Auxin treatment of grapevine (Vitis vinifera L.) berries delays ripening onset by inhibiting cell expansion.

KEY MESSAGE: Auxin treatment of grape (Vitis vinifera L.) berries delays ripening by inducing changes in gene expression and cell wall metabolism and could combat some deleterious climate change effects. Auxins are inhibitors of grape berry ripening and their application may be useful to delay harvest to counter effects of climate change. However, little is known about how this delay occurs. The expression of 1892 genes was significantly changed compared to the control during a 48 h time-course where the auxin 1-naphthaleneacetic acid (NAA) was applied to pre-veraison grape berries. Principal component analysis showed that the control and auxin-treated samples were most different at 3 h post-treatment when approximately three times more genes were induced than repressed by NAA. There was considerable cross-talk between hormone pathways, particularly between those of auxin and ethylene. Decreased expression of genes encoding putative cell wall catabolic enzymes (including those involved with pectin) and increased expression of putative cellulose synthases indicated that auxins may preserve cell wall structure. This was confirmed by immunochemical labelling of berry sections using antibodies that detect homogalacturonan (LM19) and methyl-esterified homogalacturonan (LM20) and by labelling with the CMB3a cellulose-binding module. Comparison of the auxin-induced changes in gene expression with the pattern of these genes during berry ripening showed that the effect on transcription is a mix of changes that may specifically alter the progress of berry development in a targeted manner and others that could be considered as non-specific changes. Several lines of evidence suggest that cell wall changes and associated berry softening are the first steps in ripening and that delaying cell expansion can delay ripening providing a possible mechanism for the observed auxin effects.

Grapevine field experiments reveal the contribution of genotype, the influence of environment and the effect of their interaction (G×E) on the berry transcriptome.

Changes in the performance of genotypes in different environments are defined as genotype × environment (G×E) interactions. In grapevine (Vitis vinifera), complex interactions between different genotypes and climate, soil and farming practices yield unique berry qualities. However, the molecular basis of this phenomenon remains unclear. To dissect the basis of grapevine G×E interactions we characterized berry transcriptome plasticity, the genome methylation landscape and within-genotype allelic diversity in two genotypes cultivated in three different environments over two vintages. We identified, through a novel data-mining pipeline, genes with expression profiles that were: unaffected by genotype or environment, genotype-dependent but unaffected by the environment, environmentally-dependent regardless of genotype, and G×E-related. The G×E-related genes showed different degrees of within-cultivar allelic diversity in the two genotypes and were enriched for stress responses, signal transduction and secondary metabolism categories. Our study unraveled the mutual relationships between genotypic and environmental variables during G×E interaction in a woody perennial species, providing a reference model to explore how cultivated fruit crops respond to diverse environments. Also, the pivotal role of vineyard location in determining the performance of different varieties, by enhancing berry quality traits, was unraveled.

Timing and Order of the Molecular Events Marking the Onset of Berry Ripening in Grapevine.

Grapevine (Vitis vinifera) is a model for the investigation of physiological and biochemical changes during the formation and ripening of nonclimacteric fleshy fruits. However, the order and complexity of the molecular events during fruit development remain poorly understood. To identify the key molecular events controlling berry formation and ripening, we created a highly detailed transcriptomic and metabolomic map of berry development, based on samples collected every week from fruit set to maturity in two grapevine genotypes for three consecutive years, resulting in 219 samples. Major transcriptomic changes were represented by coordinated waves of gene expression associated with early development, veraison (onset of ripening)/midripening, and late-ripening and were consistent across vintages. The two genotypes were clearly distinguished by metabolite profiles and transcriptional changes occurring primarily at the veraison/midripening phase. Coexpression analysis identified a core network of transcripts as well as variations in the within-module connections representing varietal differences. By focusing on transcriptome rearrangements close to veraison, we identified two rapid and successive shared transitions involving genes whose expression profiles precisely locate the timing of the molecular reprogramming of berry development. Functional analyses of two transcription factors, markers of the first transition, suggested that they participate in a hierarchical cascade of gene activation at the onset of ripening. This study defined the initial transcriptional events that mark and trigger the onset of ripening and the molecular network that characterizes the whole process of berry development, providing a framework to model fruit development and maturation in grapevine.

Ripening Transcriptomic Program in Red and White Grapevine Varieties Correlates with Berry Skin Anthocyanin Accumulation.

