Model-assisted analysis for tuning anthocyanin composition in grape berries.

Anthocyanin composition is responsible for the red colour of grape berries and wines, and contributes to their organoleptic quality. However, anthocyanin biosynthesis is under genetic, developmental and environmental regulation, making its targeted fine-tuning challenging. We constructed a mechanistic model to simulate the dynamics of anthocyanin composition throughout grape ripening in Vitis vinifera, employing a consensus anthocyanin biosynthesis pathway. The model was calibrated and validated using six datasets from eight cultivars and 37 growth conditions. Tuning the transformation and degradation parameters allowed us to accurately simulate the accumulation process of each individual anthocyanin under different environmental conditions. The model parameters were robust across environments for each genotype. The coefficients of determination (R2) for the simulated versus observed values for the six datasets ranged from 0.92 to 0.99, while the relative root mean square errors (RRMSEs) were between 16.8 and 42.1 %. The leave-one-out cross-validation for three datasets showed R2 values of 0.99, 0.96 and 0.91, and RRMSE values of 28.8, 32.9 and 26.4 %, respectively, suggesting a high prediction quality of the model. Model analysis showed that the anthocyanin profiles of diverse genotypes are relatively stable in response to parameter perturbations. Virtual experiments further suggested that targeted anthocyanin profiles may be reached by manipulating a minimum of three parameters, in a genotype-dependent manner. This model presents a promising methodology for characterizing the temporal progression of anthocyanin composition, while also offering a logical foundation for bioengineering endeavours focused on precisely adjusting the anthocyanin composition of grapes.

Metabolite analysis reveals distinct spatio-temporal accumulation of anthocyanins in two teinturier variants of cv. 'Gamay' grapevines (Vitis vinifera L.).

MAIN CONCLUSION: White-fleshed grape cv. 'Gamay' and its two teinturier variants presented distinct spatial-temporal accumulation of anthocyanins, with uncoupled accumulation of sugars and anthocyanins in 'Gamay Fréaux'. In most red grape cultivars, anthocyanins accumulate exclusively in the berry skin, while 'teinturier' cultivars also accumulate anthocyanins in the pulp. Here, we investigated the teinturier cvs. 'Gamay de Bouze' and 'Gamay Fréaux' (two somatic variants of the white-fleshed cv. 'Gamay') through metabolic and transcript analysis to clarify whether these two somatic variants have the same anthocyanin accumulation pattern in the skin and pulp, and whether primary metabolites are also affected. The skin of the three cultivars and the pulp of 'Gamay de Bouze' begun to accumulate anthocyanins at the onset of berry ripening. However, the pulp of 'Gamay Fréaux' exhibited a distinct anthocyanin accumulation pattern, starting as early as fruit set with very low level of sugars. The highest level of anthocyanins was found in 'Gamay Fréaux' skin, followed by 'Gamay de Bouze' and 'Gamay'. Consistently, the transcript abundance of genes involved in anthocyanin biosynthesis were in line with the anthocyanin levels in the three cultivars. Despite no evident differences in pulp sugar content, the concentration of glucose and fructose in the skin of 'Gamay Fréaux' was only half of those in the skin of 'Gamay' and 'Gamay de Bouze' throughout all berry ripening, suggesting an uncoupled accumulation of sugars and anthocyanins in 'Gamay Fréaux'. The study provides a comprehensive view of metabolic consequences in grape somatic variants and the three almost isogenic genotypes can serve as ideal reagents to further uncover the mechanisms underlying the linkage between sugar and anthocyanin accumulation.

Modelling predicts tomatoes can be bigger and sweeter if biophysical factors and transmembrane transports are fine-tuned during fruit development.

The trade-off between yield and quality, a major problem for the production of fleshy fruits, involves fruit expansive growth and sugar metabolism. Here we developed an integrative model by coupling a biophysical model of fleshy fruit growth processes, including water and carbon fluxes and organ expansion, with an enzyme-based kinetic model of sugar metabolism to better understand the interactions between these two processes. The integrative model was initially tested on tomato fruit, a model system for fleshy fruit. The integrative model closely simulated the biomass and major carbon metabolites of tomato fruit developing under optimal or stress conditions. The model also performed robustly when simulating the fruit size and sugar concentrations of different tomato genotypes including wild species. The validated model was used to explore ways of uncoupling the size-sweetness trade-off in fruit. Model-based virtual experiments suggested that larger sweeter tomatoes could be obtained by simultaneously manipulating certain biophysical factors and transmembrane transports. The integrative fleshy fruit model provides a promising tool to facilitate the targeted bioengineering and breeding of tomatoes and other fruits.

