In both 2020 and 2021, there were HWs that occurred mid to late August. Looking at anthocyanin concentration, there was more variation in concentrations among treatments in 2020. Additionally, concentrations were higher in 2021 among the three treatments. The higher anthocyanin concentrations may be due to less extreme HW temperatures, thus less anthocyanin degradation. Looking at Figure 5, in 2020, there were no significant differences between the treatments during HW2, but looking at HW3, the 60% ET treatment suffered the most significant losses. At the start of HW4 the 120% ET treatment had significantly higher flavonol concentrations than the 60% or the 180% treatments. Nevertheless, at harvest there were no significant differences between the three treatments. In 2021 as seen in Figure 5, there were no significant differences in flavonol concentration until HW2 where there were differences between the 60% and the 120% ET treatments. Interestingly, there was a major decline in concentration for the 120% ET treatment at the start of HW2, and by commercial harvest, plant plastic pots this treatment has the lowest concentration. This may be due to differences in light exposure because flavonols develop when exposed to light.
The 120% treatment may have had the lowest flavonol concentration at harvest because these vines had the most water applied during the growing season, so there was likely more foliage covering the berries. Similar to anthocyanin concentration, flavonol concentrations reached higher peaks in the 2021 growing season than in the 2020 growing season. Flavonol concentration went beyond 0.20 mg/B for the 60% ET treatment in 2021, but did not exceed 0.15 mg/B in 2020. This may be due to less extreme HW temperatures in 2021 and more direct sunlight exposure that allowed for increased flavonol development. As seen in Figure 6, the 2020 results show that, following HW2, there was a significant difference between the 60% and 120% treatments. This is also seen at the start HW3, and all treatments saw a marked decrease in PAs leading to no significance between them for the rest of the season until at harvest where there was a difference between the 60% and 180% treatments. Also seen in Figure 6, in 2021, there was a significant difference between the 90% and 120% ET treatments at the start of HW1 and at the start of HW2. After HW2, significant differences were seen between the 60% and the other two treatments. Overall, there were more significant differences between the treatments during the 2021 growing season compared to the 2020 growing season. In 2020, PA concentrations were on a similar trend among the three treatments. The larger variations in PA concentrations between treatments in 2021 may be attributed to the severity of the HWs and the impact it had on the grapevines.
During the 2021 growing season, the berries got sunburnt due to high exposure, and therefore more irrigation was applied in order to preserve quality. Table 1 shows the summary statistics of the RNA sequencing. Sample 1_NW_B3R2 was an outlier and removed from the analysis due to having a higher number of reads compared to the other samples. For each irrigation treatment, the berry transcriptomes of two consecutive dates of collection were compared to evaluate the number of differentially expressed genes . The purple represents the 60% ET treatment, the teal represents the 120% ET treatment, and the yellow represents the 180% ET treatment. As seen in the figure, there were significantly more genes differentially expressed between the HW vs. pre-HW dates than the HW and post-HW dates for the 60% ET and 120% ET treatments. Interestingly, it was the opposite for the 180% ET treatment. For each pairwise comparison of collection dates, the DEGs detected for the three irrigation treatments were compared . Looking at the top two diagrams, there is a larger number of shared genes unique to the 60% and the 120% treatments compared to those uniquely shared with the 180% treatment. Specifically, there were 550 up-regulated genes shared only between the 60% and 120% treatments versus the 96 and 38 genes only shared with the 180% treatment. Looking at the bottom two diagrams, there is an overall decrease in the number of genes being differentially expressed when comparing the post-HW date relative to the HW date. There is also a higher number of genes being differentially expressed for the 180% treatment with 478 being up-regulated and 605 being down-regulated. In terms of berry temperature, significant differences among the treatments were only observed on August 13th, which is the pre-HW date for the August 2020 HW. Measurements recorded throughout the remaining HW dates showed that berry temperature remained similar among the three treatments. These results may be due to the berries being in the shade. In a study done by Ponce de Leon and Bailey , they found that shaded berries tended to have similar temperatures to the ambient air temperature and could reach over 10 °C above ambient temperature when in direct sunlight. There is currently a lack of published literature that explores the effects of HWs and irrigation on berry temperature, and the results from this study suggest that the two factors do not play a significant role in berry temperature. There were major declines in anthocyanin concentration in 2020 and 2021 for the 60% ET treatment, which suggests a suppression of anthocyanin synthesis and promotion of degradation at such high temperatures. This finding remains consistent with published literature, such as the work done in Yan et al. . In their study, three experiments were conducted on Vitis vinifera L. cv. Merlot, cl. 347 in which the grapevines were exposed to three different temperature regimes.
