TY - JOUR
AU - Simonneau, Thierry
AB - Abstract Plants evolved different strategies to cope with water stress. While isohydric species maintain their midday leaf water potential (ΨM) under soil water deficit by closing their stomata, anisohydric species maintain higher stomatal aperture and exhibit substantial reductions in ΨM. It was hypothesized that isohydry is related to a locally higher sensitivity of stomata to the drought-hormone abscisic acid (ABA). Interestingly, recent lines of evidence in Arabidopsis (Arabidopsis thaliana) suggested that stomatal responsiveness is also controlled by an ABA action on leaf water supply upstream from stomata. Here, we tested the possibility in grapevine (Vitis vinifera) that different genotypes ranging from near isohydric to more anisohydric may have different sensitivities in these ABA responses. Measurements on whole plants in drought conditions were combined with assays on detached leaves fed with ABA. Two different methods consistently showed that leaf hydraulic conductance (Kleaf) was down-regulated by exogenous ABA, with strong variations depending on the genotype. Importantly, variation between isohydry and anisohydry correlated with Kleaf sensitivity to ABA, with Kleaf in the most anisohydric genotypes being unresponsive to the hormone. We propose that the observed response of Kleaf to ABA may be part of the overall ABA regulation of leaf water status. Maintaining adequate tissue water content through efficient controls of water supplies and losses is a key requirement for crop performance and plant survival in dry environments. Accordingly, plants evolved with varied capacities to close stomata in response to soil drying, thereby limiting the drop of water potential along the transpiration path yet at the expense of carbon assimilation and growth. Optimization between carbon gain and water loss has resulted in the evolution of a continuum of strategies among species, ranging from isohydry to anisohydry. Anisohydric species exhibit a substantial decrease in their midday leaf water potential (ΨM) as soil water deficit develops, while isohydric species maintain higher ΨM through stomatal closure at incipient stages of soil drying (Tardieu et al., 1996). A wide spectrum of behaviors has also been observed between varieties of the same species, as in apple tree (Malus; Massonnet et al., 2007) and grapevine (Vitis vinifera; Schultz, 2003; Soar et al., 2006; Prieto et al., 2010). In grapevine, genomic regions (QTLs) have been identified that control the maintenance of ΨM under moderate water deficit (Coupel-Ledru et al., 2014). Although dependent on environmental conditions (Franks et al., 2007), variation from iso- to aniso-hydry has therefore a clear genetic basis. How stomata coordinate with plant hydraulics to optimize ΨM in response to drought and how this may vary between species remain a matter of debate. Yet, it has been frequently reported that stomatal response parallels the dynamics of hydraulic conductance in roots (Zufferey and Smart, 2012; Vandeleur et al., 2014), leaves (Cochard et al., 2002), or whole plants (Meinzer, 2002; Zufferey and Smart, 2012). This observation has been mostly interpreted as the result of a biological coupling between water supply (hydraulic conductance) and water demand (transpiration), preventing water potential along the transpiration path from dropping to damaging levels (Cochard et al., 1996; Sack and Holbrook, 2006; Franks et al., 2007; Simonin et al., 2015). On the one hand, water deficit may impact on water supply via either the development of cavitation in xylem conduits (Tyree and Sperry, 1989), thereby reducing hydraulic conductance from the inner root tissues to the leaf petiole, or down-regulation of aquaporin activity, which controls water transfer through membranes of living cells in both roots and leaves (Chaumont and Tyerman, 2014). On the other hand, stomata primarily control the response of transpiration to water deficit. The physiological mechanisms underlying stomatal response most likely involve both hydraulics and biochemistry with the accumulation of the drought hormone abscisic acid (ABA). Water deficit draws down water potential in all plant tissues and may directly impair turgor pressure in the guard cells surrounding the stomatal pores, thus reducing stomatal aperture (Buckley, 2005; Peak and Mott, 2011). In parallel, accumulation of ABA, whether synthesized in roots (Simonneau et al., 1998; Borel et al., 2001) or leaves (Christmann et al., 2005, 2007; Ikegami et al., 2009; McAdam et al., 2016), directly impacts on guard cells to close stomata (Kim et al., 2010; Joshi-Saha et al., 2011). The relative contribution of ABA signaling and hydraulics to drought-induced stomatal closure varies depending on species (Tardieu et al., 1996; Brodribb and Jordan, 2011; Brodribb and McAdam, 2013; McAdam and Brodribb, 2013, 2014; Brodribb et al., 2014). Interestingly, leaf water potential appears to sensitize guard cells to the effect of ABA, thus resulting in a feedforward effect on stomatal closure upon water stress (Tardieu and Davies, 1992). Moreover, this feedforward effect is only observed in isohydric species (Tardieu and Simonneau, 1998). Although such apparent interaction between hydraulics and ABA accounts for the distinction between isohydric and anisohydric behaviors, the biological basis for this observation remains unknown. Recent studies on the isohydric species Arabidopsis (Arabidopsis thaliana) challenged the classical view that ABA induces stomatal closure by solely acting at the guard cell level. First, ABA reduces leaf hydraulic conductance (Kleaf) through the down-regulation of aquaporin activity in the bundle sheath around leaf veins (Shatil-Cohen et al., 2011). Second, xylem-fed ABA induces parallel reductions in Kleaf and stomatal conductance in leaves of mutants that are insensitive to ABA at the guard cell level (Pantin et al., 2013). These results gave rise to the proposal that ABA promotes stomatal closure in a dual way, via its local, biochemical effect on the guard cells, but also via a remote, hydraulic impact of a drop in water permeability within the bundle sheath. Bundle sheath cells were thus assigned a role of “control center” for water flow, able to convert ABA signaling into feedforward hydraulic signals up to guard cells. We surmised that such hydraulic effect of ABA may underlie the apparent interaction between hydraulics and ABA on stomatal control of isohydric species (Tardieu and Simonneau, 1998) and thus originate the genetic differences between isohydric and anisohydric behaviors. Here, we tested this hypothesis by examining the relationship between Kleaf sensitivity to ABA and (an)isohydric behavior of grapevine genotypes obtained from a cross between two contrasting cultivars, the near-anisohydric Syrah and the near-isohydric Grenache (Schultz, 2003; Soar et al., 2006; Prieto et al., 2010). A wide range of variation for (an)isohydry was evidenced within the offspring, ranking far beyond the parental behaviors (Coupel-Ledru et al., 2014). We selected a panel of contrasting genotypes and combined experiments on whole plants under two watering regimes with measurements on detached leaves fed with various ABA concentrations. The inhibiting effect of ABA on Kleaf was observed only in those genotypes that showed strong stomatal closure and typical isohydric behavior upon water deficit. By contrast, Kleaf of more anisohydric genotypes was insensitive to ABA. These results support a major role for genetic variation in Kleaf sensitivity to ABA in determining (an)isohydric behavior in grapevine. RESULTS Two Independent Methods Reveal Differential Sensitivity of Kleaf to ABA between Syrah and Grenache To investigate the putative link between ABA, leaf hydraulics, and plant (an)isohydric behavior, we assessed the effect of ABA on Kleaf on two grapevine cultivars reputed to be isohydric (Grenache) and anisohydric (Syrah). Kleaf response to ABA was first characterized using the Evaporative Flux Method (EFM) on detached leaves that were xylem-fed for 1 h with a control solution or with a solution of exogenous ABA at a concentration of 2, 4, 8, 16, or 32 mmol m−3. Kleaf displayed a strong sensitivity to ABA in Grenache, declining from 12 mmol m−2 s−1 MPa−1 in the control solution to 5 mmol m−2 s−1 MPa−1 in the 32 mmol m−3 ABA solution (Fig. 1A). By contrast, Kleaf of Syrah was much less sensitive to ABA (Fig. 1B), with a slight decrease that was not found significant (P > 0.1). Figure 1. Open in new tabDownload slide Effect of ABA on hydraulic conductances of detached leaves (Kleaf), laminas (Klamina) and petioles (Kpetiole) sampled on cultivars Grenache and Syrah. Hydraulic conductance expressed as a function of ln([ABA]) in the feeding sap. A and B, Excised leaves from potted plants were perfused for 1 h in artificial sap containing no ABA, or ABA at concentrations of 2, 4, 8, 16, or 32 mmol m−3 (+)-ABA prior to the determination of Kleaf with the EFM. C and D, Shoots were cut from a vineyard and rehydrated overnight prior to leaf excision, perfusion in artificial sap containing no ABA, or ABA at concentrations of 2, 4, 8, 16, or 32 mmol m−3 (+)-ABA, and measurement of Kleaf with the HPFM. E and F, The lamina was then removed by excising at the petiole junction, and Kpetiole was determined with the HPFM. G and H, Klamina was derived from Kleaf and Kpetiole according to the Ohm’s law analogy considering that petiole and lamina transport water in series: 1/Kleaf = 1/Kpetiole + 1/Klamina. Figure 1. Open in new tabDownload slide Effect of ABA on hydraulic conductances of detached leaves (Kleaf), laminas (Klamina) and petioles (Kpetiole) sampled on cultivars Grenache and Syrah. Hydraulic conductance expressed as a function of ln([ABA]) in the feeding sap. A and B, Excised leaves from potted plants were perfused for 1 h in artificial sap containing no ABA, or ABA at concentrations of 2, 4, 8, 16, or 32 mmol m−3 (+)-ABA prior to the determination of Kleaf with the EFM. C and D, Shoots were cut from a vineyard and rehydrated overnight prior to leaf excision, perfusion in artificial sap containing no ABA, or ABA at concentrations of 2, 4, 8, 16, or 32 mmol m−3 (+)-ABA, and measurement of Kleaf with the HPFM. E and F, The lamina was then removed by excising at the petiole junction, and Kpetiole was determined with the HPFM. G and H, Klamina was derived from Kleaf and Kpetiole according to the Ohm’s law analogy considering that petiole and lamina transport water in series: 1/Kleaf = 1/Kpetiole + 1/Klamina. To consolidate these results, we measured Kleaf sensitivity to ABA with a second, independent method. We used the High Pressure Flowmeter (HPFM) to measure Kleaf in detached leaves