Grapevine (Vitis vinifera) berry development involves a succession of physiological and biochemical changes reflecting the transcriptional modulation of thousands of genes. Although recent studies have investigated the dynamic transcriptome during berry development, most have focused on a single grapevine variety, so there is a lack of comparative data representing different cultivars. Here, we report, to our knowledge, the first genome-wide transcriptional analysis of 120 RNA samples corresponding to 10 Italian grapevine varieties collected at four growth stages. The 10 varieties, representing five red-skinned and five white-skinned berries, were all cultivated in the same experimental vineyard to reduce environmental variability. The comparison of transcriptional changes during berry formation and ripening allowed us to determine the transcriptomic traits common to all varieties, thus defining the core transcriptome of berry development, as well as the transcriptional dynamics underlying differences between red and white berry varieties. A greater variation among the red cultivars than between red and white cultivars at the transcriptome level was revealed, suggesting that anthocyanin accumulation during berry maturation has a direct impact on the transcriptomic regulation of multiple biological processes. The expression of genes related to phenylpropanoid/flavonoid biosynthesis clearly distinguished the behavior of red and white berry genotypes during ripening but also reflected the differential accumulation of anthocyanins in the red berries, indicating some form of cross talk between the activation of stilbene biosynthesis and the accumulation of anthocyanins in ripening berries.

Disclosing the Molecular Basis of the Postharvest Life of Berry in Different Grapevine Genotypes.

The molecular events that characterize postripening grapevine berries have rarely been investigated and are poorly defined. In particular, a detailed definition of changes occurring during the postharvest dehydration, a process undertaken to make some particularly special wine styles, would be of great interest for both winemakers and plant biologists. We report an exhaustive survey of transcriptomic and metabolomic responses in berries representing six grapevine genotypes subjected to postharvest dehydration under identical controlled conditions. The modulation of phenylpropanoid metabolism clearly distinguished the behavior of genotypes, with stilbene accumulation as the major metabolic event, although the transient accumulation/depletion of anthocyanins and flavonols was the prevalent variation in genotypes that do not accumulate stilbenes. The modulation of genes related to phenylpropanoid/stilbene metabolism highlighted the distinct metabolomic plasticity of genotypes, allowing for the identification of candidate structural and regulatory genes. In addition to genotype-specific responses, a core set of genes was consistently modulated in all genotypes, representing the common features of berries undergoing dehydration and/or commencing senescence. This included genes controlling ethylene and auxin metabolism as well as genes involved in oxidative and osmotic stress, defense responses, anaerobic respiration, and cell wall and carbohydrate metabolism. Several transcription factors were identified that may control these shared processes in the postharvest berry. Changes representing both common and genotype-specific responses to postharvest conditions shed light on the cellular processes taking place in harvested berries stored under dehydrating conditions for several months.

Integrated network analysis identifies fight-club nodes as a class of hubs encompassing key putative switch genes that induce major transcriptome reprogramming during grapevine development.

We developed an approach that integrates different network-based methods to analyze the correlation network arising from large-scale gene expression data. By studying grapevine (Vitis vinifera) and tomato (Solanum lycopersicum) gene expression atlases and a grapevine berry transcriptomic data set during the transition from immature to mature growth, we identified a category named "fight-club hubs" characterized by a marked negative correlation with the expression profiles of neighboring genes in the network. A special subset named "switch genes" was identified, with the additional property of many significant negative correlations outside their own group in the network. Switch genes are involved in multiple processes and include transcription factors that may be considered master regulators of the previously reported transcriptome remodeling that marks the developmental shift from immature to mature growth. All switch genes, expressed at low levels in vegetative/green tissues, showed a significant increase in mature/woody organs, suggesting a potential regulatory role during the developmental transition. Finally, our analysis of tomato gene expression data sets showed that wild-type switch genes are downregulated in ripening-deficient mutants. The identification of known master regulators of tomato fruit maturation suggests our method is suitable for the detection of key regulators of organ development in different fleshy fruit crops.

Distinct transcriptome responses to water limitation in isohydric and anisohydric grapevine cultivars.

