The transcription factor VviNAC60 regulates senescence- and ripening-related processes in grapevine.

Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit crops because the winemaking industry has huge economic relevance worldwide. Uncovering the molecular mechanisms controlling the developmental progression of plant organs will prove essential for maintaining high-quality grapes, expressly in the context of climate change, which impairs the ripening process. Through a deep inspection of transcriptomic data, we identified VviNAC60, a member of the NAC transcription factor family, as a putative regulator of grapevine organ maturation. We explored VviNAC60 binding landscapes through DNA affinity purification followed by sequencing and compared bound genes with transcriptomics datasets from grapevine plants stably and transiently overexpressing VviNAC60 to define a set of high-confidence targets. Among these, we identified key molecular markers associated with organ senescence and fruit ripening. Physiological, metabolic, and promoter activation analyses showed that VviNAC60 induces chlorophyll degradation and anthocyanin accumulation through the upregulation of STAY-GREEN PROTEIN 1 (VviSGR1) and VviMYBA1, respectively, with the latter being upregulated through a VviNAC60-VviNAC03 regulatory complex. Despite sharing a closer phylogenetic relationship with senescence-related homologs to the NAC transcription factor AtNAP, VviNAC60 complemented the nonripening(nor) mutant phenotype in tomato (Solanum lycopersicum), suggesting a dual role as an orchestrator of both ripening- and senescence-related processes. Our data support VviNAC60 as a regulator of processes initiated in the grapevine vegetative- to mature-phase organ transition and therefore as a potential target for enhancing the environmental resilience of grapevine by fine-tuning the duration of the vegetative phase.

VviNAC33 promotes organ de-greening and represses vegetative growth during the vegetative-to-mature phase transition in grapevine.

Plants undergo several developmental transitions during their life cycle. In grapevine, a perennial woody fruit crop, the transition from vegetative/green-to-mature/woody growth involves transcriptomic reprogramming orchestrated by a small group of genes encoding regulators, but the underlying molecular mechanisms are not fully understood. We investigated the function of the transcriptional regulator VviNAC33 by generating and characterizing transgenic overexpressing grapevine lines and a chimeric repressor, and by exploring its putative targets through a DNA affinity purification sequencing (DAP-seq) approach combined with transcriptomic data. We demonstrated that VviNAC33 induces leaf de-greening, inhibits organ growth and directly activates the expression of STAY-GREEN PROTEIN 1 (SGR1), which is involved in Chl and photosystem degradation, and AUTOPHAGY 8f (ATG8f), which is involved in the maturation of autophagosomes. Furthermore, we show that VviNAC33 directly inhibits AUXIN EFFLUX FACILITATOR PIN1, RopGEF1 and ATP SYNTHASE GAMMA CHAIN 1T (ATPC1), which are involved in photosystem II integrity and activity. Our results show that VviNAC33 plays a major role in terminating photosynthetic activity and organ growth as part of a regulatory network governing the vegetative-to-mature phase transition.

The grapevine VviPrx31 peroxidase as a candidate gene involved in anthocyanin degradation in ripening berries under high temperature.

Anthocyanin levels decline in some red grape berry varieties ripened under high-temperature conditions, but the underlying mechanism is not yet clear. Here we studied the effects of two different temperature regimes, representing actual Sangiovese (Vitis vinifera L.) viticulture regions, on the accumulation of mRNAs and enzymes controlling berry skin anthocyanins. Potted uniform plants of Sangiovese were kept from veraison to harvest, in two plastic greenhouses with different temperature conditions. The low temperature (LT) conditions featured average and maximum daily air temperatures of 20 and 29 °C, respectively, whereas the corresponding high temperature (HT) conditions were 22 and 36 °C, respectively. The anthocyanin concentration at harvest was much lower in HT berries than LT berries although their profile was similar under both conditions. Under HT conditions, the biosynthesis of anthocyanins was suppressed at both the transcriptional and enzymatic levels, but peroxidase activity was higher. This suggests that the low anthocyanin content of HT berries reflects the combined impact of reduced biosynthesis and increased degradation, particularly the direct role of peroxidases in anthocyanin catabolism. Overexpression of VviPrx31 decreased anthocyanin contents in Petunia hybrida petals under heat stress condition. These data suggest that high temperature can stimulate peroxidase activity thus anthocyanin degradation in ripening grape berries.

DNA-free genome editing in grapevine using CRISPR/Cas9 ribonucleoprotein complexes followed by protoplast regeneration.