of Grenache and Syrah fed for 1 h with control or ABA solutions (Fig. 1, C and D). Overall, whatever the method used, Kleaf was strongly and significantly reduced by ABA feeding in the near-isohydric cultivar Grenache (P < 0.01) but not in the near-anisohydric Syrah. Kleaf values obtained in control conditions with the HPFM were highly consistent with those previously reported by Pou et al. (2013), who operated similarly. However, Kleaf values were 3-fold higher when measured with the HPFM as compared to the EFM, which suggests that Kleaf might be overestimated by the HPFM because of the higher hydrostatic pressure imposed to water within the leaf (Prado and Maurel, 2013) while negative pressures develops in transpiring leaves. For this reason, the EFM most likely mimicked the natural pathway of water in leaves through the transpiration flow (Sack and Scoffoni, 2012), even though the flow rate was of the same order of magnitude whatever the method used (between 0.5 and 3 mmol m−2 s−1; see also Supplemental Fig. S2). The HPFM method also made it possible to distinguish the conductance between petiole and lamina. For that purpose, just after Kleaf measurement with the HPFM, the leaf was cut at the junction between petiole and lamina, and hydraulic conductance of the petiole (Kpetiole) was determined. The hydraulic conductance of the lamina (Klamina) could then be derived considering that water pathways in petiole and lamina operate in series. Irrespective of the cultivar, the conductance in the petiole was much stronger (about 10-fold higher) than in the lamina when calculated at the whole organ level, indicating that most of the resistance to water transfer takes place in the lamina. No effect of ABA was observed for Syrah on either part of the leaf (Fig. 1, F and H) as could be expected from the absence of effect on overall Kleaf (Fig. 1D). By contrast, in the nearly isohydric Grenache, ABA feeding markedly reduced Klamina (Fig. 1G; P < 0.01) while Kpetiole was not significantly affected (Fig. 1E). Measurements at the leaf and lamina levels also revealed a significant difference in K values recorded before ABA perfusion: Grenache displayed higher initial Kleaf (and Klamina) than Syrah (Fig. 1). Kleaf at maximum ABA concentration in Grenache reached about the same value as the initial Kleaf in Syrah. Stomatal responses to ABA (measured by porometry) were much closer to each other between Syrah and Grenache (Supplemental Fig. S1) than differences in Kleaf response to ABA that were detected using the HPFM method. Variability in (An)isohydry and ABA Accumulation for Ten Selected Offspring Genotypes and the Parental Cultivars Analysis was then extended to a panel of genotypes with contrasting (an)isohydric behaviors. Ten offspring genotypes were selected in the Syrah × Grenache population based on the change in ΨM previously observed between well-watered (WW) and water-deficit (WD) conditions (Coupel-Ledru et al., 2014). ΨM measured in the selected genotypes under WD and controlled atmospheric conditions ranged from −1.1 to −0.85 MPa (Fig. 2A). The drop in leaf ΨM (ƊΨM) between WW and WD regimes displayed a highly significant effect of the genotype (P < 0.001), ranging from −0.5 MPa for the most anisohydric genotype to −0.1 MPa for the most isohydric one (Fig. 2B). Change in ΨM induced by drought in the parents was intermediate, with a slightly better maintenance in Grenache (ƊΨM of −0.15 MPa) compared to the more anisohydric Syrah (ƊΨM of −0.25 MPa). Figure 2. Open in new tabDownload slide Characterization of (an)isohydric behaviors for a selection of 10 offspring genotypes as compared to the whole progeny and the parents Syrah and Grenache. Distribution of the genotypic means recorded in the whole Syrah × Grenache population for: (A) leaf water potential measured in the daytime under WD conditions (ΨM WD), and (B) reduction in leaf water potential in the daytime under WD as compared to WW conditions (ƊΨM), as an indicator of the (an)isohydric behavior. A and B, Data are genotypic means calculated for the experiment conducted in 2012 presented in Coupel-Ledru et al. (2014) for n = 188 genotypes. Values for the panel of 10 offspring genotypes and for the parental genotypes Syrah and Grenache (notified as ‘Syr’ and ‘Gre’) are indicated with black arrows. Figure 2. Open in new tabDownload slide Characterization of (an)isohydric behaviors for a selection of 10 offspring genotypes as compared to the whole progeny and the parents Syrah and Grenache. Distribution of the genotypic means recorded in the whole Syrah × Grenache population for: (A) leaf water potential measured in the daytime under WD conditions (ΨM WD), and (B) reduction in leaf water potential in the daytime under WD as compared to WW conditions (ƊΨM), as an indicator of the (an)isohydric behavior. A and B, Data are genotypic means calculated for the experiment conducted in 2012 presented in Coupel-Ledru et al. (2014) for n = 188 genotypes. Values for the panel of 10 offspring genotypes and for the parental genotypes Syrah and Grenache (notified as ‘Syr’ and ‘Gre’) are indicated with black arrows. We also assessed the variability in ABA accumulation in response to soil drying. For this purpose, ABA was assayed in xylem sap that was collected on leaves of 2 intact plants per genotype (10 offspring and 2 parental) under soil water deficit and standardized transpiring conditions. Genotype had a significant effect on ABA concentration in the xylem sap of WD plants (P < 0.01). Across genotypes, ABA concentration in the xylem sap ranged from 0.5 to 2.8 mmol m−3 (Fig. 3A). Syrah displayed much lower ABA concentration in the xylem sap (about 0.8 mmol m−3) than Grenache (1.9 mmol m−3). ABA concentration in the xylem sap did not match with the ranking of genotypes according to (an)isohydry level and did not correlate with ƊΨM (Fig. 3B). This rules out a simple role of genetic variation of ABA accumulation induced by water deficit in the determinism of (an)isohydry. We also examined whether higher ABA content could be responsible for reduced Kleaf, even under well-watered conditions, specifically in those genotypes where Kleaf did not respond to further treatment with exogenous ABA. ABA concentration was therefore determined in xylem sap extruded from leaves of Syrah and Grenache grown under well-watered conditions. Average ABA content was lower in Syrah (0.5 ± 0.2 mmol m−3, n = 4) than in Grenache (1.1 ± 0.5 mmol m−3, n = 4). This result rules out a possible role of high initial ABA levels in leaves on the absence of Kleaf response for genotypes like Syrah. Figure 3. Open in new tabDownload slide Concentration of ABA in the xylem sap ([ABA] xylem sap, midday) of 10 offspring genotypes selected in the Syrah × Grenache population and the parents, and relationship with the (an)isohydric behavior. A, ABA concentration in the xylem sap expressed at midday under controlled transpiring conditions from leaves of intact plants submitted to a moderate soil water deficit. ABA was quantified by LCMS. Means ± se for n = 2 leaves sampled on two different plants per genotype. Genotypes are ordered according to their (an)isohydric behavior as estimated by the difference in leaf ƊΨM between water deficit and well-watered conditions measured under controlled transpiring conditions (like in Fig. 2B). Genotypes without common letters above their respective bars have significantly different [ABA]xylem sap, midday (P < 0.05). B, Correlation between ABA concentration in the xylem sap and (an)isohydry level (ƊΨM). The correlation is not significant (P = 1). Figure 3. Open in new tabDownload slide Concentration of ABA in the xylem sap ([ABA] xylem sap, midday) of 10 offspring genotypes selected in the Syrah × Grenache population and the parents, and relationship with the (an)isohydric behavior. A, ABA concentration in the xylem sap expressed at midday under controlled transpiring conditions from leaves of intact plants submitted to a moderate soil water deficit. ABA was quantified by LCMS. Means ± se for n = 2 leaves sampled on two different plants per genotype. Genotypes are ordered according to their (an)isohydric behavior as estimated by the difference in leaf ƊΨM between water deficit and well-watered conditions measured under controlled transpiring conditions (like in Fig. 2B). Genotypes without common letters above their respective bars have significantly different [ABA]xylem sap, midday (P < 0.05). B, Correlation between ABA concentration in the xylem sap and (an)isohydry level (ƊΨM). The correlation is not significant (P = 1). Genetic Variation in Kleaf Sensitivity to ABA Correlates with the Degree of Isohydry Kleaf response to xylem-fed exogenous ABA was characterized on detached leaves of the 10 offspring genotypes and the parents using the EFM. In the absence of ABA, genetic variability was observed for Kleaf (P < 0.001), ranging from 2.5 to 13.5 mmol m−2 s−1 MPa−1 (Figs. 4 and 5A). Feeding with ABA solutions at various concentrations had contrasting effects depending on the genotype (Fig. 4). Grenache and Syrah responses were consistent with those reported in the previous experiment (Fig. 1). Most of the change in Kleaf was observed for xylem ABA varying between 0 and 5 mmol m−3, corresponding to actual concentrations reported in the xylem sap of grapevines under various soil water conditions (e.g. Rogiers et al., 2012). Five genotypes exhibited a Grenache-like response to ABA, with a strong, significant (P < 0.001) reduction of Kleaf when ABA concentration was increased (Fig. 4, A–E). By contrast, the seven other genotypes showed a Syrah-like response, with nonsignificant effect of ABA on Kleaf (Fig. 4, F–L). Figure 4. Open in new tabDownload slide Sensitivity of Kleaf to ABA for 10 offspring genotypes of the Syrah × Grenache population and the parents. Kleaf was measured on detached leaves fed with artificial sap containing variable ABA concentration. Each panel (A–L) represents a genotype, and genotypes are ordered from the highest (A) to the lowest (L) sensitivity of Kleaf to ABA. Kleaf was measured according to the EFM. Mean ± se for n = 5 leaves taken from two to three plants measured at each concentration for each genotype. Fitting of linear regression models was performed on semi-ln transformed data (Supplemental Fig. S3) and is represented here by the lines in the nontransformed coordinates. Figure 4. Open in new tabDownload slide Sensitivity of Kleaf to ABA for 10 offspring genotypes of the Syrah × Grenache population and the parents. Kleaf was measured on detached leaves fed with artificial sap containing variable ABA concentration. Each panel (A–L) represents a genotype, and genotypes are ordered from the highest (A) to the lowest (L) sensitivity of Kleaf