Dissecting the control of shoot development in grapevine: genetics and genomics identify potential regulators.

BACKGROUND: Grapevine is a crop of major economic importance, yet little is known about the regulation of shoot development in grapevine or other perennial fruits crops. Here we combine genetic and genomic tools to identify candidate genes regulating shoot development in Vitis spp. RESULTS: An F2 population from an interspecific cross between V. vinifera and V. riparia was phenotyped for shoot development traits, and three Quantitative Trait Loci (QTLs) were identified on linkage groups (LGs) 7, 14 and 18. Around 17% of the individuals exhibited a dwarfed phenotype. A transcriptomic study identified four candidate genes that were not expressed in dwarfed individuals and located within the confidence interval of the QTL on LG7. A deletion of 84,482 bp was identified in the genome of dwarfed plants, which included these four not expressed genes. One of these genes was VviCURLY LEAF (VviCLF), an orthologue of CLF, a regulator of shoot development in Arabidopsis thaliana. CONCLUSIONS: The phenotype of the dwarfed grapevine plants was similar to that of clf mutants of A. thaliana and orthologues of the known targets of CLF in A. thaliana were differentially expressed in the dwarfed plants. This suggests that CLF, a major developmental regulator in A. thaliana, also controls shoot development in grapevine.

Proteomic and metabolomic profiling underlines the stage- and time-dependent effects of high temperature on grape berry metabolism.

Climate change scenarios predict an increase in mean air temperatures and in the frequency, intensity, and length of extreme temperature events in many wine-growing regions worldwide. Because elevated temperature has detrimental effects on berry growth and composition, it threatens the economic and environmental sustainability of wine production. Using Cabernet Sauvignon fruit-bearing cuttings, we investigated the effects of high temperature (HT) on grapevine berries through a label-free shotgun proteomic analysis coupled to a complementary metabolomic study. Among the 2,279 proteins identified, 592 differentially abundant proteins were found in berries exposed to HT. The gene ontology categories "stress," "protein," "secondary metabolism," and "cell wall" were predominantly altered under HT. High temperatures strongly impaired carbohydrate and energy metabolism, and the effects depended on the stage of development and duration of treatment. Transcript amounts correlated poorly with protein expression levels in HT berries, highlighting the value of proteomic studies in the context of heat stress. Furthermore, this work reveals that HT alters key proteins driving berry development and ripening. Finally, we provide a list of differentially abundant proteins that can be considered as potential markers for developing or selecting grape varieties that are better adapted to warmer climates or extreme heat waves.

On a Cold Night: Transcriptomics of Grapevine Flower Unveils Signal Transduction and Impacted Metabolism.

Low temperature is a critical environmental factor limiting plant productivity, especially in northern vineyards. To clarify the impact of this stress on grapevine flower, we used the Vitis array based on Roche-NimbleGen technology to investigate the gene expression of flowers submitted to a cold night. Our objectives were to identify modifications in the transcript levels after stress and during recovery. Consequently, our results confirmed some mechanisms known in grapes or other plants in response to cold stress, notably, (1) the pivotal role of calcium/calmodulin-mediated signaling; (2) the over-expression of sugar transporters and some genes involved in plant defense (especially in carbon metabolism), and (3) the down-regulation of genes encoding galactinol synthase (GOLS), pectate lyases, or polygalacturonases. We also identified some mechanisms not yet known to be involved in the response to cold stress, i.e., (1) the up-regulation of genes encoding G-type lectin S-receptor-like serine threonine-protein kinase, pathogen recognition receptor (PRR5), or heat-shock factors among others; (2) the down-regulation of Myeloblastosis (MYB)-related transcription factors and the Constans-like zinc finger family; and (3) the down-regulation of some genes encoding Pathogen-Related (PR)-proteins. Taken together, our results revealed interesting features and potentially valuable traits associated with stress responses in the grapevine flower. From a long-term perspective, our study provides useful starting points for future investigation.