BACKGROUND: Grapevine (Vitis vinifera L.) is an economically important crop with a wide geographical distribution, reflecting its ability to grow successfully in a range of climates. However, many vineyards are located in regions with seasonal drought, and these are often predicted to be global climate change hotspots. Climate change affects the entire physiology of grapevine, with strong effects on yield, wine quality and typicity, making it difficult to produce berries of optimal enological quality and consistent stability over the forthcoming decades. RESULTS: Here we investigated the reactions of two grapevine cultivars to water stress, the isohydric variety Montepulciano and the anisohydric variety Sangiovese, by examining physiological and molecular perturbations in the leaf and berry. A multidisciplinary approach was used to characterize the distinct stomatal behavior of the two cultivars and its impact on leaf and berry gene expression. Positive associations were found among the photosynthetic, physiological and transcriptional modifications, and candidate genes encoding master regulators of the water stress response were identified using an integrated approach based on the analysis of topological co-expression network properties. In particular, the genome-wide transcriptional study indicated that the isohydric behavior relies upon the following responses: i) faster transcriptome response after stress imposition; ii) faster abscisic acid-related gene modulation; iii) more rapid expression of heat shock protein (HSP) genes and iv) reversion of gene-expression profile at rewatering. Conversely, that reactive oxygen species (ROS)-scavenging enzymes, molecular chaperones and abiotic stress-related genes were induced earlier and more strongly in the anisohydric cultivar. CONCLUSIONS: Overall, the present work found original evidence of a molecular basis for the proposed classification between isohydric and anisohydric grapevine genotypes.

Pectins, Hemicelluloses and Celluloses Show Specific Dynamics in the Internal and External Surfaces of Grape Berry Skin During Ripening.

Grapevine berry skin is a complex structure that contributes to the final size and shape of the fruit and affects its quality traits. The organization of cell wall polysaccharides in situ and their modification during ripening are largely uncharacterized. The polymer structure of Corvina berry skin, its evolution during ripening and related modifying genes were determined by combing mid-infrared micro-spectroscopy and multivariate statistical analysis with transcript profiling and immunohistochemistry. Spectra were acquired in situ using a surface-sensitive technique on internal and external sides of the skin without previous sample pre-treatment, allowing comparison of the related cell wall polymer dynamics. The external surface featured cuticle-related bands; the internal surface showed more adsorbed water. Application of surface-specific normalization revealed the major molecular changes related to hemicelluloses and pectins in the internal surface and to cellulose and pectins in the external surface and that they occur between mid-ripening and full ripening in both sides of the skin. Transcript profiling of cell wall-modifying genes indicated a general suppression of cell wall metabolism during ripening. Genes related to pectin metabolism-a ß-galactosidase, a pectin(methyl)esterase and a pectate lyase-and a xyloglucan endotransglucosylase/hydrolase, involved in hemicellulose modification, showed enhanced expression. In agreement with Fourier transform infrared spectroscopy, patterns due to pectin methyl esterification provided new insights into the relationship between pectin modifications and the associated transcript profile during skin ripening. This study proposes an original description of polymer dynamics in grape berries during ripening, highlighting differences between the internal and external sides of the skin.

miRVine: a microRNA expression atlas of grapevine based on small RNA sequencing.

BACKGROUND: miRNAs are the most abundant class of small non-coding RNAs, and they are involved in post-transcriptional regulations, playing a crucial role in the refinement of genetic programming during plant development. Here we present a comprehensive picture of miRNA regulation in Vitis vinifera L. plant during its complete life cycle. Furthering our knowledge about the post-transcriptional regulation of plant development is fundamental to understand the biology of such an important crop. RESULTS: We analyzed 70 small RNA libraries, prepared from berries, inflorescences, tendrils, buds, carpels, stamens and other samples at different developmental stages. One-hundred and ten known and 175 novel miRNAs have been identified and a wide grapevine expression atlas has been described. The distribution of miRNA abundance reveals that 22 novel miRNAs are specific to stamen, and two of them are, interestingly, involved in ethylene biosynthesis, while only few miRNAs are highly specific to other organs. Thirty-eight miRNAs are present in all our samples, suggesting a role in key regulatory circuit. On the basis of miRNAs abundance and distribution across samples and on the estimated correlation, we suggest that miRNA expression define organ identity. We performed target prediction analysis and focused on miRNA expression analysis in berries and inflorescence during their development, providing an initial functional description of the identified miRNAs. CONCLUSIONS: Our findings represent a very extensive miRNA expression atlas in grapevine, allowing the definition of how the spatio-temporal distribution of miRNAs defines organ identity. We describe miRNAs abundance in specific tissues not previously described in grapevine and contribute to future targeted functional analyses. Finally, we present a deep characterization of miRNA involvement in berry and inflorescence development, suggesting a role for miRNA-driven hormonal regulation.

