Arabidopsis leaf hydraulic conductance is regulated by xylem sap pH, controlled, in turn, by a P-type H+ -ATPase of vascular bundle sheath cells.

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 (Kleaf ), and the effect of pH on the water osmotic permeability (Pf ) of isolated BSCs protoplasts. We found that AHA2 is necessary for xylem sap acidification, and in turn, for elevating Kleaf . Conversely, AHA2 knockdown, which alkalinized the xylem sap, or, buffering its pH to 7.5, reduced Kleaf , and elevating external pH to 7.5 decreased the BSCs Pf . All these showed a causative link between AHA2 activity in BSCs and leaf radial hydraulic water conductance.

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential.

Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration.

Bundle-sheath aquaporins play a role in controlling Arabidopsis leaf hydraulic conductivity.

The role of molecular mechanisms in the regulation of leaf hydraulics (K(leaf)) is still not well understood. We hypothesized that aquaporins (AQPs) in the bundle sheath may regulate K(leaf). To examine this hypothesis, AQP genes were constitutively silenced using artificial microRNAs and recovery was achieved by targeting the expression of the tobacco AQP (NtAQP1) to bundle-sheath cells in the silenced plants. Constitutively silenced PIP1 plants exhibited decreased PIP1 transcript levels and decreased K(leaf). However, once the plants were recovered with NtAQP1, their K(leaf) values were almost the same as those of WT plants. We also demonstrate the important role of ABA, acting via AQP, in that recovery and K(leaf) regulation. These results support our previously raised hypothesis concerning the role of bundle-sheath AQPs in the regulation of leaf hydraulics.