Unraveling the key molecular events of Pinot noir berry ripening under varying crop load

Aligned to exploring the physiological and molecular complexity of grape berry development, there is a need to characterize the influence of the source:sink relationships on the genetic regulation of fruit composition. Crop load, as defined by the amount of fruit produced per unit vegetative growth at dormancy, is a common measure of source:sink relationships used to evaluate vineyard production efficiency. We studied the impact of varying crop load on the transcriptome and metabolome of Pinot noir grape berries by comparing the development and ripening of fruit grown on vines with either 50 % or 75 % of their grape clusters removed immediately following fruit set compared to unthinned vines for three consecutive vintages. A clear impact on the general phenylpropanoid pathway resulting in a redistribution between stilbenes and anthocyanins was revealed under varying crop loads and consistent with the transcriptomic profiles of the corresponding branches. Moreover, we identified genes, such as LBDIa3 and AG2, modulated by crop load around veraison, representing putative transcriptional key triggers of the berry ripening phase responding to differences in the vine source:sink ratio generated by the application of cluster thinning. Genes, specifically EXPA1 and EXPA18, involved in softening and other crucial events of ripening initiation responded to crop load and likely influenced the progression of the ripening process. Beyond the major impacts represented by a shift of the onset and completion of ripening, we were able to highlight more subtle effects of the crop load, related to the rate at which the molecular and metabolic changes occur. This study asserts that grape metabolism and transcriptome are remarkably flexible, and that manipulations such as cluster thinning induce extensive, genome-wide changes in expression during berry development. The insights gained here pave the way to progress towards the construction of robust models depicting the molecular network that characterizes berry development and the impact of crop load on its molecular regulation

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

Physiological and molecular responses of harvested wine grapes to low temperature conditioning.

Vitis vinifera is an important fruit crop species which, in different world areas, is mainly used for wine production. Nowadays, several challenges characterize the wine industry and have a marked impact on the production chain, the final quality of the wines, the environmental issues, and the consumer acceptance. Global warming which affects, among different pheno/physiological processes, the berry developmental cycle often results in anticipated harvests with high temperatures of the air and of the berries. High temperatures at harvest can induce undesirable biochemical changes in the fruit such as aroma losses and oxidative processes which negatively affect the grape composition and the wine quality. This thesis was aimed at evaluating at physiological and molecular level the effects of a postharvest practices, preprocessing cooling, intended as positive stress conditions that can alter the berry metabolism (in particular the profile of the volatile organic compounds, VOCs) and possibly improve grapes and wine quality. Two postharvest low temperature treatments (4 and 10 °C up to 48 h) were tested on white-skinned grapes of cv Vermentino while only the temperature of 4°C (up to 48 h) was applied to the red-skinned grapes of cv. Sangiovese. Control grapes of both varieties were held for the same sampling times (24 and 48 h) at room temperature (about 22 °C). Samples were collected at harvest (time zero) and after the treatments. Specific expression analyses (RT-qPCR) have been performed considering selected genes involved in the biosynthesis/metabolism of both phenylpropanoids/flavonoids and main VOCs, recognized to be highly reactive to stress conditions in grape berries. Further, the metabolic profile of the VOCs in grapes and wine samples was determined using the HP-SPME-GC-MS technique. Both molecular and metabolomic analyses were first optimized and standardized to suit the specific varieties and the tested experimental conditions. In addition, grape technological parameters have been measured (weight loss, pH, titratable acidity, total soluble solids and total polyphenols). In Vermentino grapes, the cooling treatment effected significantly on the expression of genes involved in the biosynthesis of the VOCs, while for genes involved in the biosynthesis of the phenylpropanoids/flavonoids, no significant changes were observed. Considering the cooling treatments, the expression level of both lipoxygenase A (VvLOXA) and alcohol dehydrogenase (VvADH2), which are involved in the Green leaf volatile (GLVs) biosynthesis, was significantly upregulated in the control grapes (22 °C) after 48 h, whereas in grapes kept at 4 °C it remained similar to the grapes sampled at time zero. The expression of terpineol synthase (VvTER), which is involved in the biosynthesis of volatile terpenes, remained similar to time zero in control grapes after 24 h, while, following both treatments at 4 and 10 °C for the same time period, VvTER appeared downregulated. As far as berry VOCs profiling is concerned, a trend of sesquiterpenes accumulation was evident in samples cooled at both 4 and 10 °C for 48 h. The same accumulation pattern was observed in Vermentino wine, following treatment at 4 °C for 48 h. As observed in Vermentino, also in Sangiovese berries the cooling treatment had a marked effect on VvADH2 expression level. While in control grapes VvADH2 was upregulated both after 24 and 48 h when compared to time zero, following cooling at 4 °C 24 h the VvADH2 expression level was significantly lower compared to control grapes. Phenylalanine ammonia lyase (VvPAL) and stilbene synthase (VvSTS), which encode for key enzymes in the phenylpropanoid pathway, were upregulated after treatment at 4 °C for 24 h in comparison with grapes at time zero. Flavonol synthase (VvFLS1), which is involved in a specific step of the flavonoids pathway, maintained similar expression level to time zero following cooling at 4 °C for 24 and 48 h, whereas in control grapes this gene was downregulated. A multivariate analysis on all dataset (PLS-DA) showed that hexanol and ethyl derivatives (e.g. ethyl acetate) were reduced after 4 °C treatment both at 24 and 48 h. Moreover, these compounds clustered together with VvADH2 expression data, revealing a high correlation level between hexanol and ethyl derivatives and the expression level of this gene. In conclusion, lowering the temperature of wine grapes prior to vinification induce specific molecular responses which can be responsible for or related to some of the observed changes in grape and wine composition. The physiological/molecular integrated approach, applied to study for the first time wine berries subjected to preprocessing cooling treatment, indicates that the appropriate handling of harvested grapes may be a tool to modulate wine quality and composition