The grapevine expression atlas reveals a deep transcriptome shift driving the entire plant into a maturation program.

We developed a genome-wide transcriptomic atlas of grapevine (Vitis vinifera) based on 54 samples representing green and woody tissues and organs at different developmental stages as well as specialized tissues such as pollen and senescent leaves. Together, these samples expressed ∼91% of the predicted grapevine genes. Pollen and senescent leaves had unique transcriptomes reflecting their specialized functions and physiological status. However, microarray and RNA-seq analysis grouped all the other samples into two major classes based on maturity rather than organ identity, namely, the vegetative/green and mature/woody categories. This division represents a fundamental transcriptomic reprogramming during the maturation process and was highlighted by three statistical approaches identifying the transcriptional relationships among samples (correlation analysis), putative biomarkers (O2PLS-DA approach), and sets of strongly and consistently expressed genes that define groups (topics) of similar samples (biclustering analysis). Gene coexpression analysis indicated that the mature/woody developmental program results from the reiterative coactivation of pathways that are largely inactive in vegetative/green tissues, often involving the coregulation of clusters of neighboring genes and global regulation based on codon preference. This global transcriptomic reprogramming during maturation has not been observed in herbaceous annual species and may be a defining characteristic of perennial woody plants.

Transcriptomic analysis of the late stages of grapevine (Vitis vinifera cv. Cabernet Sauvignon) berry ripening reveals significant induction of ethylene signaling and flavor pathways in the skin.

BACKGROUND: Grapevine berry, a nonclimacteric fruit, has three developmental stages; the last one is when berry color and sugar increase. Flavors derived from terpenoid and fatty acid metabolism develop at the very end of this ripening stage. The transcriptomic response of pulp and skin of Cabernet Sauvignon berries in the late stages of ripening between 22 and 37 °Brix was assessed using whole-genome micorarrays. RESULTS: The transcript abundance of approximately 18,000 genes changed with °Brix and tissue type. There were a large number of changes in many gene ontology (GO) categories involving metabolism, signaling and abiotic stress. GO categories reflecting tissue differences were overrepresented in photosynthesis, isoprenoid metabolism and pigment biosynthesis. Detailed analysis of the interaction of the skin and pulp with °Brix revealed that there were statistically significantly higher abundances of transcripts changing with °Brix in the skin that were involved in ethylene signaling, isoprenoid and fatty acid metabolism. Many transcripts were peaking around known optimal fruit stages for flavor production. The transcript abundance of approximately two-thirds of the AP2/ERF superfamily of transcription factors changed during these developmental stages. The transcript abundance of a unique clade of ERF6-type transcription factors had the largest changes in the skin and clustered with genes involved in ethylene, senescence, and fruit flavor production including ACC oxidase, terpene synthases, and lipoxygenases. The transcript abundance of important transcription factors involved in fruit ripening was also higher in the skin. CONCLUSIONS: A detailed analysis of the transcriptome dynamics during late stages of ripening of grapevine berries revealed that these berries went through massive transcriptional changes in gene ontology categories involving chemical signaling and metabolism in both the pulp and skin, particularly in the skin. Changes in the transcript abundance of genes involved in the ethylene signaling pathway of this nonclimacteric fruit were statistically significant in the late stages of ripening when the production of transcripts for important flavor and aroma compounds were at their highest. Ethylene transcription factors known to play a role in leaf senescence also appear to play a role in fruit senescence. Ethylene may play a bigger role than previously thought in this non-climacteric fruit.

The Role of Terroir on the Ripening Traits of V. vinifera cv 'Glera' in the Prosecco Area.

The grapevine (Vitis vinifera L.) is widely cultivated worldwide owing to the substantial commercial value of the grapes and other products derived from their processing, wines in particular. The grapevine is characterized by a remarkable phenotypic plasticity within the same variety, which shapes the final berry quality attributes hence reflecting the complex interactions between the plant and the environment leading to the expression of wine typicity. In this study, we explored the metabolomic and transcriptomic basis of the plasticity of Glera, a white berry grapevine variety particularly renowned for the production of wine Prosecco. The two selected vineyards varied for site altitude and pedoclimatic conditions. We highlighted that these environments determined different berry ripening dynamics at the level of both technological parameters and the total abundance and intrafamily distribution of phenolic compounds. Moreover, a clear impact on the grape aroma profile was observed. The genome-wide gene expression analysis of the berries revealed remarkable differences in the ripening transcriptomic program, reflecting the differences in water status, light exposure, and temperature experienced by the plants while growing at the two sites. Overall, this survey portrayed how the quality attributes of the cv 'Glera' grape berries may be affected by different environmental conditions within the typical area of Prosecco wine production.