to ABA. Kleaf was measured according to the EFM. Mean ± se for n = 5 leaves taken from two to three plants measured at each concentration for each genotype. Fitting of linear regression models was performed on semi-ln transformed data (Supplemental Fig. S3) and is represented here by the lines in the nontransformed coordinates. Figure 5. Open in new tabDownload slide Variability in Kleaf max, in sensitivity of Kleaf to ABA, and its relationships with the (an)isohydric behavior estimated at the whole plant level for 10 offspring genotypes of the Syrah × Grenache population and the parents. A, Variability in maximum value of Kleaf without ABA treatment (means ± SE of values measured for n = 5 leaves per genotype). Comparison using Kruskal-Wallis test revealed a significant effect of the genotype in Kleaf max (P < 0.05). Genotypes without common letters above their respective bars have significantly different Kleaf max. B, Variability in sensitivity of Kleaf to ABA, calculated as the opposite of the slope of the linearized relationship between Kleaf in mmol m−2 s−1 MPa−1 and ln([ABA] in the feeding sap expressed in mmol m−3), as presented in Supplemental Figure S3. A higher sensitivity represents a stronger down-regulation by ABA. Bars indicate the confidence interval. Genotypes without common letters above their respective bar have significantly different Kleaf sensitivity to ABA. C, Relationship between Kleaf sensitivity to ABA and Kleaf max. D, Relationship between Kleaf sensitivity to ABA and the (an)isohydric behavior estimated at the whole plant level as the difference in leaf water potential measured in the daytime between water deficit and well-watered conditions (ƊΨM) as in Figure 2B. C and D, Pearson’s determination coefficient is indicated with its significance level: **P < 0.001. Figure 5. Open in new tabDownload slide Variability in Kleaf max, in sensitivity of Kleaf to ABA, and its relationships with the (an)isohydric behavior estimated at the whole plant level for 10 offspring genotypes of the Syrah × Grenache population and the parents. A, Variability in maximum value of Kleaf without ABA treatment (means ± SE of values measured for n = 5 leaves per genotype). Comparison using Kruskal-Wallis test revealed a significant effect of the genotype in Kleaf max (P < 0.05). Genotypes without common letters above their respective bars have significantly different Kleaf max. B, Variability in sensitivity of Kleaf to ABA, calculated as the opposite of the slope of the linearized relationship between Kleaf in mmol m−2 s−1 MPa−1 and ln([ABA] in the feeding sap expressed in mmol m−3), as presented in Supplemental Figure S3. A higher sensitivity represents a stronger down-regulation by ABA. Bars indicate the confidence interval. Genotypes without common letters above their respective bar have significantly different Kleaf sensitivity to ABA. C, Relationship between Kleaf sensitivity to ABA and Kleaf max. D, Relationship between Kleaf sensitivity to ABA and the (an)isohydric behavior estimated at the whole plant level as the difference in leaf water potential measured in the daytime between water deficit and well-watered conditions (ƊΨM) as in Figure 2B. C and D, Pearson’s determination coefficient is indicated with its significance level: **P < 0.001. Linear models were then fitted to semilogarithmic transformed data (Supplemental Fig. S3), and Kleaf sensitivity to ABA was calculated as the slope of this regression, giving the expected change in Kleaf for any e-fold increase in ABA concentration. The more negative the slope, the more sensitive Kleaf was to ABA. Sensitivity was thus calculated as the opposite of the slope. Although confidence intervals on slopes were quite large (Fig. 5B), analysis of covariance revealed a significant effect of the genotype on Kleaf sensitivity to ABA (P < 0.01). Moreover, Kleaf sensitivity to ABA strongly correlated with the initial level of Kleaf before ABA inhibition (Kleaf max; Fig. 5C). We next tested the relationship between Kleaf sensitivity to ABA and (an)isohydry by examining the correlation with ƊΨM for all selected genotypes. The correlation was highly significant and positive (Fig. 5D). This confirmed that the genotypes with the most sensitive Kleaf to ABA had a better capacity to maintain ΨM at high level under soil water deficit. By contrast, the genotypes with Kleaf being hardly responsive to ABA exhibited a substantial drop in ΨM under dry soil conditions. It is somewhat counter-intuitive that a stronger reduction in Kleaf may result in a better maintenance of leaf water potential upon water deficit. A drop in Kleaf is rather expected to lower leaf water potential, provided leaf transpiration rate (Eleaf) is constant. However, ABA feeding also induced a strong reduction in Eleaf in detached leaves (Supplemental Figs. S4–S6). This result supports the assumption that a drop in Kleaf upon ABA increase could be responsible for a substantial stomatal closure, which could dominate on moderating the drop in leaf water potential (Supplemental Fig. S7). Predicting Genotypic Response to Water Deficit from ABA Accumulation and Sensitivity to ABA We then examined the hypothesis that genetic variation in Kleaf sensitivity to ABA correlated with the reduction of Eleaf in intact plants submitted to water deficit. For this purpose, we combined the sensitivities of Kleaf and Eleaf to ABA, as estimated in detached leaves of each genotype (Fig. 5B; Supplemental Figs. S3, S5, and S6), with the native ABA concentration that was measured in the xylem sap (Fig. 3A) of plants under soil water deficit. In addition, Eleaf and Kleaf of well-watered plants were estimated as the maximum values observed in detached leaves fed with ABA-free solution assuming that they were representative of leaves attached on well-watered plants. Changes in Eleaf and Kleaf induced by water deficit were then related to these maximum values determined for each genotype, yielding percent reduction in Kleaf (% reduction Kleaf) and in Eleaf (% reduction Eleaf). The correlation between % reduction Eleaf and % reduction Kleaf could then be examined (Fig. 6A). Overall, Eleaf predicted from ABA concentrations showed higher sensitivity to water deficit than Kleaf, yet the predicted changes in Eleaf (% reduction Eleaf) covered a smaller range of genetic variation within the panel of genotypes (between 38% for the less responsive genotype and 50%; Fig. 6A) compared to the genetic range observed for the % reduction Kleaf (between 0 and 38%; Fig. 6A). Despite this difference, % reduction Eleaf positively correlated with % reduction Kleaf (Fig. 6A). Similar results were obtained when plotting % reduction Kleaf against relative changes in transpiration rate directly measured on the whole plants (Fig. 6B) during the high-throughput experiment (% reduction EPlant). This suggested that the more Kleaf was reduced by ABA accumulation under water deficit, the more Eleaf was reduced. In agreement with our initial assumption, a better maintenance of plant water potential could be expected for those genotypes with Kleaf and thus Eleaf more sensitive to ABA. Figure 6. Open in new tabDownload slide Correlation between % reduction Kleaf and % reduction Eleaf (A)predicted from assays on detached leaves, or (B) measured directly on intact plants (% reduction Eplant) for the 10 offspring genotypes selected in the Syrah × Grenache population and the parents. Predicted changes in Kleaf and Eleaf between well-watered and water deficit conditions were calculated assuming maximum Kleaf in well-watered plants and combining ABA concentration measured in xylem sap of intact plants for each genotype under water deficit (Fig. 3) with response to ABA assayed on detached leaves (Fig. 5). Differences between well-watered and water deficit condition are expressed as a fraction of values observed under well-watered or no-ABA conditions. EPlant was measured on leaves of three intact plants under well-watered conditions and on leaves of three other intact plants under water deficit conditions. Pearson’s determination coefficients are indicated with their significance level: **P < 0.001, *P < 0.1. The dashed gray line corresponds to the 1:1 relationship. Figure 6. Open in new tabDownload slide Correlation between % reduction Kleaf and % reduction Eleaf (A)predicted from assays on detached leaves, or (B) measured directly on intact plants (% reduction Eplant) for the 10 offspring genotypes selected in the Syrah × Grenache population and the parents. Predicted changes in Kleaf and Eleaf between well-watered and water deficit conditions were calculated assuming maximum Kleaf in well-watered plants and combining ABA concentration measured in xylem sap of intact plants for each genotype under water deficit (Fig. 3) with response to ABA assayed on detached leaves (Fig. 5). Differences between well-watered and water deficit condition are expressed as a fraction of values observed under well-watered or no-ABA conditions. EPlant was measured on leaves of three intact plants under well-watered conditions and on leaves of three other intact plants under water deficit conditions. Pearson’s determination coefficients are indicated with their significance level: **P < 0.001, *P < 0.1. The dashed gray line corresponds to the 1:1 relationship. DISCUSSION This study demonstrates that ABA may down-regulate Kleaf in grapevine with a variable effect depending on the genotype. Previous works reported a role for ABA on plant hydraulics in Arabidopsis by means of mutants (Shatil-Cohen et al., 2011; Pantin et al., 2013). Here, we used two grapevine cultivars contrasting for their water use strategies in drought conditions (i.e. Syrah and Grenache), plus 10 offspring from a population obtained from their cross (Coupel-Ledru et al., 2014). Natural variations for Kleaf sensitivity to ABA could thus be detected within a species largely cultivated in drought-prone areas (Schultz, 2000; Jones et al., 2005). We observed that genetic variation in the sensitivity of Kleaf to ABA correlated with variation in (an)isohydric behavior in grapevine. We propose that the dual effect of ABA on stomata, via its direct biochemical effect on guard cells and the indirect consequence of Kleaf down-regulation on guard cell turgor, may underlie part of this genetic variation. Differential Kleaf Sensitivity to ABA: A Physiological Process Involved in the Variability of Plant Response to Drought In our study, genetic variation was found for ABA-induced responses to drought at two levels: (1) ABA accumulation in the xylem sap of intact plants exposed to drought, and (2) leaf sensitivity to the hormone by using detached leaves. On the one hand, ABA accumulation in the xylem sap of intact grapevine