Spatial Variability of Grape Berry Maturation Program at the Molecular Level

The application of sensors in viticulture is a fast and efficient method to monitor grapevine vegetative, yield, and quality parameters and determine spatial intra-vineyard variability. Molecular analysis at the gene expression level can further contribute to the understanding of the observed variability by elucidating how pathways contributing to different grape quality traits behave in zones diverging on any of these parameters. The intra-vineyard variability of a Cabernet Sauvignon vineyard was evaluated through a Normalized Difference Vegetation Index (NDVI) map calculated from a multispectral image and detailed ground-truthing (e.g., vegetative, yield, and berry ripening compositional parameters). The relationships between NDVI and ground measurements were explored by correlation analysis. Moreover, berries were investigated by microarray gene expression analysis performed at five time points from fruit set to full ripening. Comparison between the transcriptomes of samples taken from locations with the highest and lowest NDVI values identified 968 differentially expressed genes. Spatial variability maps of the expression level of key berry ripening genes showed consistent patterns aligned with the vineyard vigor map. These insights indicate that berries from different vigor zones present distinct molecular maturation programs and suggest that transcriptome analysis may be a valuable tool for the management of vineyard variability

Pre-processing Cooling of Harvested Grapes Induces Changes in Berry Composition and Metabolism, and Affects Quality and Aroma Traits of the Resulting Wine

Due to the greenhouse gas increase, grapes are often exposed to high temperatures in several growing areas especially during the final developmental stages, and this is particularly true when early ripening cultivars are harvested. This may cause undesirable effects on berry metabolism and composition and wine quality, particularly concerning the aroma profile. Harvesting at night or keeping the harvested grapes in cold rooms before vinification are empirical protocols applied in specific viticultural areas. To study the effects of decreasing berry temperature after harvest, white-skinned berries (cv Vermentino) were maintained at 4 or 10°C for 24 or 48 h before processing (pre-cooling). Control grapes were kept at 22°C. Grapes cooled at 10°C for 24 and 48 h resulted richer in polyphenols and showed a significant up-regulation of genes involved in polyphenols biosynthesis (i.e., VvPAL, VvSTS2, and VvFLS1). Similar behavior was observed in samples kept at 4°C for 48 h. Pre-cooling induced specific changes in the volatile organic compound (VOC) profiles. In particular, higher amounts of a specific subcategory of terpenes, namely sesquiterpenes, were detected in cooled samples. The induction of the expression of key genes involved in terpenoids biosynthesis (VvHDR, VvDX3, VvTER, VvGT14) was detected in cooled grapes, with variable effects depending on temperature and treatment duration. In both cooled samples, the evolution of alcoholic fermentation followed a regular trend but ended earlier. Higher phenolic content was detected in wines obtained from the 10°C-treated grapes. Higher residual concentration of malic acid at the end of fermentation was detected in wine samples from grapes pre-cooled at 4°C. Sesquiterpenes also showed a general increase in wines from cooled grapes, especially after pre-cooling at 10°C for 48 h. Different sensory profiles characterized the wine samples, with the best scores in terms of general pleasantness obtained by the wine produced from grapes pre-cooled at 4°C for 24 h. These results demonstrate that pre-cooling harvested grapes induces specific effect on the VOC profile and other quality parameters of Vermentino wine, and this appears to be the result of specific metabolic and compositional changes occurring in the berries

DataSheet_1_Temperature affects organic acid, terpene and stilbene metabolisms in wine grapes during postharvest dehydration.docx

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.</p

Table_1_Temperature affects organic acid, terpene and stilbene metabolisms in wine grapes during postharvest dehydration.xlsx

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.</p