The mixture was centrifuged at 20,000 g for 15 minutes, after which 0.4 ml of supernatant was extracted, mixed with 0.4 ml of 2:1 chloroform:methanol mixture, and centrifuged at 12,000 g for 5 minutes. 50ul of supernatant was combined with 50ul of Amplex®Red working solution, which was prepared according to manufacturer instructions, in a black 96-well plate with a transparent flat bottom. Absorbance was read using an Epoch-2 microplate reader at 560nm. For H2O2 quantification, a standard curve was generated following the manufacturer’s instructions. Frozen grape powder was homogenized for 10min in 0.1% trichloroacetic acid and then centrifuged at 10,000 g for 5 minutes. 1mL of the supernatant was combined with 4mL 20% trichloroacetic acid containing 0.5% thiobarbituric acid, then the mixture was heated for 15 minutes at 95°C then immediately cooled in an ice bath for 5 minutes. After centrifugation at 10,000g for 10 minutes, 100 of supernatant was plated into a Corning® black-walled, clear, flat-bottomed 96-well plate and absorbance were read at 532 and 600 nm. Concentration was calculated using the MDA molar extinction coefficient 155,000 M-1 cm-1 with the following equation: MDA concentration = •1010, accounting for aliquot volumes and dilution factors.The effect of the irrigation treatments on indicators of vine water stress, hydrogen peroxide/lipid oxidation products, and osmotic potential in the berry was examined through one-way analysis of variance for each sample date. We determined the onset and rate of berry cell death, shrivel, and ROS accumulation by fitting piece wise linear regression models between time and percent cell vitality, the berry shrivel index, and H2O2 concentrations. The onset is the date these variables transitioned from relatively constant to rapidly changing, defined as the fitted breakpoints of the piece wise regression models, plastic plants pots and the rate is the slope of these variables over time after onset. We tested for significant irrigation treatment effects on onset and rate by examining their 95% confidence intervals for overlap.

Furthermore, Correlation between shrivel and % living tissue and peroxide and % living tissue was determined through the Standardized Major Axis Tests and Routines package in R using the sma function. We also tested for significant treatment differences between treatments by one-way analysis of variance for TSS, TA, and pH at harvest, when they are most relevant to growers. Because 7/25 was not used for perimeter analysis, nor was 8/1 for cell vitality, both sets of data were omitted in order to achieve a date-by-date correlation. Out of 540 berries sampled, 248 cross sections were viable for image analysis due to damage incurred in the mesocarp from the razor blade hitting and dislodging seeds. Measurements of percent living tissue and perimeter/total area were taken on different yet mostly overlapping subsets of images due to a fraction of images lacking a smooth perimeter or consistent dye stain. For cell vitality analysis, n = 68, 65, and 72 for treatments 1, 2, and 3 respectively; and for the shrivel index analysis, n = 74, 65, and 78 for treatments 1, 2, and 3 respectively. In tracing representative areas of the berry for cell vitality analysis, the selected area routinely excluded the pedicel residue, which did not typically take up the stain. Due to the violations of equal variance caused by these asymmetries, the non-parametric One-Way test in R was used for both percent living tissue and shrivel index on the last sampling date, and, likewise, the Games-Howell test was used as a posthoc test to compare treatment groups.Our augmented irrigation regimen commencing on the estimated date of onset, i.e. the late treatment, was effective in significantly reducing the rate but not the onset of cell death. Onset was defined as the fitted break point of the piece wise linear regression models, when the rate of change in cell vitality significantly increased. The onset of cell death occurred 92 ± 1 DAA for the control, 89 ± 8 DAA for the early treatment, and 83 ± 4 DAA for the late treatment . The 95% confidence intervals for these estimates overlapped, indicating that onset was not significantly different between treatments. The fitted slope after the break point indicates the rate of cell death after onset. Post-onset slopes were -2.06 ± 0.22 for the control, -1.21 ± 0.53 for the early treatment, and -0.90 ± 0.11 for the late treatment .

The significance of the late treatment’s effect on the rate of cell death is demonstrated by the non-overlapping 95% confidence intervals between the control and late treatment. There were no final treatment differences found on cell vitality for the last sampling date. Figure 6 displays the cell vitality means while Figure 7 shows the segmented fit over the means.Our late irrigation treatment, a 40% augmented pulse starting at the beginning of cell death, generated two significant effects in our experiment: it reduced the rate of cell death in the berry mesocarp tissue and produced less shriveled, more turgid berries at harvest. We also hypothesized that the early treatment would preemptively reduce water stress and postpone cell death. We correctly anticipated the date of cell death onset , yet despite the optimal timing of our treatments the early irrigation regime was not successful in reducing water stress or cell death. While the late pulse reduced the rate of cell death, its effect on shrivel at harvest was not present for the rest of the experiment. This asymmetry is highlighted by the lack of overall correlation between shrivel and cell death for the late pulse, and the lack of a steady decline in turgidity . It was only for the control that significant correlation was observed, which may be explained by the lower error observed in the piece wise linear regression model for the control than the late or early treatments . Unexpectedly, the greater turgidity in the late treatment berries was not reflected in the osmotic potential, °Brix, or TA measurements at harvest, when we would have expected solutes to be more dilute than treatments with greater shrivel . While significant, difference in shrivel were not great enough to affect solute concentrations. The cell death onset and rates we found were largely within the range observed in other studies, blueberry pot but it can be difficult to draw definitive conclusions from comparing the onset and rate of cell death among experiments with many changing variables . Cabernet Sauvignon has only been measured in one other study with cell death beginning at 100 DAA and 40 days after veraison for field-grown vines in Napa Valley , compared to 92 DAA and 34 DAV for our control vines in Davis .