De novo transcriptome characterization of Vitis vinifera cv. Corvina unveils varietal diversity.

BACKGROUND: Plants such as grapevine (Vitis spp.) display significant inter-cultivar genetic and phenotypic variation. The genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors. RESULTS: We have shown that cDNA sequencing by RNA-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar (Corvina) and the PN40024 reference genome. We detected 15,161 known genes including 9463 with novel splice isoforms, and identified 2321 potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. We also discovered 180 apparent private genes in the Corvina genome which were missing from the reference genome. CONCLUSIONS: The de novo assembly approach allowed a substantial amount of the Corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. The private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. Therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes.

Selective defoliation affects plant growth, fruit transcriptional ripening program and flavonoid metabolism in grapevine.

BACKGROUND: The selective removal of grapevine leaves around berry clusters can improve the quality of ripening fruits by influencing parameters such as the berry sugar and anthocyanin content at harvest. The outcome depends strongly on the timing of defoliation, which influences the source-sink balance and the modified microclimate surrounding the berries. We removed the basal leaves from Vitis vinifera L. cv Sangiovese shoots at the pre-bloom and veraison stages, and investigated responses such as shoot growth, fruit morphology and composition compared to untreated controls. Moreover, we performed a genome-wide expression analysis to explore the impact of these defoliation treatments on berry transcriptome. RESULTS: We found that pre-bloom defoliation improved berry quality traits such as sugar and anthocyanin content, whereas defoliation at veraison had a detrimental effect, e.g. less anthocyanin and higher incidence of sunburn damage. Genome-wide expression analysis during berry ripening revealed that defoliation at either stage resulted in major transcriptome reprogramming, which slightly delayed the onset of ripening. However, a closer investigation of individual gene expression profiles identified genes that were specifically modulated by defoliation at each stage, reflecting the uncoupling of metabolic processes such as flavonoid biosynthesis, cell wall and stress metabolism, from the general ripening program. CONCLUSIONS: The specific transcriptional modifications we observed following defoliation at different time points allow the identification of the developmental or metabolic processes affected in berries thus deepening the knowledge of the mechanisms by which these agronomical practices impact the final berry ripening traits.

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.

A molecular phenology scale of grape berry development.

Fruit growth and development consist of a continuous succession of physical, biochemical, and physiological changes driven by a genetic program that dynamically responds to environmental cues. Establishing recognizable stages over the whole fruit lifetime represents a fundamental requirement for research and fruit crop cultivation. This is especially relevant in perennial crops like grapevine (Vitis vinifera L.) to scale the development of its fruit across genotypes and growing conditions. In this work, molecular-based information from several grape berry transcriptomic datasets was exploited to build a molecular phenology scale (MPhS) and to map the ontogenic development of the fruit. The proposed statistical pipeline consisted of an unsupervised learning procedure yielding an innovative combination of semiparametric, smoothing, and dimensionality reduction tools. The transcriptomic distance between fruit samples was precisely quantified by means of the MPhS that also enabled to highlight the complex dynamics of the transcriptional program over berry development through the calculation of the rate of variation of MPhS stages by time. The MPhS allowed the alignment of time-series fruit samples proving to be a complementary method for mapping the progression of grape berry development with higher detail compared to classic time- or phenotype-based approaches.

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.

Genome-wide analysis of the grapevine stilbene synthase multigenic family: genomic organization and expression profiles upon biotic and abiotic stresses.