Vine nitrogen status and volatile thiols and their precursors from plot to transcriptome level.

BACKGROUND: Volatile thiols largely contribute to the organoleptic characteristics and typicity of Sauvignon blanc wines. Among this family of odorous compounds, 3-sulfanylhexan-1-ol (3SH) and 4-methyl-4-sulfanylpentan-2-one (4MSP) have a major impact on wine flavor. These thiols are formed during alcoholic fermentation by the yeast from odorless, non-volatile precursors found in the berries and the must. The present study investigates the effects of vine nitrogen (N) status on 3SH and 4MSP content in Sauvignon blanc wine and on the glutathionylated and cysteinylated precursors of 3SH (Glut-3SH and Cys-3SH) in the berries and the must. This is paralleled by a RNA-seq analysis of gene expression in the berries. The impact of N supply on the expression of the glutathione-S-transferase 3 and 4 (VviGST3 and VviGST4) and the γ-glutamyltranspeptidase (VviGGT), considered as key genes in their biosynthesis, was also evaluated. RESULTS: N supply (N100 treatment) increased the 3SH content in wine while no effect was noticed on 4MSP level. Furthermore, N supply increased Glut-3SH levels in grape berries at late berry ripening stages, and this effect was highly significant in must at harvest. No significant effect of N addition was noticed on Cys-3SH concentration. The transcript abundance of the glutathione-S-transferases VviGST3 and VviGST4 and the γ-glutamyltranspeptidase (VviGGT), were similar between the control and the N100 treatment. New candidate genes which might be implicated in the biosynthetic pathway of 3SH precursors were identified by whole transcriptome shotgun sequencing (RNA-seq). CONCLUSIONS: High vine N status has a positive effect on 3SH content in wine through an increase of Glut-3SH levels in grape berries and must. Candidate GSTs and glutathione-S-conjugates type transporters involved in this stimulation were identified by RNA-seq analysis.

Anthocyanin biosynthesis is differentially regulated by light in the skin and flesh of white-fleshed and teinturier grape berries.

MAIN CONCLUSION: Light exclusion reduces the concentration and modifies the composition of grape anthocyanins, by altering the expression of genes involved in anthocyanin biosynthesis and transport, in a cultivar- and tissue-specific manner. Unlike most grapes, teinturier grapes accumulate anthocyanins both in skin and flesh. However, the concentration and composition of anthocyanins in both tissues differ, providing a valuable system to study tissue-specific regulation of anthocyanin synthesis. Furthermore, little is known about the mechanisms controlling the sensitivity of anthocyanin accumulation to light. Here, light was excluded from Gamay (white-fleshed) and Gamay Fréaux (teinturier mutant) berries throughout berry development. Under light-exposed conditions, the skin of Gamay Fréaux accumulated the highest level of anthocyanins, followed by the skin of Gamay, while the pulp of Gamay Fréaux had much lower anthocyanins than the skins. Network analysis revealed the same order on the number of significant correlations among metabolites and transcripts in the three colored tissues, indicating a higher connectivity that reflects a higher efficiency of the anthocyanin pathway. Compared to light conditions, light exclusion reduced the total amount of anthocyanins, most severely in the skin of Gamay and to a lesser extent in the flesh and skin of Gamay Fréaux. Coordinated decrease in the transcript abundance of structural, regulatory and transporter genes by light exclusion correlated with the reduced anthocyanin concentration in a cultivar- and tissue-specific manner. Moreover, light exclusion increased the ratio of dihydroxylated to trihydroxylated anthocyanins, in parallel with F3'H and F3'5'H transcript amounts. Sugars and ABA only play a limited role in the control of anthocyanin synthesis in the berries, in contrast with what has been described in cell suspensions. This study provides novel insights into the regulation of anthocyanin in wild type and teinturier cultivars.

Ultraviolet-B radiation and water deficit interact to alter flavonol and anthocyanin profiles in grapevine berries through transcriptomic regulation.