plants was highly dependent on the genotype, suggesting that ABA biosynthesis capacity or catabolism varied across genotypes. These variations may be due to differential expression of genes associated with ABA synthesis, such as NCED1 and NCED2, or genes coding for enzymes involved in ABA degradation to inactive compounds, including the ABA 8’-hydroxylases (Riahi et al., 2013; Speirs et al., 2013). On the other hand, exogenous ABA application to detached leaves using a high concentration (32 mmol m−3 (+)-ABA) reduced Kleaf by up to 50% for the most sensitive grapevine genotype (Fig. 4). This reduction in Kleaf is comparable to that observed for similarly high ABA concentrations on Arabidopsis leaves (Pantin et al., 2013). A plateau was observed at the highest concentrations, indicating that maximal effect of the hormone on Kleaf was reached at an ABA concentration far above the physiological range observed even under severe drought (Rogiers et al., 2012). Most importantly, a strong variability in Kleaf sensitivity to ABA was observed between genotypes. Combining these values of Kleaf sensitivity to ABA as observed on detached leaves with native ABA concentration made it possible to predict changes in Kleaf (% reduction Kleaf) induced for each genotype by the water deficit scenario. Overall, genetic differences in Kleaf response to drought (% reduction Kleaf) were much more influenced by differences in sensitivity of Kleaf to ABA than in ABA accumulation. A wide range of genetic variation was obtained for a drought-induced drop in Kleaf, going from no reduction in the less sensitive genotypes to a decline by up to 38% for the most responsive ones under the moderate water deficit (Fig. 6). The initial, endogenous ABA content (prior to exogenous ABA feeding) was lower in Syrah than in Grenache (0.5 ± 0.2 mmol m−3 and 1.1 ± 0.5 mmol m−3, respectively), while Syrah was less sensitive to addition of exogenous ABA than Grenache. This rules out a possible role of initial ABA concentration as responsible for a basal down-regulation of Kleaf in the less sensitive genotype Syrah. The range of Kleaf sensitivities observed within the panel of genotypes significantly correlated with their capacities to maintain leaf water potential in conditions of water deficit (Fig. 5D). The more sensitive Kleaf was to ABA, the better water potential was maintained in leaves under water deficit conditions, that is, the more isohydric was the genotype. By contrast, in more anisohydric genotypes Kleaf was hardly sensitive to ABA. Although variation from iso- to aniso-hydry has been shown to depend on environmental conditions, making their genetic origin debated (e.g. Franks et al., 2007), the genetic contrast between Syrah and Grenache was consistently reinforced across three independent experiments in our study (on whole potted plants in greenhouse, on leaves detached from plants cultivated in the vineyard, and leaves detached from potted plants cultivated outdoors). Assuming that the ABA-induced reduction in Kleaf was stronger in isohydric than anisohydric genotypes, it was possible to generate the different behaviors in silico by amending a simple model of leaf hydraulics with the observed effect of ABA on Kleaf (see Methods and Supplemental Methods S8–10). The model predicted that the water potential of guard cells decreased more rapidly when Kleaf was more responsive to ABA, leading to stomatal closure and maintenance of bulk leaf water potential. Figure 7 presents the results of two simulations during progressive soil drying, where hypothetical, isohydric, and anisohydric genotypes were built with all parameters maintained identical apart from Kleaf sensitivity to ABA, based on observed values within the whole set of 12 genotypes. The simulations were run with the most extreme values combining the highest sensitivity observed and the widest range of variation for Kleaf between min and max values. They confirm that differential down-regulation of Kleaf by ABA can account for part of the contrasted (an)isohydric behaviors, where genotypes with the most responsive Kleaf to ABA better control their transpiration rate, thus reducing the drop in leaf water potential under drought corresponding to an isohydric behavior. Figure 7. Open in new tabDownload slide Modeling confirms the influence of differential down-regulation of Kleaf by ABA on the overall (an)isohydric behavior of the plant. Two simulations were performed with all parameters maintained identical in both cases apart from Kleaf sensitivity to ABA and Kleaf maximum value without ABA (A and D). Assumptions made to simulate the effects of a progressive soil drying, characterized by the drop in the predawn leaf water potential (Ψpd in B and E), are described in “Materials and Methods.” Left part: high sensitivity of Kleaf to ABA (A) drives a drop in the guard cell leaf water potential Ψgc (B), resulting in a decrease in stomatal conductance (C) and transpiration rate, which, in turn, reduces the drop in the leaf water potential Ψleaf (B) and confers a more isohydric behavior. Right: lower sensitivity of Kleaf to ABA (D) leads to a higher stomatal conductance (F) as compared to (C), not preventing the drop in the overall leaf water potential (E), typical of a more anisohydric behavior. Figure 7. Open in new tabDownload slide Modeling confirms the influence of differential down-regulation of Kleaf by ABA on the overall (an)isohydric behavior of the plant. Two simulations were performed with all parameters maintained identical in both cases apart from Kleaf sensitivity to ABA and Kleaf maximum value without ABA (A and D). Assumptions made to simulate the effects of a progressive soil drying, characterized by the drop in the predawn leaf water potential (Ψpd in B and E), are described in “Materials and Methods.” Left part: high sensitivity of Kleaf to ABA (A) drives a drop in the guard cell leaf water potential Ψgc (B), resulting in a decrease in stomatal conductance (C) and transpiration rate, which, in turn, reduces the drop in the leaf water potential Ψleaf (B) and confers a more isohydric behavior. Right: lower sensitivity of Kleaf to ABA (D) leads to a higher stomatal conductance (F) as compared to (C), not preventing the drop in the overall leaf water potential (E), typical of a more anisohydric behavior. Stronger down-regulation of Kleaf by ABA occurred in those cultivars with higher Kleaf max (Figure 5C). Genetic variation in Kleaf max may arise from difference in intrinsic activity of aquaporins among genotypes or from variation in the vascular relative to the transmembrane, extra vascular water pathways. Multiple leaf and vein traits can influence Kleaf (Sack and Scoffoni, 2013). As an example, larger conduit lumens might provide greater xylem conductivity and Kleaf, while the whole vein diameters also promote differences in transport capacity when they contain greater sizes and numbers of xylem vessels (Russin and Evert, 1985; Coomes et al., 2008; Taneda and Terashima, 2012). Assuming that ABA downregulates Kleaf by lowering aquaporin activity, those genotypes with higher aquaporin activity or a greater proportion of extra-vascular pathways would consistently display higher Kleaf sensitivity to ABA. Other processes that occur during dehydration may modulate the responses of Kleaf to ABA and create genetic variability. Several factors result in a strong decline of Kleaf during drought, including cavitation, collapse of xylem conduits, and loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage (Trifilò et al., 2003; Cochard et al., 2004; Blackman et al., 2010). Xylem resistance to cavitation has been shown to vary in a series of conifer species and to correlate with prolonged stomatal opening after a period of 30 d without water (Brodribb et al., 2014). This suggests that variation between anisohydric and isohydric behaviors may also be ruled by differences in xylem resistance to cavitation. Cavitation unlikely occurred here on detached leaves that had their petiole immersed in water. Endogenous ABA that accumulates in leaves under water stress may have different effects from the one observed in our study with exogenous ABA fed to leaves of irrigated plants. Embolism repair, triggered by ABA, does not apply for water-fed leaves of irrigated plants but could occur in leaves of plants under water deficit (Kaldenhoff et al., 2008; Perrone et al., 2012). This possible action of ABA may introduce some discrepancies between what we observed in leaves detached from irrigated plants and what governs leaf hydraulics and indirectly influences stomatal response in plants under water deficit. Other discrepancies may originate in up-regulation of certain aquaporin gene expression when ABA accumulates in leaves under water deficit conditions (Kaldenhoff et al., 1996). Overall, ABA-triggered decrease in Kleaf, as described in our work for early stages of soil drying, may combine with further decrease in Kleaf due to cavitation under more severe drought, with possible overlapping of mechanisms. This may explain why the genetic variation in Kleaf sensitivity to ABA as determined on detached leaves did not strictly correlate with the genetic variation in (an)isohydry as determined on whole plants under water deficit. The Variable Sensitivity of Kleaf to ABA Was Confirmed by Two Independent Measurement Methods and Two Independent Experiments Measuring Kleaf with the EFM as the flow rate (i.e. transpiration rate Eleaf) divided by the leaf water potential (Sack et al., 2002) has long been a matter of debate when exploring the relationship between Eleaf and Kleaf (Flexas et al., 2013). By contrast with other methods that use pressurized water to force water flow through the leaf, the EFM respects the native paths of water flow together with the range of negative values for water potentials within the leaf (Sack and Scoffoni, 2012). However, calculation of Kleaf rests on Eleaf that causes some partial correlation between variables and hinders the analysis of their relationship. A second method was therefore used in an independent experiment to provide alternative evidence of ABA acting as a regulator of Kleaf. We reiterated our ABA-feeding experiment on detached leaves of the two parental cultivars, Syrah and Grenache, and measured Kleaf with the HPFM. This method forces water flow through the leaf by pushing water out of the lamina with a controlled pressure gradient across the leaf (Sack et al., 2002). Kleaf values that were obtained in control conditions with this second method were highly consistent with those previously reported by Pou et al. (2013) who operated similarly (Syrah displayed Kleaf of about 20 mmol m−2 s−1 MPa−1 in both studies). However, Kleaf measured with the HPFM