A sub-sample of shoots and fine roots was collected for organ-dried biomass estimation and sugar and starch analysis. Harvest index was calculated after oven drying the samples.Subsamples of leaves, shoots, and roots were oven-dried at 70◦C to a constant weight. Dried tissues were ground with a tissue lyser . Thirty milligrams of the resultant powder was extracted in ethanol:water solution. Briefly, 1.5 ml was added to each sample and extracted for 10 min at 90◦C in a water bath. Then, they were centrifuged at 10,000 rpm for a minute, and the supernatant was collected for sugar determination. Total soluble sugars and individual sugars were determined in the shoot, leaf, and root ethanolic extracts and in the diluted berry must samples . Samples were filtered with PTFE membrane filters and transferred into high-performance liquid chromatography vials and subjected to reversed-phase HPLC analysis. The equipment consisted of an Agilent 1100 system coupled to a diode array detector and an Infinity Refractive Index Detector . The reversed-phase column was Luna Omega Sugar with a guard column of 5 mm. The temperature of the column compartment was maintained at 40◦C and the RID flow cell was kept at 35◦C. The mobile phase consisted of isocratic elution with acetonitrile:water at a flow rate of 1.0 ml/min with a run time of 22 min. Standard solutions of 10 mg/L of D-glucose, D-fructose, Dsucrose, and D-raffinose were injected to obtain the retention time for each compound,flower buckets wholesale and detection was conducted by RID. Sugar standards were purchased from VWR International . Sugar concentration of each sample was determined by comparison of the peak area and retention time with standard sample curves.

Starch content of the roots, shoots, and leaves was conducted using the Starch Assay Kit SA-20 in accordance with the manufacturer’s instructions. Briefly, pellets of different tissues were dissolved in 1 ml DMSO and incubated for 5 min in a water bath at 100◦C. Starch digestion commenced with the addition of 10 µl α-amylase and then incubated in boiling water for another 5 min. Then, the ddH2O was added to a total volume of 5 ml. Next, 500 µl of the above sample and 500 µl of starch assay reagent were mixed and incubated for 15 min at 60◦C. Negative controls with the starch assay reagent blank, sample blank, and glucose assay reagent blank and positive controls with starch from wheat and corn were performed. Reaction started with the incubation of 500 µl of each sample and 1 ml of glucose assay reagent at 37◦C and was stopped with the addition of 1 ml of 6 M sulfuric acid after 30 min. The reaction was followed with a Cary 100 Series UVVis Spectrophotometer and starch content was expressed as percent of starch per tissue dried weight. Weather data for the 2019 and 2020 growing seasons are shown in Table 1. Compared with the 2019 growing season, 2020 had 17 days more with temperature over 30◦C, a maximum daily temperature of 1.1◦C higher, and almost 800 mm less of precipitation, leading to an ETo of 23 mm higher. On the other hand, the lower available water for grapevine growth resulted in smaller canopy development decreasing the ETc, which explained the lower irrigation amount of 2020 compared with 2019 . Petiole mineral nutrients were not affected by irrigation amounts in the 2018–2019 growing season . Conversely, total N increased in 100% ETc, while the K content in 25% ETc vines decreased in the 2019–2020 growing season. The micronutrients were not affected by the applied water amounts in either year of the study. The plant water status decreased throughout the season . In 2019, the 100% ETc treatment had the highest SWP, while 25% ETc had the lowest SWP as expected. Conversely, there were no significant differences during the 2020 season between treatments. Likewise, we measured significant differences between the different irrigation amounts in gs and AN in both growing seasons .

We measured higher gs and AN in grapevines subjected to 100% ETc treatment from the second half of July, coinciding with the veraison, to harvest,compared with 25% ETc. The gs and AN of 50% ETc were transiently lower than those of 100% ETc, but consistently greater than those of 25% ETc. The WUE differed between irrigation amounts at harvest in 2019 and at mid-ripening in 2020 with 100% ETc grapevines showing the highest WUE . The enhancement of the photosynthetic performance in 100% ETc grapevines was accompanied by increased total chlorophyll and carotenoid content in the leaves . Calculation of the seasonal integral of SWP and gas exchange variables allowed to establish the seasonal-long trend for grapevine physiological response. Thus, SWP seasonal integrals for both seasons were affected by the interaction between irrigation amount and year. During the 2019 season, there was a significant increase of SWP with 100 and 50% ETc siSWP compared with 25% ETc siSWP . However, in the 2020 growing season, no difference in seasonal pattern was measured. On the other hand, seasonal integrals of gs , AN, and WUE were significantly different between years. The AN and WUE were significantly lower in 2020 compared with 2019 . Grapevine growth was monitored for different organs as shown in Table 3 and Supplementary Table S2. Leaf, shoot, and root fresh weights increased with increased irrigation amounts . The biomass of the leaves, roots, and shoots increased in the grapevines subjected to 100% ETc irrigation compared with 50 and 25% ETc . The applied irrigation treatments affected the harvest index . The greatest harvest index was measured in 100% ETc, while the lowest was measured in 25% ETc, respectively. Cluster number was not affected by the replacement of different fractions of ETc . An increase in the yield per grapevine was observed in both seasons with a highly significant increase in yield per grapevine in 100% ETc treatment.

