Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (K leaf ), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate K leaf . To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (P f ), mesophyll conductance of CO 2 , photosynthesis, K leaf , transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS P f and decreased K leaf but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced P f , but not reduced mesophyll conductance of CO 2 , suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed.

The regulation of plant hydraulic conductance and gas conductance involves a number of different morphological, physiological and molecular mechanisms working in harmony. At the molecular level, aquaporins play a key role in the transport of water, as well as CO₂, through cell membranes. Yet, their tissue-related function, which controls whole-plant gas exchange and water relations, is less understood. In this study, we examined the tissue-specific effects of the stress-induced tobacco Aquaporin1 (NtAQP1), which functions as both a water and CO₂ channel, on whole-plant behavior. In tobacco and tomato plants, constitutive overexpression of NtAQP1 increased net photosynthesis (A(N)), mesophyll CO₂ conductance (g(m)) and stomatal conductance (g(s)) and, under stress, increased root hydraulic conductivity (L(pr)) as well. Our results revealed that NtAQP1 that is specifically expressed in the mesophyll tissue plays an important role in increasing both A(N) and g(m). Moreover, targeting NtAQP1 expression to the cells of the vascular envelope significantly improved the plants' stress response. Surprisingly, NtAQP1 expression in the guard cells did not have a significant effect under any of the tested conditions. The tissue-specific involvement of NtAQP1 in hydraulic and gas conductance via the interaction between the vasculature and the stomata is discussed.

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem sap pH below 6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates the xylem sap pH and leaf radial water fluxes. We monitored the xylem sap pH in the veins of detached leaves of wild-type Arabidopsis, AHA mutants and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor (vanadate) and stimulator (fusicoccin), and different pH buffers. We monitored their impact on the xylem sap pH and the leaf hydraulic conductance (K leaf ), and the effect of pH on the water osmotic permeability (P f ) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating K leaf . Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced K leaf , and elevating external pH to 7.5 decreased the BSCs P f . All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.

Abscisic acid (ABA) levels increase significantly in plants under stress conditions, and ABA is thought to serve as a key stressresponse regulator. However, the direct effect of ABA on photosynthesis and the effect of mesophyll ABA on yield under both well-watered and drought conditions are still the subject of debate. Here, we examined this issue using transgenic Arabidopsis (Arabidopsis thaliana) plants carrying a dominant ABA-signaling inhibitor under the control of a mesophyll-specific promoter (FBPase::abi1-1, abbreviated to fa). Under normal conditions, fa plants displayed slightly higher stomatal conductance and carbon assimilation than wild-type plants; however, these parameters were comparable following ABA treatment. These observations suggest that ABA does not directly inhibit photosynthesis in the short term. The fa plants also exhibited a variety of altered phenotypes under optimal conditions, including more vigorous initial growth, earlier flowering, smaller flowers, and delayed chlorophyll degradation. Furthermore, under optimal conditions, fa plant seed production was less than a third of that observed for the wild type. However, under drought conditions, wild-type and fa seed yields were similar due to a significant reduction in wild-type seed and no reduction in fa seed. These findings suggest that endogenous basal ABA inhibits a stress-escape response under nonstressed conditions, allowing plants to accumulate biomass and maximize yield. The lack of a correlation between flowering time and plant biomass combined with delayed chlorophyll degradation suggests that this stress-escape behavior is regulated independently and upstream of other ABA-induced effects such as rapid growth and flowering.

The leaf vascular bundle sheath cells (BSCs) that tightly envelop the leaf veins, are a selective and dynamic barrier to xylem-sap water and solutes radially entering the mesophyll cells. Under normal conditions, xylem-sap pH of <6 is presumably important for driving and regulating the transmembranal solute transport. Having discovered recently a differentially high expression of a BSC proton pump, AHA2, we now test the hypothesis that it regulates this pH and leaf radial water fluxes.We monitored the xylem-sap pH using the ratiometric fluorescent probe FITC-dextran fed into veins of detached leaves of WT Arabidopsis, AHA mutants, and aha2 mutants complemented with AHA2 gene solely in BSCs. We tested an AHA inhibitor and stimulator, and different pH buffers. We monitored their impact on the xylem-sap pH, the whole leaf hydraulic conductance (Kleaf) and the water osmotic permeability of isolated BSCs protoplasts (Pf).Our results demonstrated AHA2 indispensability for xylem-sap acidification, necessary, in turn, for elevating Pf and Kleaf. Conversely, elevating xylem-sap pH to 7.5, reduced significantly both Pf and Kleaf.All these demonstrate a causative link between AHA2 activity in BSCs and leaf water influx. This positions the BSCs as a pH-controlled transpiration valve in series with the stomata.One-sentence summaryBundle-sheath cells can control the leaf hydraulic conductance by proton-pump-regulated xylem sap pH