method was about 3-fold higher than the values we obtained for the same cultivars with the EFM (Fig. 1). Kleaf might be overestimated by the HPFM because of the higher hydrostatic pressure imposed to water within the leaf while negative pressures develop in transpiring leaves. The leaf is thus flooded with a liquid solution and leaf airspaces might rapidly become infiltrated, implying novel pathway for water movement, hence yielding Kleaf values not reflecting in vivo context (Prado and Maurel, 2013). By contrast, the EFM was proposed to more closely follow the natural pathway of water in leaves through the transpiration flow (Sack and Scoffoni, 2012). Despite differences in Kleaf magnitude, both methods and both independent experiments consistently evidenced a marked difference in Kleaf measured in control conditions between cultivars. Importantly, the HPFM allowed uncoupling Kleaf response to ABA from Eleaf response: because the leaf is flooded in water during the experiment, transpiration rate is nonexistent; thus, the observed effect of ABA is exclusively a direct one on Kleaf. ABA feeding similarly impacted Kleaf whatever the method used. The high repeatability across methods and experiments observed for the variable response of Kleaf to ABA among cultivars thus strengthens the novel outputs of this work. The use of the HPFM method made it possible to dissociate the effects of ABA on both components of Kleaf: the lamina and the petiole. Differences in petiole hydraulic conductance between grapevine cultivars have been proposed as a cause of differences in (an)isohydric behaviors between cultivars, although variation with drought was not mentioned (Schultz, 2003). In our study, Kpetiole was slightly (although not significantly) reduced by the ABA treatment in Grenache, whereas it was not in Syrah. Although this result merits further verification, it is consistent with a possible role of the petiole in causing differences in hydraulic behaviors between Syrah and Grenache. Regulation of hydraulic conductance in petioles likely rests on a similar mechanism as in the lamina, since substantial expression of plasma membrane aquaporins in petioles have been reported (Baiges et al., 2001; Chen et al., 2008), where they may facilitate transcellular water transport. However, the major resistance to water resides in the lamina, so that the petiole may play only a marginal role. By contrast, Klamina offers multiple sites of regulation from the petiole-leaf junction to the sites of evaporation, through apoplasm and symplasm pathways. A significant reduction of Klamina by ABA feeding was observed for Grenache, but not in Syrah (Fig. 1). We are now seeking the mechanism underlying these contrasting behaviors, and aquaporins are good candidates. Toward the Understanding of ABA Action on Leaf Hydraulic Conductance By which mechanism could ABA differentially affect Kleaf between genotypes or species remains a key question. Based on the major role of aquaporins in the water permeability of the bundle sheath cells, Shatil-Cohen et al. (2011) suggested that the ABA-induced decrease in Kleaf may occur via the down-regulation of aquaporin activity therein. Further work showed that silencing a family of plasma membrane intrinsic proteins (PIPs) specifically in the bundle sheath decreases Kleaf by a factor of three (Sade et al., 2014). However, experimental evidence is still missing that synchronously links ABA to the macroscopic regulation of Kleaf and to the cellular-level regulation of aquaporins. Different studies tried to ascribe physiological responses to water deficit with expression profile of aquaporins, but contrasting results have been obtained depending on both intensity and dynamics of water deficit (Tyerman et al., 2002; Galmés et al., 2007; Prado and Maurel, 2013). Pou et al. (2013) reported lower expression of aquaporin genes VvTIP2;1 and VvPIP2;1 and reduced activity of aquaporins in leaves under water deficit coinciding with a decrease of Kleaf. Decline of Kleaf in dehydrating leaves could also be correlated with low aquaporin activity in the mesophyll (Kim and Steudle, 2007). Importantly, ABA treatment could decrease C-terminal phosphorylation and thus activity of aquaporin AtPIP2;1 within 30 min of application in Arabidopsis seedlings (Kline et al., 2010). This timescale is compatible with the ABA-induced decrease in Kleaf described in our study like in previous works (Shatil-Cohen et al., 2011; Pantin et al., 2013). Altered phosphorylation of aquaporins may act on their trafficking and gating (Törnroth-Horsefield et al., 2006; Prak et al., 2008; Eto et al., 2010) to adjust leaf hydraulics in response to drought, as was described under changing light (Prado et al., 2013) and upon exposure to ABA in guard cells (Grondin et al., 2015). Other proposals have arisen that may explain the contrasts between iso- and anisohydry. Vandeleur et al. (2009) showed that Grenache strongly reduced root hydraulic conductivity under drought, contrasting with the more anisohydric Chardonnay, and that this difference was reflected in different responses to drought in transcript abundance of PIP1;1 aquaporin. Whether this relationship was due to changes in water transport and aquaporin expression in roots only or to concomitant changes in leaves as evidenced in our study remains to be elucidated. Such an action of ABA on root hydraulic conductance could have been implemented in our model instead of the direct effect of ABA on Kleaf to yield similar results. Thus, these different mechanisms still have to be deciphered together with their respective importance in the determinism of (an)isohydry. Contrary to most observations in leaves, ABA tends to increase hydraulic conductivity in roots (Ludewig et al., 1988; Zhang et al., 1995; Hose et al., 2000; Thompson et al., 2007). In spite of the opposite response to ABA, change in root hydraulic conductivity remains consistent with a role of aquaporins that are down-regulated at transcriptional and posttranscriptional levels when ABA concentration increases (Wan et al., 2004; Zhu et al., 2005; Parent et al., 2009). Opposite reactions in roots and leaves could be associated with selective action of ABA on specific members of the aquaporin family to alleviate the effects of water stress. CONCLUSION This study reveals a substantial genetic variation in the responsiveness of Kleaf to ABA and supports that it may impact isohydry in grapevine. Further studies will inform us whether this relationship is conserved in other species, and whether genetic variations in aquaporin regulation are responsible for the existing variability in isohydry. MATERIALS AND METHODS Assessing the Response to Exogenous ABA Feeding of the Cultivars Grenache and Syrah Cultivated in the Field Using Two Independent Methods Plant material and preparation This experiment was performed on Syrah and Grenache plants from the Coombe vineyard (Waite Campus, Adelaide, South Australia). Leaves were prepared as previously described in Pou et al. (2013). Briefly, shoots with mature leaves from the most exposed branches were collected on 5 to 10 plants per cultivar at the beginning of the night preceding measurements. Immediately after cutting, shoots were placed into a bucket with their cut ends immersed in distilled water, covered with black plastic bags, and taken to the laboratory. The shoots were then recut under degassed water and rehydrated overnight in full darkness until leaves were assigned to the perfusion solutions. Response of transpiration rate on detached leaves of Grenache and Syrah cultivars cultivated in the field and determination of Kleaf using the Evaporative Flux Method Mature leaves were chosen on similar position from the apex (8th–12th phytomers). On the morning preceding measurements, leaves were excised from shoots, and their petioles were immediately immersed and recut in individual 5-mL containers filled with a filtered (0.2 μm), degassed control solution [2 mol m−3 KH2PO4, 1 mol m−3 MES, 0.4 mol m−3 Ca(NO3)2] adjusted to pH 6.5. A light source was suspended above the leaves providing approximately 400 μmol m–2 s–1 photosynthetically active radiation at leaf level. Petioles were tightly sealed to the containers caps. As a precaution, initial transpiration rate was determined on each leaf by weighing leaves in their container every 20 min over 1 h 30 min. Measurements were stopped and discarded if the flow suddenly began to decline, likely due to blockage of water flow in the petiole by residual air bubbles. Synthetic (±)-ABA, solubilized with a negligible volume of ethanol, was then added to the solution to reach varying concentrations of (+)-ABA (2, 4, 8, 16, or 32 mmol m−3). Final transpiration in ABA was determined once the weight declined at a stabilized rate (which occurred about 1 hour after adding ABA). At the end of the experiment, each leaf was taken off its container and immediately set into a Schölander pressure chamber (Soil Moisture Equipment Corp) to measure its water potential (Ψleaf). Measurement of Ψleaf with the pressure chamber was assumed to be close to the value for the transpiring leaf just before it is enclosed in the chamber, according to the principle basis of the Schölander chamber. Leaf hydraulic conductance (Kleaf) was then calculated following the evaporative flux method (EFM) as the flow rate divided by the leaf water potential taken as the driving force for water flow from the solution (Sack et al., 2002). The transpiration rate for this calculation was determined as the stable rate of weight loss measured at the very end of the experiment, just before measuring Ψleaf. Kleaf was estimated on a leaf area basis as: where LA is the individual leaf area (m2). At the end of measurement, the leaf was scanned and leaf area was determined on photographs using ImageJ (Rasband WS, 2009). Response of stomatal conductance on detached leaves of Grenache and Syrah cultivars cultivated in the field and determination of Kleaf using High Pressure Flowmeter Another set of leaves were chosen and excised following the same protocol as described above. Each leaf was directly assigned to one of the ABA or control solutions (concentration of (+)-ABA 0, 2, 4, 8, 16, or 32 mmol m−3) prepared as above. After 1 h in perfusion, stomatal conductance (gs) was measured using a porometer (Delta T AP4; Delta-T Devices Ltd) on each leaf fed with a different solution. Leaf hydraulic conductance was then measured with a HPFM apparatus (HPFM-Gen3, Dynamax). This method first developed by Tyree et al. (2005) relies on pushing a solution into a plant part (here, a leaf) at a known delivery pressure. Measurements were performed using the transient method consisting in applying different pressures and recording