different genetic determinisms for grape PA composition in seeds and skin. Association results were consistent with QTL analyses in most cases: four out of nine tested candidate genes (VvLAR1, VvMYBPA2, VvCHI1, VvMYBPA1) showed at least one significant association with PA variables, especially VvLAR1 revealed as of great interest for further functional investigation. Some SNP-phenotype associations were observed only in the diversity panel. CONCLUSIONS: This study presents the first QTL analysis on grape berry PA composition with a comparison between skin and seeds, together with an association study. Our results suggest a complex genetic control for PA traits and different genetic architectures for grape PA composition between berry skin and seeds. This work also uncovers novel genomic regions for further investigation in order to increase our knowledge of the genetic basis of PA composition.

Transcriptional analysis of late ripening stages of grapevine berry.

BACKGROUND: The composition of grapevine berry at harvest is a major determinant of wine quality. Optimal oenological maturity of berries is characterized by a high sugar/acidity ratio, high anthocyanin content in the skin, and low astringency. However, harvest time is still mostly determined empirically, based on crude biochemical composition and berry tasting. In this context, it is interesting to identify genes that are expressed/repressed specifically at the late stages of ripening and which may be used as indicators of maturity. RESULTS: Whole bunches and berries sorted by density were collected in vineyard on Chardonnay (white cultivar) grapevines for two consecutive years at three stages of ripening (7-days before harvest (TH-7), harvest (TH), and 10-days after harvest (TH+10)). Microvinification and sensory analysis indicate that the quality of the wines made from the whole bunches collected at TH-7, TH and TH+10 differed, TH providing the highest quality wines.In parallel, gene expression was studied with Qiagen/Operon microarrays using two types of samples, i.e. whole bunches and berries sorted by density. Only 12 genes were consistently up- or down-regulated in whole bunches and density sorted berries for the two years studied in Chardonnay. 52 genes were differentially expressed between the TH-7 and TH samples. In order to determine whether these genes followed a similar pattern of expression during the late stages of berry ripening in a red cultivar, nine genes were selected for RT-PCR analysis with Cabernet Sauvignon grown under two different temperature regimes affecting the precocity of ripening. The expression profiles and their relationship to ripening were confirmed in Cabernet Sauvignon for seven genes, encoding a carotenoid cleavage dioxygenase, a galactinol synthase, a late embryogenesis abundant protein, a dirigent-like protein, a histidine kinase receptor, a valencene synthase and a putative S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase. CONCLUSIONS: This set of up- and down-regulated genes characterize the late stages of berry ripening in the two cultivars studied, and are indirectly linked to wine quality. They might be used directly or indirectly to design immunological, biochemical or molecular tools aimed at the determination of optimal ripening in these cultivars.

In vivo grapevine anthocyanin transport involves vesicle-mediated trafficking and the contribution of anthoMATE transporters and GST.

In cells, anthocyanin pigments are synthesized at the cytoplasmic surface of the endoplasmic reticulum, and are then transported and finally accumulated inside the vacuole. In Vitis vinifera (grapevine), two kinds of molecular actors are putatively associated with the vacuolar sequestration of anthocyanins: a glutathione-S-transferase (GST) and two MATE-type transporters, named anthoMATEs. However, the sequence of events by which anthocyanins are imported into the vacuole remains unclear. We used MYBA1 transformed hairy roots as a grapevine model tissue producing anthocyanins, and took advantage of the unique autofluorescence of anthocyanins to study their cellular trafficking. In these tissues, anthocyanins were not only visible in the largest vacuoles, but were also present at higher concentrations in several vesicles of different sizes. In the cell, small vesicles actively moved alongside the tonoplast, suggesting a vesicular trafficking to the vacuole. Subcellular localization assays revealed that anthoMATE transporters were closely related with these small vesicles, whereas GST was localized in the cytoplasm around the nucleus, suggesting an association with the endoplasmic reticulum. Furthermore, cells in hairy roots expressing anthoMATE antisense did not display small vesicles filled with anthocyanins, whereas in hairy roots expressing GST antisense, anthocyanins were accumulated in vesicles but not in the vacuole. This suggests that in grapevine, anthoMATE transporters and GST are involved in different anthocyanin transport mechanisms.

'Bois noir' phytoplasma induces significant reprogramming of the leaf transcriptome in the field grown grapevine.

BACKGROUND: Phytoplasmas are bacteria without cell walls from the class Mollicutes. They are obligate intracellular plant pathogens which cause diseases in hundreds of economically important plants including the grapevine (Vitis vinifera). Knowledge of their biology and the mechanisms of their interactions with hosts is largely unknown because they are uncultivable and experimentally inaccessible in their hosts. We detail here the global transcriptional profiling in grapevine responses to phytoplasmas. The gene expression patterns were followed in leaf midribs of grapevine cv. 'Chardonnay' naturally infected with a phytoplasma from the stolbur group 16SrXII-A, which is associated with the grapevine yellows disease 'Bois noir'. RESULTS: We established an on field experimental system in a productive vineyard that allowed application of molecular tools in a plant natural environment. Global transcription profiles of infected samples were compared with the healthy ones using microarray datasets and metabolic pathway analysis software (MapMan). The two-year-long experiment revealed that plant genes involved in primary and secondary metabolic pathways were changed in response to infection and that these changes might support phytoplasma nutrition. A hypothesis that phytoplasmas interact with the plant carbohydrate metabolism was proven and some possibilities how the products of this pathway might be utilized by phytoplasmas are discussed. In addition, several photosynthetic genes were largely down-regulated in infected plants, whereas defense genes from the metabolic pathway leading to formation of flavonoids and some PR proteins were significantly induced. Few other genes involved in defense-signaling were differentially expressed in healthy and infected plants. A set of 17 selected genes from several differentially expressed pathways was additionally analyzed with quantitative real-time PCR and confirmed to be suitable for a reliable classification of infected plants and for the characterization of susceptibility features in the field conditions. CONCLUSION: This study revealed some fundamental aspects of grapevine interactions with the stolbur 'Bois noir' phytoplasma in particular and some plant interactions with phytoplasmas in general. In addition, the results of the study will likely have an impact on grape improvement by yielding marker genes that can be used in new diagnostic assays for phytoplasmas or by identifying candidate genes that contribute to the improved properties of grape.

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 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.