UV-B radiation and water deficit may trigger flavonol and anthocyanin biosynthesis in plant tissues. In addition, previous research has showed strong qualitative effects on grape berry skin flavonol and anthocyanin profiles in response to UV-B and water deficit. The aim of this study is to identify the mechanisms leading to quantitative and qualitative changes in flavonol and anthocyanin profiles, in response to separate and combined UV-B and water deficit. Grapevines (Vitis vinifera L. cv. Tempranillo) were exposed to three levels of UV-B radiation (0, 5.98 and 9.66 kJ m(-2) day(-1)) and subjected to two water regimes. A strong effect of UV-B on flavonol and anthocyanin biosynthesis was found, resulting in an increased anthocyanin concentration and a change in their profile. Concomitantly, two key biosynthetic genes (FLS1 and UFGT) were up-regulated by UV-B, leading to increased flavonol and anthocyanin skin concentration. Changes in flavonol and anthocyanin composition were explained to a large extend by transcript levels of F3'H, F3'5'H and OMT2. A significant interaction between UV-B and water deficit was found in the relative abundance of 3'4' and 3'4'5' substituted flavonols, but not in their anthocyanin homologues. The ratio between 3'4'5' and 3'4' substituted flavonols was linearly related to the ratios of F3'5'H and FLS1 transcription, two steps up-regulated independently by water deficit and UV-B radiation, respectively. Our results indicate that changes in flavonol profiles in response to environmental conditions are not only a consequence of changes in the expression of flavonoid hydroxylases; but also the result of the competition of FLS, F3'5'H and F3'H enzymes for the same flavonol substrates.

Genetic analysis of the biosynthesis of 2-methoxy-3-isobutylpyrazine, a major grape-derived aroma compound impacting wine quality.

Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.

Long-term in vitro culture of grape berries and its application to assess the effects of sugar supply on anthocyanin accumulation.

Grape berry development and ripening are under complex regulation by the nutrients, hormones, and environment cues sensed by the berry. However, the biochemical and molecular mechanisms underlying these types of regulation are poorly understood. A simplified but realistic model system that enables fruit growth conditions to be modulated easily will facilitate the deciphering of these mechanisms. Here, an in vitro culture system of intact detached grape berries was developed by coupling the production of greenhouse fruiting-cuttings and in vitro organ culture techniques. (13)C and (15)N labelling experiments showed that this system enables the intact detached berries actively to absorb and utilize carbon and nitrogen from the culture medium. It was further used to study the effects of sugars on anthocyanin accumulation. A sucrose concentration >2% could induce anthocyanin synthesis in the absence of additional exogenous abscisic acid. The higher the sucrose concentration, the earlier was the induction of anthocyanin accumulation. Glucose, fructose, and sucrose increased anthocyanin accumulation, with glucose and fructose being more effective than sucrose. This increase was not due to an increase in its precursor level, since the phenylalanine content was decreased by a high sugar supply. Instead, genome-wide transcriptome analysis suggests that the sugar-induced enhancement of anthocyanin accumulation results from altered expression of regulatory and structural genes (especially UDP-glucose:anthocyanidin 3-O-glucosyltransferase), together with massive reprogramming in signalling transduction pathways. This in vitro system may serve to study the response of berry composition to nutrient factors and hormones, and their interaction with environmental factors (e.g. light and temperature), which can all be finely tuned and controlled.

Berry ripening: recently heard through the grapevine.

Grapevine (Vitis vinifera L.) is a non-climacteric fruit species used as table fruit, dried raisins, and for vinification (wines) and distillation (liquors). In recent years, our knowledge of the molecular basis of ripening regulation has improved. Water status, light conditions, and temperature may hasten, delay, or enhance ripening. Hormones seem to play a central role, as their concentrations change prior to and during ripening and in response to several environmental cues. The review summarizes recent data related to the molecular and hormonal control of grape berry development and ripening, with special emphasis on secondary metabolism and its response to the environment, and pinpoints some experimental limitations.

The grape berry-specific basic helix-loop-helix transcription factor VvCEB1 affects cell size.