CRISPR/Cas9 genome editing technology can overcome many limitations of traditional breeding, offering enormous potential for crop improvement and food production. Although the direct delivery of Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) complexes to grapevine (Vitis vinifera) protoplasts has been shown before, the regeneration of edited protoplasts into whole plants has not been reported. Here, we describe an efficient approach to obtain transgene-free edited grapevine plants by the transfection and subsequent regeneration of protoplasts isolated from embryogenic callus. As proof of concept, a single-copy green fluorescent protein reporter gene (GFP) in the grapevine cultivar Thompson Seedless was targeted and knocked out by the direct delivery of RNPs to protoplasts. CRISPR/Cas9 activity, guided by two independent sgRNAs, was confirmed by the loss of GFP fluorescence. The regeneration of GFP- protoplasts into whole plants was monitored throughout development, confirming that the edited grapevine plants were comparable in morphology and growth habit to wild-type controls. We report the first highly efficient protocol for DNA-free genome editing in grapevine by the direct delivery of preassembled Cas9-sgRNA RNP complexes into protoplasts, helping to address the regulatory concerns related to genetically modified plants. This technology could encourage the application of genome editing for the genetic improvement of grapevine and other woody crop plants.

Temperature affects organic acid, terpene and stilbene metabolisms in wine grapes during postharvest dehydration.

The partial dehydration of grapes after harvest is a traditional practice in several winegrowing regions that leads to the production of high quality wines. Postharvest dehydration (also known as withering) has a significant impact on the overall metabolism and physiology of the berry, yielding a final product that is richer in sugars, solutes, and aroma compounds. These changes are, at least in part, the result of a stress response, which is controlled at transcriptional level, and are highly dependent on the grape water loss kinetics and the environmental parameters of the facility where grapes are stored to wither. However, it is difficult to separate the effects driven by each single environmental factor from those of the dehydration rate, especially discerning the effect of temperature that greatly affects the water loss kinetics. To define the temperature influence on grape physiology and composition during postharvest dehydration, the withering of the red-skin grape cultivar Corvina (Vitis vinifera) was studied in two conditioned rooms set at distinct temperatures and at varying relative humidity to maintain an equal grape water loss rate. The effect of temperature was also studied by withering the grapes in two unconditioned facilities located in geographic areas with divergent climates. Technological, LC-MS and GC-MS analyses revealed higher levels of organic acids, flavonols, terpenes and cis- and trans-resveratrol in the grapes withered at lower temperature conditions, whereas higher concentrations of oligomeric stilbenes were found in the grapes stored at higher temperatures. Lower expression of the malate dehydrogenase and laccase, while higher expression of the phenylalanine ammonia-lyase, stilbene synthase and terpene synthase genes were detected in the grapes withered at lower temperatures. Our findings provide insights into the importance of the temperature in postharvest withering and its effect on the metabolism of the grapes and on the quality of the derived wines.

The Grape Gene Reference Catalogue as a Standard Resource for Gene Selection and Genetic Improvement.

Effective crop improvement, whether through selective breeding or biotech strategies, is largely dependent on the cumulative knowledge of a species' pangenome and its containing genes. Acquiring this knowledge is specially challenging in grapevine, one of the oldest fruit crops grown worldwide, which is known to have more than 30,000 genes. Well-established research communities studying model organisms have created and maintained, through public and private funds, a diverse range of online tools and databases serving as repositories of genomes and gene function data. The lack of such resources for the non-model, but economically important, Vitis vinifera species has driven the need for a standardised collection of genes within the grapevine community. In an effort led by the Integrape COST Action CA17111, we have recently developed the first grape gene reference catalogue, where genes are ascribed to functional data, including their accession identifiers from different genome-annotation versions (https://integrape.eu/resources/genes-genomes/). We present and discuss this gene repository together with a validation-level scheme based on varied supporting evidence found in current literature. The catalogue structure and online submission form provided permits community curation. Finally, we present the Gene Cards tool, developed within the Vitis Visualization (VitViz) platform, to visualize the data collected in the catalogue and link gene function with tissue-specific expression derived from public transcriptomic data. This perspective article aims to present these resources to the community as well as highlight their potential use, in particular for plant-breeding applications.

Rapid dehydration of grape berries dampens the post-ripening transcriptomic program and the metabolite profile evolution.