Their results consistently showed that the low-temperature regimes and high-temperature regimes had the highest and lowest anthocyanin levels, respectively. Further evidence of anthocyanin synthesis suppression and promotion of degradation at high temperatures is found in Mori et al. . Similar to the experimental design in Yan et al. , Kyoho grape berries in this study were grown under different temperature conditions. Anthocyanin levels were consistently lower in berries grown under high temperature conditions compared to berries grown at 25 °C or berries grown at 30 °C during the day and 15 °C at night. These results were attributed to a decrease of UFGT activities in the flavonoid pathway, as previously mentioned. Particular attention should be given to the anthocyanin results seen in 2020 . By harvest, the 120% ET treatment was significantly different from the 60%, black plastic pots but not significantly different from the 180% treatment. This is important because similar results were seen in anthocyanin concentrations with much less water used for irrigation. These results further support research that has proven the beneficial aspects of deficit irrigation. As seen in Bucchetti et al. and Roby et al. , deficit irrigation consistently increased anthocyanin concentrations by reducing berry size and thus increasing content per berry. That being said, theresults from this study show that overwatering during HWs is unnecessary when trying to compensate for berry phenolics. In terms of flavonol concentration, particular attention should be given to the 120% ET treatment in the 2021 growing season . By harvest, there was a major decline in concentration for this treatment. As previously mentioned, flavonol synthesis is light-dependent, and shading can have notable effects on flavonol concentration . Since sunlight is a key influencing factor, the decline in flavonol concentration for the 120% ET treatment may be attributed to a denser canopy that provided more shading to the fruit, which inhibited flavonol synthesis. In the 2020 and 2021 growing seasons, total PA concentration was significantly lower in the 60% ET treatment than the other two treatments by harvest . Since HWs occurred in both seasons pre-veraison, the treatment differences likely happened during PA synthesis, which occurs from flowering to veraison. The pre-veraison HWs could have impacted PA synthesis in the 60% ET treatment, which was exposed to higher water and heat stress, thus decreasing PA concentration. Although studies have shown variable results on the effects of heat events on PA concentration, PAs are unlikely to degrade due to the high stability of their chemical structure . A common trend seen throughout both years is that the 180% and 120% treatments from 2020 and 2021 respectively resulted in lower phenolic concentrations than the 120% and 90% treatments. This is important to note because these results suggest that irrigation prior to HWs can be beneficial in maintaining fruit quality, but excessive watering may negate the beneficial aspects of deficit irrigation. In terms of the lower anthocyanin biosynthesis in the 3x and 2.5x treatments, it may be related to plant water status since moderate deficit irrigation has been shown to increase anthocyanin biosynthesis and promote ripening . Additionally, this may also be due to insufficient light exposure of the clusters since the canopies were more dense with foliage.
As seen in Figure 7 and Figure 8, there were significantly more genes differentially expressed between the HW vs. pre-HW dates than the HW and post-HW dates for the 60% ET and 120% ET treatments, and it was the opposite for the 180% ET treatment. This may potentially be a combined response to the heat stress and too much irrigation being applied. The RNA sequencing results showed that there were several down-regulated genes from the phenylpropanoid and flavonoid pathways shared between the pre-, during, and post-HW dates and irrigation treatments. What is potentially being observed is a general repression of core and peripheral phenylpropanoid pathways, which are normally triggered in red-skinned grape berries throughout ripening . Some of the genes that deserve particular attention include trans-cinnamate 4-monooxygenase , 4-coumarate-CoA ligase , chalcone synthase , flavonol synthase, F3’5’H, and UFGT. C4H and 4CL play key roles in the phenylpropanoid pathway. C4H catalyzes the reaction that forms p-coumaric acid, and 4CL converts p-coumaric acid to 4-Coumaroyl-CoA . The down-regulation of C4H and 4CL are important to note because 4-Coumaroyl-CoA is an important precursor for compounds produced in the flavonoid pathway, and decreased production of this precursor may lead to overall decreased levels of phenolic compounds. Chalcone synthase is the first key enzyme in the flavonoid pathway, and therefore plays a central role in initiating flavonoid biosynthesis . The down-regulation of this gene may lead to reduced flavonol levels in grape berries, which is crucial in times of excessive heat and light exposure due to their nature as UV protectants. Flavonol synthase plays an important role in converting dihydroflavonols to flavonols . F3’5’H catalyzes flavonoid hydroxylation, which leads to the formation of flavonols, anthocyanins, and PAs . UFGT converts anthocyanidins to anthocyanins . The down-regulation of these genes lead to decreased production of phenolic compounds, and such effects have been well-documented . Implementation of differential irrigation prior to and during HWs were shown to have a major impact on berry composition and gene expression. Damaging effects on berry quality were observed from underwatering and overwatering. Significant changes in berry composition occurred throughout the HWs, including a suppression of anthocyanin synthesis and promotion of degradation, and lower flavonol and PA concentrations in highly irrigated treatments. The down-regulation of key enzymes involved in the phenylpropanoid and flavonoid pathways, suggest a possible mechanism for the lower phenolic concentrations. The frequency and duration of extreme heat events predicted for the upcoming decades indicates the need for further field research looking at potential strategies to mitigate the negative effects of heat waves on berry composition and gene expression. Misting, shade cloths, cover crops, canopy management, and trellis systems that protect fruit exposure remain as options to help cope with the effects. Furthermore, studying acclimation within and across seasons of different cultivars to extreme heat events will help guide the grape and wine industry towards cultivars with better adaptations and ultimately higher quality fruit and wine. Obesity has become a major health problem worldwide and in the absence of an effective universal medical approach to tackle this epidemic, many patients use over-the-counter weight loss supplements to help them lose weight. Numerous products are available that promote weight loss and appetite suppression and are sold as fat burners. These supplements are often sold without any regulatory process or having undergone any Food and Drug Administration approved clinical trial showing safety and efficacy.