Likewise, the linear increase in yield was evident from 25% ETc to 50% ETc as well, in both years. We also measured linear increases in leaf area to fruit ratio and berry size as the amount of irrigation increased from 25% ETc to 100% ETc. There was a significant increase of SS and starch content in the leaves as affected by the applied water amount . This increase in leaf SS was attributed to the increases in glucose, fructose, and raffinose content of the leaves . The total sugar and starch content of the shoots were not affected by the applied water amount . However, sucrose and raffinose in the shoots increased in 50 and 100% ETc treatments compared with 25% ETc . Root carbohydrate content and composition were not affected by irrigation treatments, with sucrose being the main soluble sugar found in root tissues . Our analysis of the different carbohydrates found in grapevine tissues indicated that starch was the main NSC in the shoots and roots, which accounted for >50% regardless of the applied water affecting their proportions . In the leaves, starch content was the less abundant NSC, but a significant effect of irrigation treatments was observed with the 100% ETc treatment reaching the highest amount. Finally, the proportions of sucrose and raffinose in the shoots decreased when water application was restricted to 25% ETc . Regarding the sugar composition of the must, fructose and glucose were the main sugars found , and their ratio ranged between 0.62 and 0.78 with no difference between treatments . In spite of the warming trends recorded for the study area within the two growing seasons covered by this study, the plant water status recorded in both growing seasons was optimal for grapevine growth as indicated by the midday SWP and the gs . Thus, seasonal integrals of SWP ranged between -0.8 and -1.1 MPa, while gs ranged between 150 and 250 mmol m−2 s −1 , in accordance to the midday SWP and gs values considered as well-watered conditions . Moreover,flower harvest buckets water status of the grapevines subjected to less applied water amount never reached values lower than -1.5 MPa for SWP and/or 50 mmol m−2 s −1 for gs , which have been reported to impair grapevine performance and berry ripening . As Keller et al. reported before, in warmer years, 100% ETc treatment may suffer from mild water deficit. Thus, under our experimental conditions, at the end of the season, especially in 2020, grapevines reached SWP values to ca. -1.2 MPa; however, they are not sufficient to impair grapevine physiology and metabolism in warm climates . Previous studies highlighted that plant water status is closely related to leaf gas exchange parameters . Thus, low values of SWP were related to decreased gs likely because plants subjected to mild to moderate water deficit close their stomata as an early response to water scarcity to diminish water loss and carbon assimilation . Accordingly, in both growing seasons, a higher SWP promoted increased stomatal conductance and, consequently, net carbon assimilation rates in grapevines subjected to 100% ETc. AN and gs peaked around veraison and then declined in all the treatments similar to several studies conducted in a warm climate before . Thus, previous studies have pointed out that limited photosynthetic performance, hence lower gs and AN values, may be triggered by passive or active signals .

Nevertheless, AN in 50% ETc treatment was not severely decreased presumably by increases in WUE, which have been related to improvements in stomatal sensitivity to water loss and vapor pressure despite the hormonal signaling from roots to shoots . Likewise, Tortosa et al. suggested that differences in WUE between Tempranillo grapevine clones were more explanatory of the variations in carbon assimilation rather than a different stomatal control. Finally, it is worth mentioning that WUE was significantly lower in the driest and hotter growing season regardless of the irrigation treatment as previously reported . Regarding intrinsic WUE , no effect due to growing conditions was observed in contrast to previous studies on vines subjected to mild water stress . The water deficits applied in this study were from moderate to severe based on SWP values; thus, it is expected that the vegetative and reproductive growth of vines will be impacted accordingly. Thus, in previous studies, higher water deficits resulted in reductions of yield and berry size . The reduction in berry mass has been associated with the inhibition of cell expansion and the diminution of inner mesocarp cell sap . The detrimental effects of 25% ETc were reported previously, suggesting that this applied water amount Vegetative growth was also impaired by water deficits applied in this study, as indicated in the decrease of leaf and root dry biomasses measured in 25 and 50% ETc treatments. Diminution of root growth under water stress has been related to the loss of cell turgor and increased penetration resistance of dried soils . In addition, a recent study suggested that the loss of leaves could decrease the supply of carbohydrates and/or growth hormones to meristematic regions, thereby inhibiting growth . In accordance with previous studies, severe water deficits led to lower shoot to root ratio because root growth is generally less affected than shoot growth in drought-stressed grapevines . Given that grapevine vegetative growth occurs soon after bud break in springtime, our results corroborated the crucial role of water availability during that period on vine development, physiological performance, and yield components reported in previous studies . Thus, irrigation of grapevines during summer could not be sufficient to fulfill water requirements when rainfall has been scarce in spring , and precipitation amounts prior to bud break result in cascading effects for the rest of the growing season that cannot be overcome with supplemental irrigation . The allocation of NSC varied between organs for which roots accounted 30%, shoots 25%, and leaves 40% of the whole plant NSCs at harvest, slightly differing from those reported for several fruit trees but similar to the works in grapevine . The NSC composition was highly dependent on the grapevine organs, with starch being the main NSC in the roots and shoots. Previous studies reported that roots accumulated the largest amounts of starch in plastids, namely amyloplasts, which is fundamental to allow rapid vegetative development during the next spring .