The postharvest dehydration of grape berries allows the concentration of sugars and other solutes and promotes the synthesis of metabolites and aroma compounds unique to high-quality raisin wines such as the passito wines made in Italy. These dynamic changes are dependent on environmental parameters such as temperature and relative humidity, as well as endogenous factors such as berry morphology and genotype, but the contribution of each variable is not well understood. Here, we compared berries subjected to natural or accelerated dehydration, the latter driven by forced air flow. We followed the evolution of transcript and metabolite profiles and found that accelerated dehydration clearly dampened the natural transcriptomic and metabolomic programs of postharvest berries. We found that slow dehydration over a prolonged duration is necessary to induce gene expression and metabolite accumulation associated with the final quality traits of dehydrated berries. The accumulation of key metabolites (particularly stilbenoids) during postharvest dehydration is inhibited by rapid dehydration conditions that shorten the berry life time.

Transcriptional Responses to Pre-flowering Leaf Defoliation in Grapevine Berry from Different Growing Sites, Years, and Genotypes.

Leaf removal is a grapevine canopy management technique widely used to modify the source-sink balance and/or microclimate around berry clusters to optimize fruit composition. In general, the removal of basal leaves before flowering reduces fruit set, hence achieving looser clusters, and improves grape composition since yield is generally curtailed more than proportionally to leaf area itself. Albeit responses to this practice seem quite consistent, overall vine performance is affected by genotype, environmental conditions, and severity of treatment. The physiological responses of grape varieties to defoliation practices have been widely investigated, and just recently a whole genome transcriptomic approach was exploited showing an extensive transcriptome rearrangement in berries defoliated before flowering. Nevertheless, the extent to which these transcriptomic reactions could be manifested by different genotypes and growing environments is entirely unexplored. To highlight general responses to defoliation vs. different locations, we analyzed the transcriptome of cv. Sangiovese berries sampled at four development stages from pre-flowering defoliated vines in two different geographical areas of Italy. We obtained and validated five markers of the early defoliation treatment in Sangiovese, an ATP-binding cassette transporter, an auxin response factor, a cinnamyl alcohol dehydrogenase, a flavonoid 3-O-glucosyltransferase and an indole-3-acetate beta-glucosyltransferase. Candidate molecular markers were also obtained in another three grapevine genotypes (Nero d'Avola, Ortrugo, and Ciliegiolo), subjected to the same level of selective pre-flowering defoliation (PFD) over two consecutive years in their different areas of cultivation. The flavonol synthase was identified as a marker in the pre-veraison phase, the jasmonate methyltransferase during the transition phase and the abscisic acid receptor PYL4 in the ripening phase. The characterization of transcriptome changes in Sangiovese berry after PFD highlights, on one hand, the stronger effect of environment than treatment on the whole berry transcriptome rearrangement during development and, on the other, expands existing knowledge of the main molecular and biochemical modifications occurring in defoliated vines. Moreover, the identification of candidate genes associated with PFD in different genotypes and environments provides new insights into the applicability and repeatability of this crop practice, as well as its possible agricultural and qualitative outcomes across genetic and environmental variability.

Plasticity of the Berry Ripening Program in a White Grape Variety.

Grapevine (Vitis vinifera L.) is considered one of the most environmentally sensitive crops and is characterized by broad phenotypic plasticity, offering important advantages such as the large range of different wines that can be produced from the same cultivar, and the adaptation of existing cultivars to diverse growing regions. The uniqueness of berry quality traits reflects complex interactions between the grapevine plant and the combination of natural factors and human cultural practices which leads to the expression of wine typicity. Despite the scientific and commercial importance of genotype interactions with growing conditions, few studies have characterized the genes and metabolites directly involved in this phenomenon. Here, we used two large-scale analytical approaches to explore the metabolomic and transcriptomic basis of the broad phenotypic plasticity of Garganega, a white berry variety grown at four sites characterized by different pedoclimatic conditions (altitudes, soil texture, and composition). These conditions determine berry ripening dynamics in terms of sugar accumulation and the abundance of phenolic compounds. Multivariate analysis unraveled a highly plastic metabolomic response to different environments, especially the accumulation of hydroxycinnamic and hydroxybenzoic acids and flavonols. Principal component analysis (PCA) revealed that the four sites strongly affected the berry transcriptome allowing the identification of environmentally-modulated genes and the plasticity of commonly-modulated transcripts at different sites. Many genes that control transcription, translation, transport, and carbohydrate metabolism showed different expression depending on the environmental conditions, indicating a key role in the observed transcriptomic plasticity of Garganega berries. Interestingly, genes representing the phenylpropanoid/flavonoid pathway showed plastic responses to the environment mirroring the accumulation of the corresponding metabolites. The comparison of Garganega and Corvina berries showed that the metabolism of phenolic compounds is more plastic in ripening Garganega berries under different pedoclimatic conditions.

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.