BACKGROUND: Plant stilbenes are a small group of phenylpropanoids, which have been detected in at least 72 unrelated plant species and accumulate in response to biotic and abiotic stresses such as infection, wounding, UV-C exposure and treatment with chemicals. Stilbenes are formed via the phenylalanine/polymalonate-route, the last step of which is catalyzed by the enzyme stilbene synthase (STS), a type III polyketide synthase (PKS). Stilbene synthases are closely related to chalcone synthases (CHS), the key enzymes of the flavonoid pathway, as illustrated by the fact that both enzymes share the same substrates. To date, STSs have been cloned from peanut, pine, sorghum and grapevine, the only stilbene-producing fruiting-plant for which the entire genome has been sequenced. Apart from sorghum, STS genes appear to exist as a family of closely related genes in these other plant species. RESULTS: In this study a complete characterization of the STS multigenic family in grapevine has been performed, commencing with the identification, annotation and phylogenetic analysis of all members and integration of this information with a comprehensive set of gene expression analyses including healthy tissues at differential developmental stages and in leaves exposed to both biotic (downy mildew infection) and abiotic (wounding and UV-C exposure) stresses. At least thirty-three full length sequences encoding VvSTS genes were identified, which, based on predicted amino acid sequences, cluster in 3 principal groups designated A, B and C. The majority of VvSTS genes cluster in groups B and C and are located on chr16 whereas the few gene family members in group A are found on chr10. Microarray and mRNA-seq expression analyses revealed different patterns of transcript accumulation between the different groups of VvSTS family members and between VvSTSs and VvCHSs. Indeed, under certain conditions the transcriptional response of VvSTS and VvCHS genes appears to be diametrically opposed suggesting that flow of carbon between these two competing metabolic pathways is tightly regulated at the transcriptional level. CONCLUSIONS: This study represents an overview of the expression pattern of each member of the STS gene family in grapevine under both constitutive and stress-induced conditions. The results strongly indicate the existence of a transcriptional subfunctionalization amongst VvSTSs and provide the foundation for further functional investigations about the role and evolution of this large gene family. Moreover, it represents the first study to clearly show the differential regulation of VvCHS and VvSTS genes, suggesting the involvement of transcription factors (TFs) in both the activation and repression of these genes.

Co-evolution between Grapevine rupestris stem pitting-associated virus and Vitis vinifera L. leads to decreased defence responses and increased transcription of genes related to photosynthesis.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a widespread virus infecting Vitis spp. Although it has established a compatible viral interaction in Vitis vinifera without the development of phenotypic alterations, it can occur as distinct variants that show different symptoms in diverse Vitis species. The changes induced by GRSPaV in V. vinifera cv 'Bosco', an Italian white grape variety, were investigated by combining agronomic, physiological, and molecular approaches, in order to provide comprehensive information about the global effects of GRSPaV. In two years, this virus caused a moderate decrease in physiological efficiency, yield performance, and sugar content in berries associated with several transcriptomic alterations. Transcript profiles were analysed by a microarray technique in petiole, leaf, and berry samples collected at véraison and by real-time RT-PCR in a time course carried out at five grapevine developmental stages. Global gene expression analyses showed that transcriptomic changes were highly variable among the different organs and the different phenological phases. GRSPaV triggers some unique responses in the grapevine at véraison, never reported before for other plant-virus interactions. These responses include an increase in transcripts involved in photosynthesis and CO(2) fixation, a moderate reduction in the photosynthesis rate and some defence mechanisms, and an overlap with responses to water and salinity stresses. It is hypothesized that the long co-existence of grapevine and GRSPaV has resulted in the evolution of a form of mutual adaptation between the virus and its host. This study contributes to elucidating alternative mechanisms used by infected plants to contend with viruses.

Overexpression of PhEXPA1 increases cell size, modifies cell wall polymer composition and affects the timing of axillary meristem development in Petunia hybrida.

• Expansins are cell wall proteins required for cell enlargement and cell wall loosening during many developmental processes. The involvement of the Petunia hybrida expansin A1 (PhEXPA1) gene in cell expansion, the control of organ size and cell wall polysaccharide composition was investigated by overexpressing PhEXPA1 in petunia plants. • PhEXPA1 promoter activity was evaluated using a promoter-GUS assay and the protein's subcellular localization was established by expressing a PhEXPA1-GFP fusion protein. PhEXPA1 was overexpressed in transgenic plants using the cauliflower mosaic virus (CaMV) 35S promoter. Fourier transform infrared (FTIR) and chemical analysis were used for the quantitative analysis of cell wall polymers. • The GUS and GFP assays demonstrated that PhEXPA1 is present in the cell walls of expanding tissues. The constitutive overexpression of PhEXPA1 significantly affected expansin activity and organ size, leading to changes in the architecture of petunia plants by initiating premature axillary meristem outgrowth. Moreover, a significant change in cell wall polymer composition in the petal limbs of transgenic plants was observed. • These results support a role for expansins in the determination of organ shape, in lateral branching, and in the variation of cell wall polymer composition, probably reflecting a complex role in cell wall metabolism.