Given Davis is a warmer climate than Oakville, this may explain why our vines exhibited cell death onset earlier. These differences could also reflect a lack of precision in estimating onset dates, since fluctuations in %living tissue measurements can weaken trends with time, as in this study . This could also reflect differences in growing conditions or typical variation within cultivars. The reported ranges of cell death onset dates for any given cultivar vary by as much as 12 days. Syrah onset tends to fall between 87-96 DAA after which tissue vitality sharply declines while Chardonnay’s cell death much slower at an approximately constant rate. . Our rates of cell death fell within the reported range of living tissue analysis performed thus far on grape berries. The Cabernet in Krasnow et al. , between 100-150DAA, lost cellvitality at a rate of approximately -0.83 %LT/DAA while our control and early treatments for Cabernet had a rate of -1.21 and -2.06 respectively. Metabolic processes sensitive to temperature have been connected to cell death, however Xiao et al. and Bonada et al. have both observed no impact of elevated temperature on cell death rate. For Bonada et al. , within the range of 80-120DAA, Syrah lost living tissue at a rate of -0.62 %LT/DAA and Chardonnay at -0.53. The greatest rate of cell death was reported by Xiao et al with living tissue in Syrah diminishing at rates as high as -5.17 %LT/DAA. Interestingly, Xiao et al. found that irrigation postponed cell death onset much later than the non-irrigated vines, yet the irrigated vines crashed twice as fast as the non-irrigated vines , which may have developed more extensive root systems with greater access to water. Conversely, our late treatment vines exhibited the slowest cell death and the earliest onset, highlighting the efficacy in slowing cell death. Our results for cell vitality and shrivel analysis accord with the reported role that xylem backflow plays in the relationship between cell death and LSD. Cell death occurs prior to LSD by reducing turgor pressure and accelerating water loss through xylem backflow, thus a more negative ψx would enhance the pulling force for water out of the berry . Since ψmd and berry osmotic potentials were nearly the same across treatments, the observed differences in cell death rate and shrivel index cannot be attributed to differences in xylem water potential or the berry solute concentrations. In the late treatment berries cell death was slower and cell vitality remained greater from 92DAA onwards , which indicates more water retention and less backflow. Less water loss in berries from cell death supports our finding that the late treatment berries maintained the greatest turgidity for almost all dates during that same period, from 92DAA to harvest. Another factor to consider affecting xylem backflow is pedicel hydraulic conductivity.

Xylem blockages that are known to develop post-veraison in grapevines could hinder pedicel hydraulic conductivity and slow down backflow out of the berry , though there is no basis for such a physiological response to our late irrigation pulse. There are established differences in pedicel hydraulic conductivity between grape cultivars post-veraison and it has been reported that grapevines produce tylose blockages in response to pruning wounds and bacterial infection . Yet, there is no reported evidence of grapevines producing xylem gelblockages in relation to water stress during our period of ripening. For future studies, it may be of interest to track phloem inflow to the berries in response to extra water availability during cell death. Post-veraison the phloem remains the main source of water influx into the berry, remaining functional until 25-26 Brix, a range our berries did not exceed during the late treatment pulse, which suggests the phloem likely remained functional . Our results have shown vines receiving additional irrigation during the onset of cell death, despite having an earlier onset of cell death, retained water better than vines with later onset. Since phloem functionality declines steadily during ripening , an earlier onset of cell death comes at a time of potentially greater phloem hydraulic conductivity, rendering an irrigation pulse more effective at providing more phloem inflow to compensate for xylem backflow. Greater sap influx into the berry would introduce more dissolved oxygen and alleviate hypoxic stress due to respiration and ethanolic fermentation, though this has not been experimentally determined.We expected that H2O2 levels to begin to rise at cell death and continue throughout the experiment. Our H2O2 concentrations saw an uptick during the onset of cell death and, with exception of one outlying date, steadily increased throughout cell death proliferation at similar levels for all treatments . Across the three treatments cell death onset coincided with the onset of H2O2 accumulation at 88 DAA . This is evidenced by the significant correlation found between the two variables for the late treatment and the control. It should be noted that it is only coincidental that those same two treatment groups were significantly different in our cell death rate analysis—matching significant H2O2 correlations and cell death rates would not be causally related findings. Whether the concentrations found in our study, between 1-3 nmol/g FW, are great enough to cause the degree of cell death we observed is not clear. Gowder measured H2O2 in combined skin and pulp samples finding concentrations in the range of 4-10 nmol/gFW in Chardonnay, 12-9 in Grenache, and 15-40 nmol/gFW in Syrah between 90-120 DAA with similar percentages of living mesocarp tissue to this experiment. We would expect to find lower values in the mesocarp only given that the skin is known to contain higher levels of H2O2, yet Pilati et al. found values in range of 18-27 nmol/gFW for the mesocarp only of Pinot Noir berries between 70-84 DAA. Interestingly, also in Gowder the three cultivars expressed no clear relationships between H2O2 accumulation and cell death, indicating there is much greater complexity to be addressed in the sources of H2O2, its scavenging, and alternative causes of cell death such as lipoxygenases.