MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Alternative splicing regulation appears to play a crucial role in grape berry development and is also potentially involved in adaptation responses to the environment.

BACKGROUND: Alternative splicing (AS) produces transcript variants playing potential roles in proteome diversification and gene expression regulation. AS modulation is thus essential to respond to developmental and environmental stimuli. In grapevine, a better understanding of berry development is crucial for implementing breeding and viticultural strategies allowing adaptation to climate changes. Although profound changes in gene transcription have been shown to occur in the course of berry ripening, no detailed study on splicing modifications during this period has been published so far. We report here on the regulation of gene AS in developing berries of two grapevine (Vitis vinifera L.) varieties, Gewurztraminer (Gw) and Riesling (Ri), showing distinctive phenotypic characteristics. Using the software rMATS, the transcriptomes of berries at four developmental steps, from the green stage to mid-ripening, were analysed in pairwise comparisons between stages and varieties. RESULTS: A total of 305 differential AS (DAS) events, affecting 258 genes, were identified. Interestingly, 22% of these AS events had not been reported before. Among the 80 genes that underwent the most significant variations during ripening, 22 showed a similar splicing profile in Gw and Ri, which suggests their involvement in berry development. Conversely, 23 genes were subjected to splicing regulation in only one variety. In addition, the ratios of alternative isoforms were different in Gw and Ri for 35 other genes, without any change during ripening. This last result indicates substantial AS differences between the two varieties. Remarkably, 8 AS events were specific to one variety, due to the lack of a splice site in the other variety. Furthermore, the transcription rates of the genes affected by stage-dependent splicing regulation were mostly unchanged, identifying AS modulation as an independent way of shaping the transcriptome. CONCLUSIONS: The analysis of AS profiles in grapevine varieties with contrasting phenotypes revealed some similarity in the regulation of several genes with developmental functions, suggesting their involvement in berry ripening. Additionally, many splicing differences were discovered between the two varieties, that could be linked to phenotypic specificities and distinct adaptive capacities. Together, these findings open perspectives for a better understanding of berry development and for the selection of grapevine genotypes adapted to climate change.

Genetic variations of acidity in grape berries are controlled by the interplay between organic acids and potassium.

KEY MESSAGE: In a grapevine segregating population, genomic regions governing berry pH were identified, paving the way for breeding new grapevine varieties best adapted to a warming climate. As a consequence of global warming, grapevine berry acidity is expected to dramatically decrease. Adapting grapevine (Vitis vinifera L.) varieties to the climatic conditions of the future requires a better understanding of the genetic architecture of acidity-related traits. For this purpose, we studied during five growing seasons 120 individuals from a grapevine biparental cross. Each offspring was genotyped by simple sequence repeats markers and by hybridization on a 20-K Grapevine Illumina® SNP chip. Quantitative trait loci (QTLs) for pH colocalized with QTLs for the ratio between potassium and tartaric acid concentrations, on chromosomes 10, 11 and 13. Strong QTLs for malic acid concentration or for the malic acid-to-tartaric acid ratio, on chromosomes 6 and 8, were not associated with variations of pH but can be useful for controlling pH stability under high temperatures. Our study highlights the interdependency between acidity parameters and consequently the constraints and degrees of freedom for designing grapevine genotypes better adapted to the expected warmer climatic conditions. In particular, it is possible to create grapevine genotypes with a high berry acidity as the result of both high tartaric acid concentrations and low K+ accumulation capacities.

A grapevine cytochrome P450 generates the precursor of wine lactone, a key odorant in wine.

Monoterpenes are important constituents of the aromas of food and beverages, including wine. Among monoterpenes in wines, wine lactone has the most potent odor. It was proposed to form via acid-catalyzed cyclization of (E)-8-carboxylinalool during wine maturation. It only reaches very low concentrations in wine but its extremely low odor detection threshold makes it an important aroma compound. Using LC-MS/MS, we show here that the (E)-8-carboxylinalool content in wines correlates with their wine lactone content and estimate the kinetic constant for the very slow formation of wine lactone from (E)-8-carboxylinalool. We show that (E)-8-carboxylinalool is accumulated as a glycoside in grape (Vitis vinifera) berries and that one of the cytochrome P450 enzymes most highly expressed in maturing berries, CYP76F14, efficiently oxidizes linalool to (E)-8-carboxylinalool. Our analysis of (E)-8-carboxylinalool in Riesling × Gewurztraminer grapevine progeny established that the CYP76F14 gene co-locates with a quantitative trait locus for (E)-8-carboxylinalool content in grape berries. Our data support the role of CYP76F14 as the major (E)-8-carboxylinalool synthase in grape berries and the role of (E)-8-carboxylinalool as a precursor to wine lactone in wine, providing new insights into wine and grape aroma metabolism, and new methods for food and aroma research and production.