flow rates to calculate the conductance as the slope of the regression line between flow rate and pressure (Sack et al., 2002). Leaves were attached to the flowmeter through the petiole using compression fittings. Filtered watered was forced into the leaves at increasing pressure (P) up to 0.4 MPa, while measuring the instantaneous flow rate (F) every 2 s (Supplemental Fig. S2). Corresponding hydraulic conductances (Kleaf) were computed from the slope of the plot water flux versus pressure as: where LA is the leaf area (m2). The lamina was then removed by excising the leaf at the junction with the petiole, and the leaf-specific petiole conductivity (Kpetiole) was measured and computed according to (Sack et al., 2002) as: Klamina was then calculated from Kleaf and Kpetiole according to the Ohm’s law analogy considering petiole’s and lamina’s pathways in series: 1/Kleaf = 1/(Kpetiole × petiole length) + 1/Klamina. During HPFM measurements, the leaves were submerged in a container filled with water to maintain constant leaf temperature and prevent transpiration. The temperature in the compartment was adjusted to 23°C with a regulated bath (Ministat, Peter Huber Kälte maschinenbau GmbH) which was continuously aerated. The HPFM apparatus corrects the hydraulic conductance for possible changes in temperature to account for corresponding changes in water viscosity. The leaves were exposed to the same light as the one used during perfusion. At the end of measurement, the leaf was scanned and leaf area was determined on photographs using ImageJ (Rasband WS, 2009). Experiments on 10 Grapevine Offspring, and the Parents, from a Syrah × Grenache Mapping Population with Contrasting (An)isohydric Behaviors Plant material and preparation A panel of 10 offspring was selected within the pseudo-F1 population of 186 two-year-old genotypes obtained as the first generation from a reciprocal cross between the grapevine cultivars Syrah and Grenache (Adam-Blondon et al., 2004; Coupel-Ledru et al., 2014; Coupel-Ledru et al., 2016). Offspring plus the two parental genotypes were grafted on 110 Richter rootstocks in 2010 and grown outside in 3-L pots for 2 years in Montpellier (France). In winter 2012 (January), plants were transferred to a greenhouse in 9-L pots filled with a substrate calibrated for soil water management (Coupel-Ledru et al., 2014). In early spring 2012, the whole population was installed into a high throughput phenotyping platform (PHENOARCH platform hosted at the M3P, Montpellier Plant Phenotyping Platforms, http://bioweb.supagro.inra.fr/phenoarch) in another greenhouse where two soil water conditions were applied using automated weighing stations and daily watering. Well-watered conditions, corresponding to a soil water content of 1.5 g water g−1 dry soil, were imposed on one-half of the plants, while the other one-half was submitted to a moderate soil water deficit (1.05 g water g−1 dry soil). Detailed information of PHENOARCH platform and measurement of environmental conditions is described in (Cabrera‐Bosquet et al., 2016). At the end of the high-throughput experiment in the greenhouse, the plants were transferred outside, where they were grown for one additional year and pruned to produce one, unbranched leafy axis with their inflorescences removed. Automated ferti-irrigation completed by occasional, individual weighing and watering ensured that all the plants were maintained wellwatered (Coupel-Ledru et al., 2014). These plants were used in 2013 for experiments on detached leaves. Leaf water potential and transpiration rate of whole plants under controlled conditions in the phenotyping platform Potted plants were characterized for water relations under transpiring conditions during a 24-h cycle using a controlled environment chamber close to the phenotyping platform. For each genotype, leaf water potential was measured in the daytime (ΨM) on three plants per watering regime, using a pressure chamber (Soil Moisture Equipment Corp.) (Coupel-Ledru et al., 2014). Isohydric (respectively anisohydric) behavior was defined as the capacity (respectively inability) of a genotype to maintain leaf water potential in the daytime under water stress. The panel of 12 genotypes (10 offspring plus the 2 parents, Syrah and Grenache) was selected based on their contrasted behaviors for (an)isohydry. Transpiration rate was determined in parallel in the same transpiring conditions and as was previously described (Coupel-Ledru et al., 2014, 2016). Each pot was weighed with 0.1 g accuracy (Sartorius balance, IB 34 EDEP) at the beginning and end of the light period in the controlled-environment chamber. Weight losses were used together with the estimated whole plant leaf area to calculate average transpiration rate on a leaf area basis (EPlant). Leaf area Individual whole plant leaf area was estimated through processed images taken every 2 d in the platform imaging cabin as previously described (Coupel-Ledru et al., 2014). ABA sampling in the phenotyping platform and quantification For each of the 12 genotypes (10 offspring plus 2 parental), xylem sap was extracted from two leaves on two water deficit plants following measurement of ΨM with the pressure chamber. The same pressure chamber was used to pressurize the leaf at about 0.3 MPa above the balancing pressure that was imposed for ΨM measurement, and about 30 µL of sap was expressed from the cut petiole exposed outside the pressure chamber. Expressed sap was collected in 0.5-mL microtubes and conserved at −80°C in a dedicated deep freezer (Herafreeze, HFU T series, Thermo Fisher Scientific) pending freeze-drying. Microtubes containing frozen sap samples were centrifuged in a centrifuge (Speed Vac Plus SC110A, Fisher Scientific) connected to a benchtop freeze-drier (Christ Alpha2-4, Fisher Scientific) that imposed a partial vacuum of 0.020 mbar. The water contained in the sap samples was sublimated and trapped on the cold condenser of the freeze-drier maintained at a temperature of −80°C. Analysis of ABA abundance in xylem sap was undertaken by liquid chromatography/mass spectrometry (LC MS/MS, Agilent 6410). Dried xylem sap samples were dissolved in 30 µL 10% acetonitrile containing 0.05% acetic acid plus a deuterated internal standard mix (containing D6–3′,5′,5′,7’,7’,7’-ABA at a concentration of 10 ng mL−1) before introduction into LC MS/MS. Separation was carried out on a C18 column (Phenomenex C18(2) 75mm × 4.5mm × 5 µm) at 40°C. Solvents were nanopure water and acetonitrile, both with 0.05% acetic acid. Samples were eluted with a linear 15-min gradient starting at 10% acetonitrile and ending at 90% acetonitrile. Compounds were identified by retention times and multiple reaction monitoring. Parent and product ions were the same as previously described (Speirs et al., 2013). Response of transpiration rate on detached leaves of the 10 offspring plus 2 parental genotypes under controlled conditions and determination of Kleaf using EFM The potted plants used in the whole-plant experiment described above were further cultivated outdoors. In July 2013, one night prior to experiments, plants were transferred from outside to a controlled environment chamber to ensure initial, low-transpiring conditions. On the morning preceding measurements, leaves were excised from plants and their petioles were immediately immersed and recut in individual 5-mL containers filled with a filtered (0.2 μm), degassed control solution [2 mol m−3 KH2PO4, 1 mol m−3 MES, 0.4 mol m−3 Ca(NO3)2] adjusted to pH 6.5. Petioles were tightly sealed to the containers’ caps. Each leaf in its container was then placed in the chamber with VPD maintained at 2 ± 0.2 kPa and temperature at 27°C. Light was provided in the chamber by a bank of sodium lamps that maintained the PPFD at approximately 480 μmol m–2 s–1 at the leaf level. All leaves were chosen as well-exposed, fully expanded, generally on the eighth phytomer from the apex. The protocol then followed for ABA perfusion (determination of transpiration rate, of Kleaf with the EFM method, and of their response to ABA) was the same as described in the section “Response of stomatal conductance on detached leaves of Grenache and Syrah cultivars cultivated in the field and determination of Kleaf using High Pressure Flow Meter.” Statistical Analyses Statistical analyses were performed with R (R Development Core Team, 2012). The Kruskal-Wallis test at the 5% alpha level was used for comparison of means. Semi-ln transformations were used to fit linear models to Kleaf response to ABA and extract the slopes. Regression slopes were compared by analysis of covariance. Tests for significant differences among all pairs of slopes were further achieved using R compSlopes package with the FDR correction method to adjust P values and protect against false-positive results. Modeling the Possible Influence of Down-Regulation of Kleaf by ABA on (An)isohydric Behavior Two simulations were performed with all parameters maintained identical in both cases apart from Kleaf sensitivity to ABA and Kleaf max, values of which were chosen based on results obtained on detached leaves of representative isohydric and anisohydric genotypes. We used the most extreme values combining the highest sensitivity observed for genotype 8S034 in Figure 4 and the widest range of variation for Kleaf between min (observed for 7G082 in Fig. 4) and max values (observed for 8S034 in Fig. 4). The consequences of a progressive soil drying, characterized by a drop of predawn leaf water potential (Ψpd, a proxy for Ψsoil), were modeled using Ohm’s law analogy for water transfer from soil to leaves with constant Kplant and from leaf xylem to guard cells with Kleaf sensitivity to ABA differing between the two simulations. Stomatal response to ABA and guard cell water potential (Ψguard cell) following equations derived from the Tardieu-Davies model (Tardieu et al., 2015). Full description of the model, equations, and parameters initialization is provided in Supplemental Methods S8 to S10. The model was implemented in R programming language. Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Response of gs to ABA for the parental cultivars Syrah and Grenache. Supplemental Figure S2. Example of raw plots from the HPFM experiments. Supplemental Figure S3. Data showing effect of exogenous ABA on hydraulic conductance of detached leaves (Kleaf). Supplemental Figure S4. Response of Eleaf to exogenous ABA measured on detached leaves for the 10 grapevine offspring genotypes and the parents. Supplemental Figure S5. Eleaf of detached leaves as a function of ln([ABA]) in the feeding sap. Supplemental Figure S6. Barplots showing the genotypic variability in sensitivity of Eleaf to ABA. Supplemental Figure S7. Sensitivity of Ψleaf to ABA fed to detached leaves for 10 offspring genotypes of the Syrah × Grenache population and the parents. Supplemental Figure S8. Details of the equation and parameter values used in the modeling of ΨM response to soil drying in iso- and aniso-hydric genotypes as a function of Kleaf sensitivity to ABA. Supplemental Figure S9. Common relationship for isohydric and anisohydric genotypes used for modeling the concentration of ABA in the xylem sap at predawn. Supplemental Figure S10. Relationship used for modeling stomatal conductance as a function of concentration of ABA in guard cell apoplasts and water potential of guard cells. ACKNOWLEDGMENTS We acknowledge Philippe Hamard, Philippe Péchier, and Victorien Taudou for their technical support in plant preparation, high-throughput experiments, and measurements on detached leaves. We are also grateful to Wendy Sullivan for helping with the HPFM experiments and to Annette Boettcher for running ABA analysis with the LCMS. In memory of our dear colleague and friend Eric Lebon. LITERATURE CITED Adam-Blondon A-F , Roux C, Claux D, Butterlin G, Merdinoglu D, This P ( 2004 ) Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics . Theor Appl Genet 109 : 1017 – 1027 Google Scholar Crossref Search ADS PubMed WorldCat Baiges I , Schäffner AR, Mas A ( 2001 ) Eight cDNA encoding putative aquaporins in Vitis hybrid Richter-110 and their differential expression . J Exp Bot 52 : 1949 – 1951 Google Scholar Crossref Search ADS PubMed WorldCat Blackman CJ , Brodribb TJ, Jordan GJ ( 2010 ) Leaf hydraulic vulnerability is related to conduit dimensions and drought resistance across a diverse range of woody angiosperms . New Phytol 188 : 1113 – 1123 Google Scholar Crossref Search ADS PubMed WorldCat Borel C , Frey A, Marion-Poll A, Tardieu F, Simonneau T ( 2001 ) Does engineering abscisic acid biosynthesis in Nicotiana plumbaginifolia modify stomatal response to drought? Plant Cell Environ 24 : 477 – 489 Google Scholar Crossref Search ADS WorldCat Brodribb TJ , Jordan GJ ( 2011 ) Water supply and demand remain balanced during leaf acclimation of Nothofagus cunninghamii trees . New Phytol 192 : 437 – 448 Google Scholar Crossref Search ADS PubMed WorldCat Brodribb TJ , McAdam SAM ( 2013 ) Abscisic acid mediates a divergence in the drought response of two conifers . Plant Physiol 162 : 1370 – 1377 Google Scholar Crossref Search ADS PubMed WorldCat Brodribb TJ , McAdam SA, Jordan GJ, Martins SC ( 2014 ) Conifer species adapt to low-rainfall climates by following one of two divergent pathways . Proc Natl Acad Sci USA 111 : 14489 – 14493 Google Scholar Crossref Search ADS PubMed WorldCat Buckley TN ( 2005 ) The control of stomata by water balance . New Phytol 168 : 275 – 292 Google Scholar Crossref Search ADS PubMed WorldCat Cabrera-Bosquet L , Fournier C, Brichet N, Welcker C, Suard B, Tardieu F ( 2016 ) High-throughput estimation of incident light, light interception and radiation-use efficiency of thousands of plants in a phenotyping platform . New Phytol 212 : 269 – 281 Google Scholar Crossref Search ADS PubMed WorldCat Chaumont F , Tyerman SD ( 2014 ) Aquaporins: highly regulated channels controlling plant water relations . Plant Physiol 164 : 1600 – 1618 Google Scholar Crossref Search ADS PubMed WorldCat Chen C-X , Cai S-B, Bai G-H ( 2008 ) A major QTL controlling seed dormancy and pre-harvest sprouting resistance on chromosome 4A in a Chinese wheat landrace . Mol Breed 21 : 351 – 358 Google Scholar Crossref Search ADS WorldCat Christmann A , Hoffmann T, Teplova I, Grill E, Müller A ( 2005 ) Generation of active pools of abscisic acid revealed by in vivo imaging of water-stressed Arabidopsis . Plant Physiol 137 : 209 – 219 Google Scholar Crossref Search ADS PubMed WorldCat Christmann A , Weiler EW, Steudle E, Grill E ( 2007 ) A hydraulic signal in root-to-shoot signalling of water shortage . Plant J 52 : 167 – 174 Google Scholar Crossref Search ADS PubMed WorldCat Cochard H , Bréda N, Granier A ( 1996 ) Whole tree hydraulic conductance and water loss regulation in Quercus during drought: evidence for stomatal control of embolism? Ann Sci For 53 : 197 – 206 Google Scholar Crossref Search ADS WorldCat Cochard H , Coll L, Le Roux X, Améglio T ( 2002 ) Unraveling the effects of plant hydraulics on stomatal closure during water stress in walnut . Plant Physiol 128 : 282 – 290 Google Scholar Crossref Search ADS PubMed WorldCat Cochard H , Froux F, Mayr S, Coutand C ( 2004 ) Xylem wall collapse in water-stressed pine needles . Plant Physiol 134 : 401 – 408 Google Scholar Crossref Search ADS PubMed WorldCat Coomes DA , Heathcote S, Godfrey ER, Shepherd JJ, Sack L ( 2008 ) Scaling of xylem vessels and veins within the leaves of oak species . Biol Lett 4 : 302 – 306 Google Scholar Crossref Search ADS PubMed WorldCat Coupel-Ledru A , Lebon É, Christophe A, Doligez A, Cabrera-Bosquet L, Péchier P, Hamard P, This P, Simonneau T ( 2014 ) Genetic variation in a grapevine progeny (Vitis vinifera L. cvs Grenache×Syrah) reveals inconsistencies between maintenance of daytime leaf water potential and response of transpiration rate under drought . J Exp Bot 65 : 6205 – 6218 Google Scholar Crossref Search ADS PubMed WorldCat Coupel-Ledru A , Lebon E, Christophe A, Gallo A, Gago P, Pantin F, Doligez A, Simonneau T ( 2016 ) Reduced nighttime transpiration is a relevant breeding target for high water-use efficiency in grapevine . Proc Natl Acad Sci USA 113 : 8963 – 8968 Google Scholar Crossref Search ADS PubMed WorldCat Eto K , Noda Y, Horikawa S, Uchida S, Sasaki S ( 2010 ) Phosphorylation of aquaporin-2 regulates its water permeability . J Biol Chem 285 : 40777 – 40784 Google Scholar Crossref Search ADS PubMed WorldCat Flexas J , Scoffoni C, Gago J, Sack L ( 2013 ) Leaf mesophyll conductance and leaf hydraulic conductance: an introduction to their measurement and coordination . J Exp Bot 64 : 3965 – 3981 Google Scholar Crossref Search ADS PubMed WorldCat Franks PJ , Drake PL, Froend RH ( 2007 ) Anisohydric but isohydrodynamic: seasonally constant plant water potential gradient explained by a stomatal control mechanism incorporating variable plant hydraulic conductance . Plant Cell Environ 30 : 19 – 30 Google Scholar Crossref Search ADS PubMed WorldCat Galmés J , Pou A, Alsina MM, Tomàs M, Medrano H, Flexas J ( 2007 ) Aquaporin expression in response to different water stress intensities and recovery in Richter-110 (Vitis sp.): relationship with ecophysiological status . Planta 226 : 671 – 681 Google Scholar Crossref Search ADS PubMed WorldCat Grondin A , Rodrigues O, Verdoucq L, Merlot S, Leonhardt N, Maurel C ( 2015 ) Aquaporins contribute to ABA-triggered stomatal closure through OST1-mediated phosphorylation . Plant Cell 27 : 1945 – 1954 Google Scholar Crossref Search ADS PubMed WorldCat Hose E , Steudle E, Hartung W ( 2000 ) Abscisic acid and hydraulic conductivity of maize roots: a study using cell- and root-pressure probes . Planta 211 : 874 – 882 Google Scholar Crossref Search ADS PubMed WorldCat Ikegami K , Okamoto M, Seo M, Koshiba T ( 2009 ) Activation of abscisic acid biosynthesis in the leaves of Arabidopsis thaliana in response to water deficit . J Plant Res 122 : 235 – 243 Google Scholar Crossref Search ADS PubMed WorldCat Jones GV , White MA, Cooper OR, Storchmann K ( 2005 ) Climate change and global wine quality . Clim Change 73 : 319 – 343 Google Scholar Crossref Search ADS WorldCat Joshi-Saha A , Valon C, Leung J ( 2011 ) Abscisic acid signal off the STARting block . Mol Plant 4 : 562 – 580 Google Scholar Crossref Search ADS PubMed WorldCat Kaldenhoff R , Kölling A, Richter G ( 1996 ) Regulation of the Arabidopsis thaliana aquaporin gene AthH2 (PIP1b) . J Photochem Photobiol B 36 : 351 – 354 Google Scholar Crossref Search ADS PubMed WorldCat Kaldenhoff R , Ribas-Carbo M, Sans JF, Lovisolo C, Heckwolf M, Uehlein N ( 2008 ) Aquaporins and plant water balance . Plant Cell Environ 31 : 658 – 666 Google Scholar Crossref Search ADS PubMed WorldCat Kim T-H , Böhmer M, Hu H, Nishimura N, Schroeder JI ( 2010 ) Guard cell signal transduction network: advances in understanding abscisic acid, CO2, and Ca2+ signaling . Annu Rev Plant Biol 61 : 561 – 591 Google Scholar Crossref Search ADS PubMed WorldCat Kim YX , Steudle E ( 2007 ) Light and turgor affect the water permeability (aquaporins) of parenchyma cells in the midrib of leaves of Zea mays. J Exp Bot 58 : 4119 – 4129 Google Scholar Crossref Search ADS PubMed WorldCat Kline KG , Barrett-Wilt GA, Sussman MR ( 2010 ) In planta changes in protein phosphorylation induced by the plant hormone abscisic acid . Proc Natl Acad Sci USA 107 : 15986 – 15991 Google Scholar Crossref Search ADS PubMed WorldCat Ludewig M , Dörffling K, Seifert H ( 1988 ) Abscisic acid and water transport in sunflowers . Planta 175 : 325 – 333 Google Scholar Crossref Search ADS PubMed WorldCat Massonnet C , Costes E, Rambal S, Dreyer E, Regnard JL ( 2007 ) Stomatal regulation of photosynthesis in apple leaves: evidence for different water-use strategies between two cultivars . Ann Bot (Lond) 100 : 1347 – 1356 Google Scholar Crossref Search ADS WorldCat McAdam SAM , Brodribb TJ ( 2013 ) Ancestral stomatal control results in a canalization of fern and lycophyte adaptation to drought . New Phytol 198 : 429 – 441 Google Scholar Crossref Search ADS PubMed WorldCat McAdam SAM , Brodribb TJ ( 2014 ) Separating active and passive influences on stomatal control of transpiration . Plant Physiol 164 : 1578 – 1586 Google Scholar Crossref Search ADS PubMed WorldCat McAdam SAM , Brodribb TJ, Ross JJ ( 2016 ) Shoot-derived abscisic acid promotes root growth . Plant Cell Environ 39 : 652 – 659 Google Scholar Crossref Search ADS PubMed WorldCat Meinzer FC ( 2002 ) Co-ordination of vapour and liquid phase water transport properties in plants . Plant Cell Environ 25 : 265 – 274 Google Scholar Crossref Search ADS PubMed WorldCat Pantin F , Monnet F, Jannaud D, Costa JM, Renaud J, Muller B, Simonneau T, Genty B ( 2013 ) The dual effect of abscisic acid on stomata . New Phytol 197 : 65 – 72 Google Scholar Crossref Search ADS PubMed WorldCat Parent B , Hachez C, Redondo E, Simonneau T, Chaumont F, Tardieu F ( 2009 ) Drought and abscisic acid effects on aquaporin content translate into changes in hydraulic conductivity and leaf growth rate: a trans-scale approach . Plant Physiol 149 : 2000 – 2012 Google Scholar Crossref Search ADS PubMed WorldCat Peak D , Mott KA ( 2011 ) A new, vapour-phase mechanism for stomatal responses to humidity and temperature . Plant Cell Environ 34 : 162 – 178 Google Scholar Crossref Search ADS PubMed WorldCat Perrone I , Gambino G, Chitarra W, Vitali M, Pagliarani C, Riccomagno N, Balestrini R, Kaldenhoff R, Uehlein N, Gribaudo I, et al. ( 2012 ) The