SUMMARYThe hydraulic conductivity of the leaf vascular system (K leaf ) is dynamic and decreases rapidly under drought stress, possibly in response to the stress phytohormone ABA, which increases sharply in the xylem sap (ABA xyl ) during periods of drought. Vascular bundle-sheath cells (BSCs; a layer of parenchymatous cells tightly enwrapping the entire leaf vasculature) have been hypothesized to control K leaf via the specific activity of BSC aquaporins (AQPs). We examined this hypothesis and provide evidence for drought-induced ABA xyl diminishing BSC osmotic water permeability (P f ) via downregulated activity of their AQPs. ABA fed to the leaf via the xylem (petiole) both decreased K leaf and led to stomatal closure, replicating the effect of drought. In contrast, smearing ABA on the leaf blade, while also closing stomata, did not decrease K leaf within 2-3 h of application, demonstrating that K leaf does not depend entirely on stomatal closure. GFP-labeled BSCs showed decreased P f in response to 'drought' and ABA treatment, and a reversible decrease with HgCl 2 (an AQP blocker). These P f responses, absent in mesophyll cells, suggest stress-regulated AQP activity specific to BSCs, and imply a role for these cells in decreasing K leaf via a reduction in P f . Our results support the above hypothesis and highlight the BSCs as hitherto overlooked vasculature sensor compartments, extending throughout the leaf and functioning as 'stress-regulated valves' converting vasculature chemical signals (possibly ABA xyl ) into leaf hydraulic signals.

SUMMARYStomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO 2 for photosynthesis with the loss of water through transpiration. Increased guardcell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar-phosphorylating enzyme involved in sugar-sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback-inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.

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.

HighlightIsohydric poplars have high water-use efficiency, while anisohydric poplars show faster growth under a variable water supply, with implications for performance of the different genotypes for woody biomass production.

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.