Proanthocyanidins are found in both the skin and seeds of grape berries . Tannin synthesis in the grape berry starts at flowering, continues in the early stages of grape development, and reaches its maximum around veraison . It then decreases throughout ripening due to a reduction in the extractability of tannins related to their interactions with cell wall components, rather than a decrease related to degradation . The effect of high temperatures on skin and seed tannins have shown inconsistent results across different studies. Cohen et al., found a linear relationship between the increase of skin tannin content and heat accumulation during phase I of grape development. However, Gouot et al., found an increase of total skin tannin concentration in vines exposed to short periods of heat stress before veraison, but found no differences in skin tannin concentration and content at maturity. Studies where heat was applied after veraison have shown no effect on tannin levels, which could be associated with the stop of tannin synthesis after this point in development . Lastly, Bonada et al., conducted a field experiment where vines were exposed to elevated temperatures throughout the entire growing season and found a decrease in total tannin content and concentration at maturity. This was mainly related to a decrease of seed tannins while skin tannins remained unresponsive to elevated temperatures. The different experimental conditions as well as differences in heat treatment design and time of application could be possible explanations for the discrepancies of these findings, as well as whether concentration or content of tannins was studied. Flavonols, anthocyanins, large plastic planting pots and flavan-3-ols are derived from the same precursors and are produced from the flavonoid biosynthetic pathway .
These precursors are derived from the Shikimate and phenylpropanoid pathways. Each step of these pathways are catalyzed by specific enzyme families. Flavonoid enzymes include flavone synthase , flavanone-3ß-hydroxylase , and flavonol synthase . Two enzymes, flavonoid-3’-hydroxylase and flavonoid-3’-5’-hydroxylase , are particularly important because they catalyze the reactions that form di- and tri-hydroxylated flavonoids from monohydroxylated precursors. The genes that code for F3’H and F3’5’H are expressed in the berry skin and contribute to the production of di- and tri-hydroxylated flavonols, anthocyanins, cyanidins, and delphinidin derivatives . The main enzymes for flavan-3-ol production are anthocyanidin reductase and leucoanthocyanidin reductase . However, the enzymes involved in producing proanthocyanidins are unknown. The enzyme that converts anthocyanidins to anthocyanins is UDP-glucose flavonoid-3-O-glucosyltransferase. It is also important to note the MYB transcription factors that are involved in the regulation of the phenylpropanoid pathway. MYBF1 regulates FLS for flavonol production. MYBA1&2 and MYB4 regulate UFGT for anthocyanin synthesis, with MYBA1&2 also being involved in several other steps in the pathway . The effects of high temperatures on the phenylpropanoid and flavonoid pathways is variable, and dependent upon the phenological stage during which heat events occur. Veraison is the most sensitive stage for anthocyanins, while flowering is the most sensitive stage for tannins . Although it has been widely reported that high temperatures impact anthocyanin synthesis, gene expression does not consistently reflect concentration.
A study done by Mori et al. on the variety Darkridge showed that high night temperature resulted in lower anthocyanin concentration due to a down regulation of genes including F3’H and UFGT. Similar results were seen in Kyoho grapes in that high day and night temperatures down-regulated UFGT . However, when the berries were exposed to high temperatures only at night, there was an initial decrease then sudden increase in UFGT activity during ripening. In contrast, a study done on Cabernet Sauvignon showed that UFGT was not strongly down-regulated in grapes that were exposed to high temperatures . Studies show that the effects of high temperatures on the expression of MYB transcription factors remains inconsistent, particularly with MYBA1. Some studies have shown that the expression of MYBA1 was unaffected by high temperatures , whereas other studies showed an extreme down-regulation of this transcription factor . Currently, there is a lack of published research that investigates the potential effects of HWs on berry temperature. While there have been studies published that look at how elevated temperatures affect berry development, primary metabolism, and secondary metabolism, they did not collect data measuring temperature of the berries themselves. For example, in a study done by Cohen et al. , individual cluster temperatures were manipulated to assess the impact of temperature on phenolic metabolism. While this study provided insight to the direct effects of high temperatures, it failed to show whether heat events result in higher berry temperature under different irrigation regimes, and if so, to what extent. The objective of this study was to evaluate the impact that different irrigation practices have on berry phenolics and gene expression carried out prior to and during HWs in a commercial vineyard of Cabernet Sauvignon. The results from this study provide a better understanding of how irrigation during heat events can help mitigate the effects of extreme temperatures and lead to more efficient water use.
