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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.

The Arabidopsis (Arabidopsis thaliana) leaf veins bundle sheath cells (BSCs) – a selective barrier to water and solutes entering the mesophyll – increase the leaf radial hydraulic conductance (Kleaf) by acidifying the xylem sap by their plasma membrane H+-ATPase, AHA2. Based on this and on the BSCs’ expression of phototropins PHOT1 and PHOT2, and the known blue-light (BL)-induced Kleaf increase, we hypothesized that, resembling the guard cells, BL perception by the BSCs’ phots activates its H+-ATPase, which, consequently, upregulates Kleaf. Indeed, under BL, the Kleaf of the knockout mutant lines phot1-5, phot2-1, phot1-5 phot2-1, and aha2-4 was lower than that of the wild type (WT). BSC-only-directed complementation of phot1-5 or aha2-4 by PHOT1 or AHA2, respectively, restored the BL-induced Kleaf increase. BSC-specific silencing of PHOT1 or PHOT2 prevented such Kleaf increase. A xylem-fed kinase inhibitor (tyrphostin 9) replicated this also in WT plants. White light – ineffective in the phot1-5 mutant – acidified the xylem sap (relative to darkness) in WT and in the PHOT1-complemented phot1-5. These results, supported by BL increase of BSC protoplasts’ water permeability and cytosolic pH and their hyperpolarization by BL, identify the BSCs as a second phot-controlled water conductance element in leaves, in series with stomatal conductance. Through both, BL regulates the leaf water balance.

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