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, 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 UVV is Spectrophotometer and starch content was expressed as percent of starch per tissue dried weight.In spite of the warming trends recorded for the study area within the two growing seasons covered by this study, 30 planter pot 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, 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 may not be adequate for hot climates with very little or no summer precipitation . Vegetative growth was also impaired by water deficits applied in this study, as indicated in the decrease of leaf and root dry bio-masses 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 theworks 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 . Our results also corroborated with this finding. Our results indicated that, apart from fruits, SS were mainly accumulated in the leaves at harvest, which accounted for about 90% of the total leaf NSC. Thus, the allocation of NSCs in different organs allowed the plants to persist when respiration rate was higher than photo assimilation in annual events, but also aided in responding to abiotic stresses such as drought . Our results indicated that plants that received 100% ETc had higher NSC content. Similarly, a previous study with potted grapevines reported increased starch and SS contents in the leaves from the grapevines with higher leaf area to fruit ratio that were well-watered . In shoots, sucrose and raffinose proportions were higher in 50 and 100% ETc treatments compared with 25% ETc. As a great part of the shoot biomass is vascular tissue, this may suggest an increase in NSC translocation in these treatments. Although sucrose is the main sugar for carbon translocation through the phloem into the sink tissues, recent research highlighted the roles of other sugars, such as raffinose, in carbon translocation and storage . On the other hand, plastic growers pots previous research reported less NSC accumulation in grapevine canes under carbon starvation at a low leaf to fruit ratios, suggesting that sucrose may control starch accumulation through adjustment of the sink strength . Furthermore, Rossouw et al. also highlighted the role of raffinose toward root carbohydrate source functioning in grapevines with significantly lower leaf to fruit ratio due to defoliation from carbon starvation . When the photosynthetic supply of carbohydrates is limited, remobilization from perennial tissues can provide an alternative carbon source . Thus, previous research conducted on potted grapevines reported a concurrent starch remobilization from roots with a rapid berry sugar accumulation . Conversely, under our experimental conditions , no effect of water deficits on NSC remobilization from roots to berries was observed despite the decreased leaf to fruit ratio. Likewise, Keller et al. did not observe higher amounts of sugars in berries from field-grown Cabernet Sauvignon subjected to 25% ETc compared with 70 or 100% ETc under field conditions.Under our experimental conditions, yield per plant was strongly related to shoot, leaf, and root BM. Similarly, Field et al. found that grapevines with the lowest shoot growth rate before veraison had significantly less fruit set than the other treatments, attributing these effects to the restoration of root carbohydrate reserves that occurred at the same time. Grapevines subjected to 25% ETc had reduced photo assimilates due to lower AN in both seasons resulting in less NSC in the source leaves available for new growth and exported to sinks. This resulted in a general lower plantBM . Contrarily, grapevines subjected to 100% ETc had higher photo assimilation rates throughout the course of the study that led to higher SS and starch content and, consequently, to the improvement of BM and, therefore, higher harvest index. Therefore, the reduced growth rate of both sink and source organs in response to water deficits indicated that the availability of carbon is a major growth constraint. The yield per plant of 50% ETc was lower than 100% ETc, but not as low as 25% ETc. However, canopy BM was greatly reduced in both 50% ETc and 25% ETc compared with 100% ETc. Accordingly, Field et al. reported that grapevine grown under warm soil conditions favored shoot and fruit development over carbohydrate reserve accumulation. In contrast, Candolfi-Vasconcelos et al. reported that a lower leaf area to fruit ratio increased the translocation of carbohydrates from permanent structures to reproductive organs to support grape ripening. The shoot to root ratio revealed a positive relationship with the total BM, leaf and root NSC, and N contents. Thus, the distribution of biomass relies on the C:N ratio as highlighted by the negative relationship between shoot to root and the sucrose:nitrogen ratios. Similarly, a linear relationship between NSC and root to shoot ratio in grapevines grown under stressful conditions was previously reported . From a molecular point of view, the alterations of source:sink ratios led to transcriptional adjustments of genes involved in starch metabolism, including the upregulation of VvGPT1 and VvNTT for lower leaf area to fruit ratios . Furthermore, enhanced root biomass in 100% ETc likely resulted from higher sugar content in the roots as our data supported. It was recently reported that increases in root elongation and hexose contents were due to the VvSWEET4 overexpression, a gene implied as a grapevine response to abiotic stress . Similarly, Medici et al. reported up- or downregulation of the genes encoding hexose transporters in grapevines subjected to water deficits corroborating this result. Therefore, although some genes may be expressed under water deficit, lack of carbon accumulation impaired the growth. The relationship between root to shoot ratio and plant nitrogen content was previously reported for grapevines, suggesting that dry matter partitioning is largely a function of the internal status of the plants . We found decreased N content in grapevines facing water deficits, which resulted in a decrease of total BM. Similarly, Romero et al. reported reductions in leaf nitrogen content when vines were subjected to water deficits. These authors suggested that nutrient uptake may be reduced due to deficits in soil water profile, and the slow root growth under these conditions consequently inhibited grapevine growth. In our study, N content was strongly related to photosynthetic pigments. Accordingly, previous studies reported lower leaf N and leaf chlorophyll in deficit-irrigated grapevines, suggesting quantitative losses in the photosynthetic apparatus and/or damage to the biochemical photosynthetic machinery, decreasing photosynthetic capacity as corroborated with the lower NSC leaf content with water deficits. Finally, molecular research over the last decades has suggested the important regulatory functions of sucrose and N metabolites in metabolism at the cellular and subcellular levels and/or in gene expression patterns, giving new insights into how plants may modulate over a longer period its growth and biomass allocation in response to fluctuating environmental conditions .