Simone Castellarin

Frequency of drought events is expected to increase in many important grape-growing regions, with implications on sustainability of wine grape production. There are large differences between grapevine cultivars that have implications on effects of drought on berries, including the level of isohydricity they display and berry transpiration rates, and traits that contribute to preventing losses in yield and metabolite changes during drought are important to determine. The primary goal of this study was to quantify the degree of isohydricity of four grapevine cultivars ('Cabernet Franc', 'Semillon', 'Grenache', and 'Riesling') using different methods – predawn water potential threshold of stomatal closure, slope of the linear regression between predawn and midday water potentials, and the daily difference between predawn and midday water potentials – and determine whether degree of isohydricity has a relationship with sensitivity of berries to size and metabolite changes during progressive drought. I also sought to determine whether any relationships (if present) were dependent on phenological stage (i.e. pre- and post-veraison). It was found that the daily difference between predawn and midday water potentials has a relationship with sensitivity of changes in berry size and metabolites as predawn water potential declined. Secondly, berry transpiration rates were compared between cultivars to determine whether differences can impact berry size changes during progressive drought. It was found that 'Cabernet Franc' berries had lower rates of transpiration compared to other cultivars, and this contributed to delayed and smaller changes to berry size changes.The recently introduced “water potential curve” was also further analyzed to determine whether it can differentiate grapevine cultivars based on predawn water potential thresholds of leaf stomatal closure and turgor loss. It was found that “water potential curves” were able to accurately differentiate grapevine cultivars based on thresholds of turgor loss, but not based on thresholds of stomatal closure.

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Grape growers use viticultural practices such as deficit irrigation and crop load management via cluster thinning to improve phenolics and aromas in red grapes and wines; however, the impact of these practices on grape terpenes – key aromatics for quality in wines – remains largely unknown. I performed two three-year studies to investigate the effect of deficit irrigation and crop load management strategies on the accumulation of terpenes in Gewürztraminer grapes grown in the Okanagan Valley (BC, Canada). Yield and grape sugars were reduced by deficit irrigation treatments; however, effects were minimal when the deficit was applied late in the season. Applying late deficit allowed to save ~30% of irrigation water compared to standard irrigation. Total free terpenes were not affected by deficit irrigation treatments, but the concentration of key terpenes for the aroma of Gewürztraminer wines, such as geraniol and citronellol, was significantly increased in grapes exposed to water deficit late in the season. This irrigation treatment did not affect the expression of terpene genes, suggesting that the increased concentration of specific terpenes was not regulated at the transcriptional level. Reducing the crop load stimulated sugar accumulation, particularly if this reduction was applied early in the season. Reducing the crop load early during the season also increased the terpene levels before the commercial harvest (20-21 oBrix), but had no effects at harvest, suggesting a faster accumulation of terpenes during ripening. The peak of expression of several terpene synthases occurred before the commercial harvest. Expression of two terpene synthases was increased in grapes of grapevines subjected to crop load reduction early in the season. My studies indicate that it is possible to modulate terpenes in vineyards by managing irrigation and crop load. However, I observed that the seasons had a stronger effect on terpenes than the treatments investigated.

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Flavonoids are an important class of plant secondary metabolites, which impart color andmouthfeel to grapes and wine. Flavonoid accumulation is genetically and environmentallycontrolled in grapes. Among the environmental factors, temperature is arguably the one that mostaffects flavonoid accumulation in grape berry. High temperatures (e.g., 30-35 °C) were shown inprevious reports to impair anthocyanin accumulation in red grape varieties. Most of the studiescompared only two or three temperature regimes and have never investigated the effect of thedifference between day and night temperatures on flavonoid accumulation. This study employedcontrolled-environment (growth chamber) experiments to investigate the effect of temperatureregimes and the difference between day and night temperatures on flavonoid accumulation ingrapes, with an emphasis on anthocyanins and flavonols. Five temperature regimes were imposedon potted Merlot grapevines during berry ripening in each experiment (day/night, °C: 20/10, 20/15,25/15, 35/25, 35/30 in Experiment 1; 20/10, 20/15, 25/15, 25/20, 30/20 in Experiment 2). In bothexperiments, high temperature regimes (i.e., 30/20, 35/25, and 35/30) decreased anthocyanin andflavonol concentrations and increased the relative concentration of 3’4’5’-substituted andmethoxylated anthocyanins and flavonols as well as acylated anthocyanins, with comparison tolow temperature regimes (i.e., 20/10 and 20/15). The difference between day and nighttemperatures exerted minor effect when regimes with the same day and different nighttemperatures were compared. When grapevines were subjected to the same night temperature, alarger difference between day and night temperatures (ΔT = 10 °C, e.g., 25/15) reduced theanthocyanin and flavonol level, with comparison to a smaller temperature difference (ΔT = 5 °C,e.g., 20/15). Gene expression analyses revealed that VviF3’H and VviF3’5’Hs integrated for theregulation of 3’4’5’-substituted anthocyanin accumulation and Vvi3AT controlled the level of anthocyanin acylation; the down-regulation VviFLS4 and VviMybF1 expression in hightemperature regimes led to the corresponding reduction of flavonol accumulation. These resultsindicate that modulation of anthocyanin and flavonol accumulation occurs partially at thetranscriptional level. Finally, this study suggests that day temperature plays a more important rolethan night temperature in determining the concentration and composition of anthocyanins andflavonols in grape berry.

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The cuticle is a layer found on the surface of plant aerial parts mostly composed of cutin polymer and aliphatic waxes, and provides protection from biotic and abiotic environmental stresses. In fruits, cuticular waxes are also rich in triterpenoids. The cuticular waxes of the grapevine (Vitis vinifera L.) berry and the related biosynthetic pathways are poorly characterized. It is not understood how the berry cuticle responds to water deficit (WD) stress, a stress that commonly occurs in vineyards. We hypothesized that under severe WD stress, the cuticular aliphatic wax biosynthetic pathway of developing grape berries would be upregulated, resulting in an increased wax load in the fruit’s cuticle and a decrease in the transpiration rate through the berry cuticle. Candidate genes for the cuticular wax biosynthetic pathway were identified by phylogenetic analyses and surveys of publicly available grapevine transcriptomic datasets. Analyses of these transcriptomes also showed that a number of wax-related genes are significantly upregulated in response to WD stress, and are also modulated by other environmental stresses. A greenhouse experiment was performed in order to test the impact of water deficit on wax composition, expression of candidate biosynthetic genes, and water transpiration in Merlot grapes. A significant increase in aliphatic wax and a decrease in the ratio of triterpenoids:aliphatic wax was observed under WD stress. The increase in aliphatic wax was due to an upregulation of the aliphatic wax biosynthetic pathway, with an increase in expression seen in fatty acid elongation, the alkane forming, and the alcohol forming genes. The transpiration rate of the berry was not significantly affected by WD; however, a marginally significant (P = 0.051) reduction of the rate was observed in WD berries. The study also revealed that cuticular aliphatic wax composition changed over the course of berry development, with very long chain (VLC)-aldehydes and VLC- primary alcohols predominating before veraison (the onset of berry ripening), and VLC-fatty acids and wax esters mainly accumulating after veraison.

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