The development of fleshy fruits involves complex physiological and biochemical changes. After fertilization, fruit growth usually begins with cell division, continues with both cell division and expansion, allowing fruit set to occur, and ends with cell expansion only. In spite of the economical importance of grapevine, the molecular mechanisms controlling berry growth are not fully understood. The present work identified and characterized Vitis vinifera cell elongation bHLH protein (VvCEB1), a basic helix-loop-helix (bHLH) transcription factor controlling cell expansion in grape. VvCEB1 was expressed specifically in berry-expanding tissues with a maximum around veraison. The study of VvCEB1 promoter activity in tomato confirmed its specific fruit expression during the expansion phase. Overexpression of VvCEB1 in grape embryos showed that this protein stimulates cell expansion and affects the expression of genes involved in cell expansion, including genes of auxin metabolism and signalling. Taken together, these data show that VvCEB1 is a fruit-specific bHLH transcription factor involved in grape berry development.

Metabolic profiling reveals coordinated switches in primary carbohydrate metabolism in grape berry (Vitis vinifera L.), a non-climacteric fleshy fruit.

Changes in carbohydrate metabolism during grape berry development play a central role in shaping the final composition of the fruit. The present work aimed to identify metabolic switches during grape development and to provide insights into the timing of developmental regulation of carbohydrate metabolism. Metabolites from central carbon metabolism were measured using high-pressure anion-exchange chromatography coupled to tandem mass spectrometry and enzymatic assays during the development of grape berries from either field-grown vines or fruiting cuttings grown in the greenhouse. Principal component analysis readily discriminated the various stages of berry development, with similar trajectories for field-grown and greenhouse samples. This showed that each stage of fruit development had a characteristic metabolic profile and provided compelling evidence that the fruit-bearing cuttings are a useful model system to investigate regulation of central carbon metabolism in grape berry. The metabolites measured showed tight coordination within their respective pathways, clustering into sugars and sugar-phosphate metabolism, glycolysis, and the tricarboxylic acid cycle. In addition, there was a pronounced shift in metabolism around veraison, characterized by rapidly increasing sugar levels and decreasing organic acids. In contrast, glycolytic intermediates and sugar phosphates declined before veraison but remained fairly stable post-veraison. In summary, these detailed and comprehensive metabolite analyses revealed the timing of important switches in primary carbohydrate metabolism, which could be related to transcriptional and developmental changes within the berry to achieve an integrated understanding of grape berry development. The results are discussed in a meta-analysis comparing metabolic changes in climacteric versus non-climacteric fleshy fruits.

Anthocyanin phytochemical profiles and anti-oxidant activities of Vitis candicans and Vitis doaniana.

INTRODUCTION: Grapes are one of the most important fruit crops in the world. The quality of red grape berries greatly depends on skin colour, mainly due to the anthocyanin profile. Today, the American Vitis species have the greatest potential for breeding work. They have multiple resistance properties in comparison with Vitis vinifera but little is known about their anthocyanin content. OBJECTIVE: To determine the anti-oxidant properties and anthocyanin profile of two American species, Vitis candicans and Vitis doaniana, by using LC-MS(n) and LC-NMR. METHODS: Grape extracts were prepared by extraction of berry skins with acidified methanol. The complete structure elucidation of the individual anthocyanins was performed with LC-MS(n) , LC-NMR and NMR experiments. Individual anthocyanins in the extracts were quantified by using malvidin glucoside as external standard. The anti-oxidant activities of grape skin extracts were evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH•) radical scavenging and oxygen radical absorbance capacity (ORAC) assays. RESULTS: By using LC-MS(n) and LC-NMR experiments, 30 anthocyanins were identified and quantified in the two Vitis species, including two new cis-p-coumaroyl derivatives. Vitis candicans and V. doaniana showed significant differences in their anthocyanin profile. These two Vitis species possess low-to-medium anti-oxidant activities in comparison with V. vinifera. CONCLUSION: The profiles of 30 anthocyanins were established unambiguously in two American Vitis species.

PlantACT! - how to tackle the climate crisis.

Greenhouse gas (GHG) emissions have created a global climate crisis which requires immediate interventions to mitigate the negative effects on all aspects of life on this planet. As current agriculture and land use contributes up to 25% of total GHG emissions, plant scientists take center stage in finding possible solutions for a transition to sustainable agriculture and land use. In this article, the PlantACT! (Plants for climate ACTion!) initiative of plant scientists lays out a road map of how and in which areas plant scientists can contribute to finding immediate, mid-term, and long-term solutions, and what changes are necessary to implement these solutions at the personal, institutional, and funding levels.