grapevine root-specific aquaporin VvPIP2;4N controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress . Plant Physiol 160 : 965 – 977 Google Scholar Crossref Search ADS PubMed WorldCat Pou A , Medrano H, Flexas J, Tyerman SD ( 2013 ) A putative role for TIP and PIP aquaporins in dynamics of leaf hydraulic and stomatal conductances in grapevine under water stress and re-watering . Plant Cell Environ 36 : 828 – 843 Google Scholar Crossref Search ADS PubMed WorldCat Prado K , Boursiac Y, Tournaire-Roux C, Monneuse J-M, Postaire O, Da Ines O, Schäffner AR, Hem S, Santoni V, Maurel C ( 2013 ) Regulation of Arabidopsis leaf hydraulics involves light-dependent phosphorylation of aquaporins in veins . Plant Cell 25 : 1029 – 1039 Google Scholar Crossref Search ADS PubMed WorldCat Prado K , Maurel C ( 2013 ) Regulation of leaf hydraulics: from molecular to whole plant levels . Front Plant Sci 4 : 255 10.3389/fpls.2013.00255 Google Scholar Crossref Search ADS PubMed WorldCat Prak S , Hem S, Boudet J, Viennois G, Sommerer N, Rossignol M, Maurel C, Santoni V ( 2008 ) Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP2;1 in response to salt stress . Mol Cell Proteomics 7 : 1019 – 1030 Google Scholar Crossref Search ADS PubMed WorldCat Prieto JA , Lebon E, Ojeda H ( 2010 ) Stomatal behavior of different grapevine cultivars in response to soil water status and air water vapor pressure deficit . J Int Sci Vigne Vin 44 : 9 – 20 Google Scholar OpenURL Placeholder Text WorldCat R Development Core Team ( 2012 ) R: a language and environment for statistical computing. R Foundation for Statistical Computing , Vienna, Austria Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Rasband WS ( 2009 ) ImageJ. National Institutes of Health , Bethesda, MD, USA Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Riahi L , Zoghlami N, Dereeper A, Laucou V, Mliki A, This P ( 2013 ) Single nucleotide polymorphism and haplotype diversity of the gene NAC4 in grapevine . Ind Crops Prod 43 : 718 – 724 Google Scholar Crossref Search ADS WorldCat Rogiers SY , Greer DH, Hatfield JM, Hutton RJ, Clarke SJ, Hutchinson PA, Somers A ( 2012 ) Stomatal response of an anisohydric grapevine cultivar to evaporative demand, available soil moisture and abscisic acid . Tree Physiol 32 : 249 – 261 Google Scholar Crossref Search ADS PubMed WorldCat Russin WA , Evert RF ( 1985 ) Studies on the leaf of Populus deltoides (Salicaceae): Quantitative aspects, and solute concentrations of the sieve-tube members . Am J Bot 72 : 487 – 500 Google Scholar Crossref Search ADS WorldCat Sack L , Holbrook NM ( 2006 ) Leaf hydraulics . Annu Rev Plant Biol 57 : 361 – 381 Google Scholar Crossref Search ADS PubMed WorldCat Sack L , Melcher PJ, Zwieniecki MA, Holbrook NM ( 2002 ) The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods . J Exp Bot 53 : 2177 – 2184 Google Scholar Crossref Search ADS PubMed WorldCat Sack L , Scoffoni C ( 2012 ) Measurement of leaf hydraulic conductance and stomatal conductance and their responses to irradiance and dehydration using the Evaporative Flux Method (EFM) . J Vis Exp 70 : 4179 Google Scholar OpenURL Placeholder Text WorldCat Sack L , Scoffoni C ( 2013 ) Leaf venation: structure, function, development, evolution, ecology and applications in the past, present and future . New Phytol 198 : 983 – 1000 Google Scholar Crossref Search ADS PubMed WorldCat Sade N , Shatil-Cohen A, Attia Z, Maurel C, Boursiac Y, Kelly G, Granot D, Yaaran A, Lerner S, Moshelion M ( 2014 ) The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics . Plant Physiol 166 : 1609 – 1620 Google Scholar Crossref Search ADS PubMed WorldCat Schultz HR ( 2000 ) Climate change and viticulture: a European perspective on climatology, carbon dioxide and UV-B effects . Aust J Grape Wine Res 6 : 2 – 12 Google Scholar Crossref Search ADS WorldCat Schultz HR ( 2003 ) Differences in hydraulic architecture account for near-isohydric and anisohydric behaviour of two field-grown Vitis vinifera L. cultivars during drought . Plant Cell Environ 26 : 1393 – 1405 Google Scholar Crossref Search ADS WorldCat Shatil-Cohen A , Attia Z, Moshelion M ( 2011 ) Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA? Plant J 67 : 72 – 80 Google Scholar Crossref Search ADS PubMed WorldCat Simonin KA , Burns E, Choat B, Barbour MM, Dawson TE, Franks PJ ( 2015 ) Increasing leaf hydraulic conductance with transpiration rate minimizes the water potential drawdown from stem to leaf . J Exp Bot 66 : 1303 – 1315 Google Scholar Crossref Search ADS PubMed WorldCat Simonneau T , Barrieu P, Tardieu F ( 1998 ) Accumulation rate of ABA in detached maize roots correlates with root water potential regardless of age and branching order . Plant Cell Environ 21 : 1113 – 1122 Google Scholar Crossref Search ADS WorldCat Soar CJ , Speirs J, Maffei SM, Penrose AB, McCarthy MG, Loveys BR ( 2006 ) Grape vine varieties Shiraz and Grenache differ in their stomatal response to VPD: apparent links with ABA physiology and gene expression in leaf tissue . Aust J Grape Wine Res 12 : 2 – 12 Google Scholar Crossref Search ADS WorldCat Speirs J , Binney A, Collins M, Edwards E, Loveys B ( 2013 ) Expression of ABA synthesis and metabolism genes under different irrigation strategies and atmospheric VPDs is associated with stomatal conductance in grapevine (Vitis vinifera L. cv Cabernet Sauvignon) . J Exp Bot 64 : 1907 – 1916 Google Scholar Crossref Search ADS PubMed WorldCat Taneda H , Terashima I ( 2012 ) Co-ordinated development of the leaf midrib xylem with the lamina in Nicotiana tabacum. Ann Bot (Lond) 110 : 35 – 45 Google Scholar Crossref Search ADS WorldCat Tardieu F , Davies WJ ( 1992 ) Stomatal response to abscisic acid is a function of current plant water status . Plant Physiol 98 : 540 – 545 Google Scholar Crossref Search ADS PubMed WorldCat Tardieu F , Lafarge T, Simonneau T ( 1996 ) Stomatal control by fed or endogenous xylem ABA in sunflower: interpretation of correlations between leaf water potential and stomatal conductance in anisohydric species . Plant Cell Environ 19 : 75 – 84 Google Scholar Crossref Search ADS WorldCat Tardieu F , Simonneau T ( 1998 ) Variability among species of stomatal control under fluctuating soil water status and evaporative demand: modelling isohydric and anisohydric behaviours . J Exp Bot 49 : 419 – 432 Google Scholar Crossref Search ADS WorldCat Tardieu F , Simonneau T, Parent B ( 2015 ) Modelling the coordination of the controls of stomatal aperture, transpiration, leaf growth, and abscisic acid: update and extension of the Tardieu-Davies model . J Exp Bot 66 : 2227 – 2237 Google Scholar Crossref Search ADS PubMed WorldCat Thompson AJ , Andrews J, Mulholland BJ, McKee JMT, Hilton HW, Horridge JS, Farquhar GD, Smeeton RC, Smillie IRA, Black CR, et al. ( 2007 ) Overproduction of abscisic acid in tomato increases transpiration efficiency and root hydraulic conductivity and influences leaf expansion . Plant Physiol 143 : 1905 – 1917 Google Scholar Crossref Search ADS PubMed WorldCat Törnroth-Horsefield S , Wang Y, Hedfalk K, Johanson U, Karlsson M, Tajkhorshid E, Neutze R, Kjellbom P ( 2006 ) Structural mechanism of plant aquaporin gating . Nature 439 : 688 – 694 Google Scholar Crossref Search ADS PubMed WorldCat Trifilò P , Nardini A, Lo Gullo MA, Salleo S ( 2003 ) Vein cavitation and stomatal behaviour of sunflower (Helianthus annuus) leaves under water limitation . Physiol Plant 119 : 409 – 417 Google Scholar Crossref Search ADS WorldCat Tyerman SD , Niemietz CM, Bramley H ( 2002 ) Plant aquaporins: multifunctional water and solute channels with expanding roles . Plant Cell Environ 25 : 173 – 194 Google Scholar Crossref Search ADS PubMed WorldCat Tyree MT , Nardini A, Salleo S, Sack L, El Omari B ( 2005 ) The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response? J Exp Bot 56 : 737 – 744 Google Scholar Crossref Search ADS PubMed WorldCat Tyree MT , Sperry JS ( 1989 ) Vulnerability of xylem to cavitation and embolism . Annu Rev Plant Biol 40 : 19 – 36 Google Scholar Crossref Search ADS WorldCat Vandeleur RK , Mayo G, Shelden MC, Gilliham M, Kaiser BN, Tyerman SD ( 2009 ) The role of plasma membrane intrinsic protein aquaporins in water transport through roots: diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine . Plant Physiol 149 : 445 – 460 Google Scholar Crossref Search ADS PubMed WorldCat Vandeleur RK , Sullivan W, Athman A, Jordans C, Gilliham M, Kaiser BN, Tyerman SD ( 2014 ) Rapid shoot-to-root signalling regulates root hydraulic conductance via aquaporins . Plant Cell Environ 37 : 520 – 538 Google Scholar Crossref Search ADS PubMed WorldCat Wan X , Steudle E, Hartung W ( 2004 ) Gating of water channels (aquaporins) in cortical cells of young corn roots by mechanical stimuli (pressure pulses): effects of ABA and of HgCl2 . J Exp Bot 55 : 411 – 422 Google Scholar Crossref Search ADS PubMed WorldCat Zhang J , Zhang X, Liang J ( 1995 ) Exudation rate and hydraulic conductivity of maize roots are enhanced by soil drying and abscisic acid treatment . New Phytol 131 : 329 – 336 Google Scholar Crossref Search ADS WorldCat Zhu C , Schraut D, Hartung W, Schäffner AR ( 2005 ) Differential responses of maize MIP genes to salt stress and ABA . J Exp Bot 56 : 2971 – 2981 Google Scholar Crossref Search ADS PubMed WorldCat Zufferey V , Smart DR ( 2012 ) Stomatal behaviour of irrigated Vitis vinifera cv. Syrah following partial root removal . Funct Plant Biol 39 : 1019 – 1027 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the French programs LACCAVE funded by the “Institut National de la Recherche Agronomique” and ANR-09-GENM-024-002. A.C.-L. received a PhD Grant from the French government. Support for S.D.T. lab is from the Australian Research Council Centre of Excellence in Plant Energy Biology (CE 140100008). 2 Deceased. 3 Address correspondence to thierry.simonneau@inra.fr. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Thierry Simonneau (thierry.simonneau@inra.fr). A.C.-L., T.S., S.D.T., A.C., and E.L. designed the research; A.C.-L. and D.M. performed the experiments; T.S. and S.D.T. supervised the experiments; E.J.E. supervised ABA analyses; A.C.-L. analyzed the data; A.C.-L. and T.S. wrote the paper with the contribution of S.D.T. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.17.00698 © 2017 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2017. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - Abscisic Acid Down-Regulates Hydraulic Conductance of Grapevine Leaves in Isohydric Genotypes Only  
JF - Plant Physiology
DO - 10.1104/pp.17.00698
DA - 2017-10-31
UR - https://www.deepdyve.com/lp/oxford-university-press/abscisic-acid-down-regulates-hydraulic-conductance-of-grapevine-leaves-ctGILIDl08
SP - 1121
EP - 1134
VL - 175
IS - 3
DP - DeepDyve
ER -