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BACKGROUND AND HYPOTHESIS•Under water deprivation, in many perennial species, the stress hormone, ABA, appears in the xylem sap in the shoot (including leaf) veins and the xylem sap pH (pHEXT) increases. This study aimed to test the hypothesis that ABA is the signal for an altered proton balance of the leaf-vein-enwrapping bundle sheath cells (BSCs).METHODS•Plant Material. We used a few Arabidopsis thaliana (L.) Heynh. genotypes: wildtype (WT) of two accessions, Landsberg erecta (Ler) and Columbia (Col), and a few mutants and transformants in these backgrounds.•H+-Pumps activities. We monitored ABA effects on the H+-pump activities in the BSCs cytosol-delimiting membranes (plasma membrane and tonoplast) by monitoring the cytosol and the xylem pH, and the membrane potential (EM), by imaging the fluorescence of pH- and membrane potential (EM)-reporting probes: (a) the BSCs’ pHEXT – with the ratiometric fluorescent dye FITC-dextran petiole-fed into detached leaves in unbuffered xylem perfusion solution (XPS), (b) the BSCs’ pHCYT – with the ratiometric dye SNARF1 loaded into BSCs isolated protoplasts, and (c) the BSCs’ EM – with the ratiometric dye di- 8-ANEPPS.RESULTS•ABA increased the pHEXT; this response was abolished in an abi1-1 mutant with impaired signaling via a PP2C (ABI1) and in an aha2-4 mutant with knocked-down AHA2;•ABA depolarized the WT BSCs;•ABA increased pHCYT irrespective of AHA2 activity (i.e., whether or not AHA was inhibited by vanadate, or in the aha2-4 mutant);•The ABA-induced cytosol alkalinization was abolished in the absence of VHA activity (i.e., when VHA was inhibited by bafilomycin A1, or in the vha-a2 vha-a3 double mutant with inactive VHA);•All these results resemble the ABA effect on GCs;•In contrast to GCs, AHA2 and not AHA1 is the ABA major target in BSCs;•Blue light (BL) enabled the response of the BSCs’ VHA to ABA;•The ABA- and BL-signaling pathways acting on both BSCs’ pumps, AHA2 and VHA, are likely to be BSCs autonomous, based on (a) the presence in the BSCs of many genes of the ABA- and BL-signaling pathways and (b) ABA responses (depolarization and pHCYT elevation) demonstrated under BL in isolated protoplasts.SIGNIFICANCE STATEMENTWe reveal here an alkalinizing effect of the plant drought-stress hormone ABA on the pH on both sides of the plasmalemma of the vein-enwrapping bundle sheath cells (BSCs), due to ABA inhibition of the BSCs’ AHA2, the plasmalemma H+- ATPase and stimulation of VHA, their vacuolar H+-ATPase. Since pH affects the BSCs’ selective regulation of solute and water fluxes into the leaf, these H+- pumps may be attractive targets for manipulations aiming to improve plant drought response.

Water-transport pathways through the leaf are complex and include several checkpoints. Some of these checkpoints exhibit dynamic behavior that may be regulated by aquaporins (AQPs). To date, neither the relative weight of the different water pathways nor their molecular mechanisms are well understood. Here, we have collected evidence to support a putative composite model of water pathways in the leaf and the distribution of water across those pathways. We describe how water moves along a single transcellular path through the parenchyma and continues toward the mesophyll and stomata along transcellular, symplastic and apoplastic paths. We present evidence that points to a role for AQPs in regulating the relative weight of each path in the overall leaf water-transport system and the movement of water between these paths as a result of the integration of multiple signals, including transpiration demand, water potential and turgor. We also present a new theory, the hydraulic fuse theory, to explain effects of the leaf turgor-loss-point on water paths alternation and the subsequent reduction in leaf hydraulic conductivity. An improved understating of leaf water-balance management may lead to the development of crops that use water more efficiently, and responds better to environmental changes.