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.

Phenotyping grapevine resistance to downy mildew: deep learning as a promising tool to assess sporulation and necrosis.

BACKGROUND: Downy mildew is a plant disease that affects all cultivated European grapevine varieties. The disease is caused by the oomycete Plasmopara viticola. The current strategy to control this threat relies on repeated applications of fungicides. The most eco-friendly and sustainable alternative solution would be to use bred-resistant varieties. During breeding programs, some wild Vitis species have been used as resistance sources to introduce resistance loci in Vitis vinifera varieties. To ensure the durability of resistance, resistant varieties are built on combinations of these loci, some of which are unfortunately already overcome by virulent pathogen strains. The development of a high-throughput machine learning phenotyping method is now essential for identifying new resistance loci. RESULTS: Images of grapevine leaf discs infected with P. viticola were annotated with OIV 452-1 values, a standard scale, traditionally used by experts to assess resistance visually. This descriptor takes two variables into account the complete phenotype of the symptom: sporulation and necrosis. This annotated dataset was used to train neural networks. Various encoders were used to incorporate prior knowledge of the scale's ordinality. The best results were obtained with the Swin transformer encoder which achieved an accuracy of 81.7%. Finally, from a biological point of view, the model described the studied trait and identified differences between genotypes in agreement with human observers, with an accuracy of 97% but at a high-throughput 650% faster than that of humans. CONCLUSION: This work provides a fast, full pipeline for image processing, including machine learning, to describe the symptoms of grapevine leaf discs infected with P. viticola using the OIV 452-1, a two-symptom standard scale that considers sporulation and necrosis. If symptoms are frequently assessed by visual observation, which is time-consuming, low-throughput, tedious, and expert dependent, the method developed sweeps away all these constraints. This method could be extended to other pathosystems studied on leaf discs where disease symptoms are scored with ordinal scales.

LiDAR Is Effective in Characterizing Vine Growth and Detecting Associated Genetic Loci.

The strong societal demand to reduce pesticide use and adaptation to climate change challenges the capacities of phenotyping new varieties in the vineyard. High-throughput phenotyping is a way to obtain meaningful and reliable information on hundreds of genotypes in a limited period. We evaluated traits related to growth in 209 genotypes from an interspecific grapevine biparental cross, between IJ119, a local genitor, and Divona, both in summer and in winter, using several methods: fresh pruning wood weight, exposed leaf area calculated from digital images, leaf chlorophyll concentration, and LiDAR-derived apparent volumes. Using high-density genetic information obtained by the genotyping by sequencing technology (GBS), we detected 6 regions of the grapevine genome [quantitative trait loci (QTL)] associated with the variations of the traits in the progeny. The detection of statistically significant QTLs, as well as correlations (R2) with traditional methods above 0.46, shows that LiDAR technology is effective in characterizing the growth features of the grapevine. Heritabilities calculated with LiDAR-derived total canopy and pruning wood volumes were high, above 0.66, and stable between growing seasons. These variables provided genetic models explaining up to 47% of the phenotypic variance, which were better than models obtained with the exposed leaf area estimated from images and the destructive pruning weight measurements. Our results highlight the relevance of LiDAR-derived traits for characterizing genetically induced differences in grapevine growth and open new perspectives for high-throughput phenotyping of grapevines in the vineyard.

An improved reference of the grapevine genome reasserts the origin of the PN40024 highly homozygous genotype.

The genome sequence of the diploid and highly homozygous Vitis vinifera genotype PN40024 serves as the reference for many grapevine studies. Despite several improvements to the PN40024 genome assembly, its current version PN12X.v2 is quite fragmented and only represents the haploid state of the genome with mixed haplotypes. In fact, being nearly homozygous, this genome contains several heterozygous regions that are yet to be resolved. Taking the opportunity of improvements that long-read sequencing technologies offer to fully discriminate haplotype sequences, an improved version of the reference, called PN40024.v4, was generated. Through incorporating long genomic sequencing reads to the assembly, the continuity of the 12X.v2 scaffolds was highly increased with a total number decreasing from 2,059 to 640 and a reduction in N bases of 88%. Additionally, the full alternative haplotype sequence was built for the first time, the chromosome anchoring was improved and the number of unplaced scaffolds was reduced by half. To obtain a high-quality gene annotation that outperforms previous versions, a liftover approach was complemented with an optimized annotation workflow for Vitis. Integration of the gene reference catalogue and its manual curation have also assisted in improving the annotation, while defining the most reliable estimation of 35,230 genes to date. Finally, we demonstrated that PN40024 resulted from 9 selfings of cv. "Helfensteiner" (cross of cv. "Pinot noir" and "Schiava grossa") instead of a single "Pinot noir". These advances will help maintain the PN40024 genome as a gold-standard reference, also contributing toward the eventual elaboration of the grapevine pangenome.