In addition, by studying how HWs and irrigation practices influence the production of metabolites in the berry at the chemical and molecular levels provides the wine industry new knowledge that can be used to direct future efforts at preserving berry and wine quality under climate change conditions. In this experiment, a HW was defined as three consecutive days with maximum temperatures at or above 38 °C . Rather than creating artificial HWs with a heating system, this study relied on the occurrence of natural HWs. There were four HWs during the 2020 growing season, and there were two HWs during the 2021 growing season. Differential irrigation treatments were applied when HWs occurred and started one or two days before each HW and continued until the last day of the HW. There were three irrigation treatments: a baseline, which was exposed to deficit irrigation and held at 60% ET, a second treatment where irrigation was double the baseline , and a third treatment where irrigation was triple the baseline . In 2021, the irrigation treatments were adjusted in order to fine tune the amount of water used to see if less water could be used while maintaining the positive aspects of increased irrigation during HWs. The baseline treatment stayed the same at 60% ET, the second treatment was 1.5x baseline , and the third treatment was 2x baseline . Four pixels per treatment were randomly selected and distributed along the site using the VRDI system to provide differential irrigation to the individual plots. ET was estimated throughout the season using Landsat data, normalized difference vegetation index and crop coefficient. Variations in pre-treatment irrigation were implemented at the beginning of the growing season on the plots to even out vigor based on NDVI and thermal imaging due to the heterogeneous soil profile of the site. After, differences in irrigation schedules were based completely on implemented treatmentsBerry temperature was measured using 0.076 mm diameter type ‘E’ thermocouples . There were a total of 20 thermocouples placed in individual berries within three of the treatment blocks. The thermocouples were inserted into the center of the berries in exposed clusters facing the east and west side of the vine, and at each side of the vine 4 thermocouples were placed in different berries within the cluster. Because berries could develop necrosis from being punctured by the thermocouple, thermocouples were relocated to adjacent exposed berries at least every 12 days to maintain relatively fresh conditions . A subset of grape samples were stored at -20 °C for phenolic extraction. Three sets of sixty berries were weighed and volume occupied in water was recorded using a graduated cylinder.Skins were prepared for extraction by separating pulp and seed by hand. The skins were homogenized using a T25 digital ULTRA-TURRAX and S 25 N-18 G Dispersing tool. The skins were homogenized for one minute at 14 speed x 1000 rpm with 100 mL of a 66% acetone and transferred to an opaque polypropylene jar. The jar’s head space was filled with nitrogen gas, closed with a screw cap lid, black plastic planting pots and sealed with parafilm. The skins were extracted for 24 hours on a orbital shaker . The next day, samples were centrifuged for 10 minutes at 10,000 rpm. Samples were then poured through a Buchner funnel into a Buchner flask and filtered through Whatman filter paper with a pore size of 1.
Each filtered sample was transferred to a round-bottom flask and put onto the rotovap for 10 minutes with the water bath at 33 °C. Acetone was removed under reduced pressure. The extracts were brought back to 50 mL with milli-q water, and volume was recorded using a graduated cylinder. The samples were transferredto centrifuge tubes and stored at -20 °C until analysis. The next day, the samples were filtered, and acetone was separated from the solution using a rotovap. The samples stayed on the rotovap for 10 minutes with the water bath at 33 °C. The concentrated extracts were diluted with milli-q water, and volume was recorded using a graduated cylinder. The samples were transferred to falcon tubes and stored at -20 °C until analysis. Triplicate grape extracts were analyzed by methyl cellulose precipitation, which is the method used for analyzing tannin in extracts and wine, as previously published . Briefly, 25 µL of extraction sample was placed into 1200 µL deep well plates and combined with 300 µL of 0.04% methyl cellulose solution or water , and mixed on an Thermomixer-C for 5 minutes at 1500 RPM and left to stand for 3 minutes. Following the mixing, 200 µL of saturated ammonium sulfate was added to the wells to prevent the re-release of proanthocyanidin material into solution following precipitation. Water was then added to the wells and again it was mixed for 5 minutes. The deep well plate was then allowed to stand for 10 minutes before being centrifuged for 5 minutes at 2,272 × g . Both the treated and control samples were taken and placed in a 96 half-area well plate . -Epicatechin was used as a quantitative standard. Proanthocyanidin quantification was conducted by calculating Δ280 nm with the linear regression from an -epicatechin standard curve. The samples were analyzed on the SpectraMax iD3 microplate reader. Endpoint analysis was at 280 nm, and a pathway correction made up for volume differences. Berry temperature was recorded across the August 2020 and September 2020 heat waves . Berry temperatures from 60% ET treatment were significantly different from the 120% ET and 180% ET treatments for the pre-HW date of August 13th . There were no significant differences in berry temperature between the three treatments for the August 2020 HW and post-HW dates. For the August 13th pre-HW date, peak berry temperature occurred at 13:00 hours for the 180% ET treatment. For the HW date, peak berry temperature was at 15:00 hours for the 120% ET treatment. For the post-HW date, peak berry temperature was at 17:00 hours for the 60% ET treatment. There were no significant differences among the three treatments during the September 2020 HW . For the pre-HW date, peak berry temperature was at 15:00 hours for the 60% ET treatment. Similarly, for the HW and post-HW dates, peak berry temperature was at 15:00 hours for the 180% ET treatment. Berries in the August 2020 HW consistently reached higher temperatures than berries in the September 2020 HW, which may be due to differences in the extremity of the respective HWs. Anthocyanins in both growing seasons were measured from pre-veraison until commercial harvest. As seen in Figure 4, in the 2020 growing season, the 60% ET treatment started out with the lowest total anthocyanin concentration and continued throughout the rest of the season. During HW3 and HW4, we see significant differences between the treatments. At harvest, there were no differences between the 120% ET and 180% ET treatments, but both treatments had significantly higher anthocyanin concentrations than the 60% ET treatment, suggesting that the additional water prior to the heat waves had mitigated the loss of anthocyanin material. Looking at Figure 4, again in 2021, the 60% ET treatment had consistently lower anthocyanin concentrations throughout the season. Unlike the 2020 growing season, the 60% ET treatment remained significantly different from the other two treatments starting at August 17th until close to harvest. However, at harvest, there were no significant differences between the three treatments.