VvGOLS1 and VvHsfA2 are involved in the heat stress responses in grapevine berries.

Among various environmental factors, temperature is a major regulator affecting plant growth, development and fruit composition. Grapevine is the most cultivated fruit plant throughout the world, and grapes are used for wine production and human consumption. The molecular mechanisms involved in grapevine tolerance to high temperature, especially at the fruit level, are poorly understood. To better characterize the sensitivity of berries to the microenvironment, high temperature conditions were locally applied to Vitis vinifera Cabernet Sauvignon clusters. Two genes, VvGOLS1 and VvHsfA2, up-regulated by this treatment, were identified and further characterized. The expression profile of VvGOLS1 correlated positively with galactinol accumulation in heat-stressed berries. However, no galactinol derivatives, such as raffinose and stachyose, accumulated upon heat stress. Heterologous expression of VvGOLS1 in Escherichia coli showed that it encodes a functional galactinol synthase. Transient expression assays showed that the heat stress factor VvHsfA2 transactivates the promoter of VvGOLS1 in a heat stress-dependent manner. Taken together, our results highlight the intrinsic capacity of grape berries to perceive heat stress and to initiate adaptive responses, suggesting that galactinol may play a signaling role in these responses.

Regulation of anthocyanin and sugar accumulation in grape berry through carbon limitation and exogenous ABA application.

To optimize vineyard management practices to adapt viticulture to climate change, knowledge of the regulation mechanism of metabolite accumulation under carbon source limitation and abscisic acid (ABA) application in grapes should be deepened. Here, carbon source limitations were imposed by reducing leaf area from 12 to 2 leaves per vine (at pea sized stage, - 2L-P; or one week prior to veraison - 2L-V) and phloem girdling between the second and third leaf from bottom to top (one week prior to veraison - 12L-girdling) were compared for their effects on berry composition. All three modalities significantly reduced sugar, anthocyanin and ABA content in comparison with berries under sufficient carbon supply (12 leaves per vine - 12L), with 2L-V being the greatest. Allowing leaf area to partially recover (2L-R) or berry ABA application (400 mg. L-1) one week before veraison increased the ratio of anthocyanin to sugar under source limitation. Combined with the analysis of berry metabolites and transcript abundances, our results indicate that source limitation and exogenous ABA co-regulated anthocyanins content through differential gene expression.

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity.

BACKGROUND: Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. RESULTS: In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. CONCLUSIONS: Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.

Molecular Tools for Adapting Viticulture to Climate Change.

Adaptation of viticulture to climate change includes exploration of new geographical areas, new training systems, new management practices, or new varieties, both for rootstocks and scions. Molecular tools can be defined as molecular approaches used to study DNAs, RNAs, and proteins in all living organisms. We present here the current knowledge about molecular tools and their potential usefulness in three aspects of grapevine adaptation to the ongoing climate change. (i) Molecular tools for understanding grapevine response to environmental stresses. A fine description of the regulation of gene expression is a powerful tool to understand the physiological mechanisms set up by the grapevine to respond to abiotic stress such as high temperatures or drought. The current knowledge on gene expression is continuously evolving with increasing evidence of the role of alternative splicing, small RNAs, long non-coding RNAs, DNA methylation, or chromatin activity. (ii) Genetics and genomics of grapevine stress tolerance. The description of the grapevine genome is more and more precise. The genetic variations among genotypes are now revealed with new technologies with the sequencing of very long DNA molecules. High throughput technologies for DNA sequencing also allow now the genetic characterization at the same time of hundreds of genotypes for thousands of points in the genome, which provides unprecedented datasets for genotype-phenotype associations studies. We review the current knowledge on the genetic determinism of traits for the adaptation to climate change. We focus on quantitative trait loci and molecular markers available for developmental stages, tolerance to water stress/water use efficiency, sugar content, acidity, and secondary metabolism of the berries. (iii) Controlling the genome and its expression to allow breeding of better-adapted genotypes. High-density DNA genotyping can be used to select genotypes with specific interesting alleles but genomic selection is also a powerful method able to take into account the genetic information along the whole genome to predict a phenotype. Modern technologies are also able to generate mutations that are possibly interesting for generating new phenotypes but the most promising one is the direct editing of the genome at a precise location.