Towards the adaptation of grapevine varieties to climate change: QTLs and candidate genes for developmental stages.

The genetic determinism of developmental stages in grapevine was studied in the progeny of a cross between grapevine cultivars Riesling and Gewurztraminer by combining ecophysiological modelling, genetic analysis and data mining of the grapevine whole genome sequence. The dates of three phenological stages, budbreak, flowering and veraison, were recorded during four successive years for 120 genotypes in the vineyard. The phenotypic data analysed were the duration of three periods expressed in thermal time (degree-days): 15 February to budbreak (Bud), budbreak to flowering (Flo) and flowering to veraison (Ver). Parental and consensus genetic maps were built using 153 microsatellite markers on 188 individuals. Six independent quantitative trait loci (QTLs) were detected for the three phases. They were located on chromosomes 4 and 19 for Bud, chromosomes 7 and 14 for Flo and chromosomes 16 and 18 for Ver. Interactions were detected between loci and also between alleles at the same locus. Using the available grapevine whole-genome sequences, candidate genes underlying the QTLs were identified. VvFT, on chromosome 7, and a CONSTANS-like gene, on chromosome 14, were found to colocalise with the QTLs for flowering time. Genes related to the abscisic acid response and to sugar metabolism were detected within the confidence intervals of QTLs for veraison time. Their possible roles in the developmental process are discussed. These results raise new hypotheses for a better understanding of the physiological processes governing grapevine phenology and provide a framework for breeding new varieties adapted to the future predicted climatic conditions.

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.

Grapevine and Wine Metabolomics-Based Guidelines for FAIR Data and Metadata Management.

In the era of big and omics data, good organization, management, and description of experimental data are crucial for achieving high-quality datasets. This, in turn, is essential for the export of robust results, to publish reliable papers, make data more easily available, and unlock the huge potential of data reuse. Lately, more and more journals now require authors to share data and metadata according to the FAIR (Findable, Accessible, Interoperable, Reusable) principles. This work aims to provide a step-by-step guideline for the FAIR data and metadata management specific to grapevine and wine science. In detail, the guidelines include recommendations for the organization of data and metadata regarding (i) meaningful information on experimental design and phenotyping, (ii) sample collection, (iii) sample preparation, (iv) chemotype analysis, (v) data analysis (vi) metabolite annotation, and (vii) basic ontologies. We hope that these guidelines will be helpful for the grapevine and wine metabolomics community and that it will benefit from the true potential of data usage in creating new knowledge being revealed.

The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication.

BACKGROUND: A key step in domestication of the grapevine was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. sativa (V. vinifera). It is known that V. sylvestris has an XY system and V. vinifera a modified Y haplotype (Yh) and that the sex locus is small, but it has not previously been precisely characterized. RESULTS: We generate a high-quality de novo reference genome for V. sylvestris, onto which we map whole-genome re-sequencing data of a cross to locate the sex locus. Assembly of the full X, Y, and Yh haplotypes of V. sylvestris and V. vinifera sex locus and examining their gene content and expression profiles during flower development in wild and cultivated accessions show that truncation and deletion of tapetum and pollen development genes on the X haplotype likely causes male sterility, while the upregulation of a Y allele of a cytokinin regulator (APRT3) may cause female sterility. The downregulation of this cytokinin regulator in the Yh haplotype may be sufficient to trigger reversal to hermaphroditism. Molecular dating of X and Y haplotypes is consistent with the sex locus being as old as the Vitis genus, but the mechanism by which recombination was suppressed remains undetermined. CONCLUSIONS: We describe the genomic and evolutionary characterization of the sex locus of cultivated and wild grapevine, providing a coherent model of sex determination in the latter and for transition from dioecy to hermaphroditism during domestication.

Annotation, classification, genomic organization and expression of the Vitis vinifera CYPome.

