• volatile organic compounds (VOCs) analysis
• relative gene expression analysis
• postharvest
• low temperature
• wine grapes (Vitis vinifera)

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.