Cytochromes P450 are enzymes that participate in a wide range of functions in plants, from hormonal signaling and biosynthesis of structural polymers, to defense or communication with other organisms. They represent one of the largest gene/protein families in the plant kingdom. The manual annotation of cytochrome P450 genes in the genome of Vitis vinifera PN40024 revealed 579 P450 sequences, including 279 complete genes. Most of the P450 sequences in grapevine genome are organized in physical clusters, resulting from tandem or segmental duplications. Although most of these clusters are small (2 to 35, median = 3), some P450 families, such as CYP76 and CYP82, underwent multiple duplications and form large clusters of homologous sequences. Analysis of gene expression revealed highly specific expression patterns, which are often the same within the genes in large physical clusters. Some of these genes are induced upon biotic stress, which points to their role in plant defense, whereas others are specifically activated during grape berry ripening and might be responsible for the production of berry-specific metabolites, such as aroma compounds. Our work provides an exhaustive and robust annotation including clear identification, structural organization, evolutionary dynamics and expression patterns for the grapevine cytochrome P450 families, paving the way to efficient functional characterization of genes involved in grapevine defense pathways and aroma biosynthesis.

Inter-Species Comparative Analysis of Components of Soluble Sugar Concentration in Fleshy Fruits.

The soluble sugar concentration of fleshy fruit is a key determinant of fleshy fruit quality. It affects directly the sweetness of fresh fruits and indirectly the properties of processed products (e.g., alcohol content in wine). Despite considerable divergence among species, soluble sugar accumulation in a fruit results from the complex interplay of three main processes, namely sugar import, sugar metabolism, and water dilution. Therefore, inter-species comparison would help to identify common and/or species-specific modes of regulation in sugar accumulation. For this purpose, a process-based mathematical framework was used to compare soluble sugar accumulation in three fruits: grape, tomato, and peach. Representative datasets covering the time course of sugar accumulation during fruit development were collected. They encompassed 104 combinations of species (3), genotypes (30), and growing conditions (19 years and 16 nutrient and environmental treatments). At maturity, grape showed the highest soluble sugar concentrations (16.5-26.3 g/100 g FW), followed by peach (2.2 to 20 g/100 g FW) and tomato (1.4 to 5 g/100 g FW). Main processes determining soluble sugar concentration were decomposed into sugar importation, metabolism, and water dilution with the process-based analysis. Different regulation modes of soluble sugar concentration were then identified, showing either import-based, dilution-based, or import and dilution dual-based. Firstly, the higher soluble sugar concentration in grape than in tomato is a result of higher sugar importation. Secondly, the higher soluble sugar concentration in grape than in peach is due to a lower water dilution. The third mode of regulation is more complicated than the first two, with differences both in sugar importation and water dilution (grape vs. cherry tomato; cherry tomato vs. peach; peach vs. tomato). On the other hand, carbon utilization for synthesis of non-soluble sugar compounds (namely metabolism) was conserved among the three fruit species. These distinct modes appear to be quite species-specific, but the intensity of the effect may significantly vary depending on the genotype and management practices. These results provide novel insights into the drivers of differences in soluble sugar concentration among fleshy fruits.

A semi-automatic non-destructive method to quantify grapevine downy mildew sporulation.

The availability of fast, reliable and non-destructive methods for the analysis of pathogen development contributes to a better understanding of plant-pathogen interactions. This is particularly true for the genetic analysis of quantitative resistance to plant pathogens, where the availability of a method allowing a precise quantification of pathogen development allows the reliable detection of different genomic regions involved in the resistance. Grapevine downy mildew, caused by the biotrophic Oomycete Plasmopara viticola, is one of the most important diseases affecting viticulture. Here we report the development of a simple image analysis-based semi-automatic method for the quantification of grapevine downy mildew sporulation, requiring just a compact digital camera and the open source software ImageJ. We confirm the suitability of the method for the analysis of the interaction between grapevine and downy mildew by performing QTL analysis of resistance to downy mildew as well as analysis of the kinetics of downy mildew infection. The non-destructive nature of the method will enable comparison between the phenotypic and molecular data obtained from the very same sample, resulting in a more accurate description of the interaction, while its simplicity makes it easily adaptable to other plant-pathogen interactions, in particular those involving downy mildews.

A grapevine (Vitis vinifera L.) deoxy-D: -xylulose synthase gene colocates with a major quantitative trait loci for terpenol content.

Linalool, geraniol, nerol, citronellol and alpha-terpineol are isoprenoid molecules responsible for specific aromas found in grapes and wines. Total concentrations (free and bound forms) of these compounds were measured in the skins of mature berries during 2 successive years in two progenies obtained from Muscat Ottonel and Gewurztraminer selfings. Partial genetic maps based on microsatellite markers were constructed and several quantitative trait loci (QTLs) related to terpenol content were detected. A major QTL on linkage group (LG) 5 colocated with a deoxy-D: -xylulose synthase gene, coding for the first enzyme of the plastidial isoprenoid biosynthesis pathway. The number of favourable alleles at this locus determined the level of terpenol synthesis. A second QTL, on LG 10, was found to determine the balance linalool versus geraniol and nerol in the Muscat self-progeny plants.