Plant plasma membrane intrinsic proteins (PIPs) cluster in two evolutionary subgroups, PIP1 and PIP2, with different aquaporin activities when expressed in Xenopus oocytes. Maize ZmPIP1;1 and ZmPIP1;2 do not increase the osmotic water permeability coefficient (P f ), whereas ZmPIP2;1, ZmPIP2;4, and ZmPIP2;5 do. Here, we show that coexpression of the nonfunctional ZmPIP1;2 and the functional ZmPIP2;1, ZmPIP2;4, or ZmPIP2;5 resulted in an increase in P f that was dependent on the amount of injected ZmPIP1;2 complementary RNA. Confocal analysis of oocytes expressing ZmPIP1;2-green fluorescent protein (GFP) alone or ZmPIP1;2-GFP plus ZmPIP2;5 showed that the amount of ZmPIP1;2-GFP present in the plasma membrane was significantly greater in coexpressing cells. Nickel affinity chromatography purification of ZmPIP2;1 fused to a His tag coeluted with ZmPIP1;2-GFP demonstrated physical interaction and heteromerization of both isoforms. Interestingly, coexpression of ZmPIP1;1 and ZmPIP2;5 did not result in a greater increase in P f than did the expression of ZmPIP2;5 alone, but coexpression of the ZmPIP1;1 and ZmPIP1;2 isoforms induced a P f increase, indicating that PIP1 isoform heteromerization is required for both of them to act as functional water channels. Mutational analysis demonstrated the important role of the C-terminal part of loop E in PIP interaction and water channel activity induction. This study has revealed a new mechanism of plant aquaporin regulation that might be important in plant water relations.

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.

Water movement across root tissues occurs by parallel apoplastic, symplastic, and transcellular pathways that the plant can control to a certain extent. Because water channels or aquaporins (AQPs) play an important role in regulating water flow, studies on AQP mRNA and protein expression in different root tissues are essential. Here, we quantified and localized the expression of Zea mays plasma membrane AQPs (ZmPIPs) in primary root tip using in situ and quantitative RT-PCR and immunodetection approaches. All ZmPIP genes except ZmPIP2;7 were expressed in primary roots. Expression was found to be dependent on the developmental stage of the root, with, in general, an increase in expression towards the elongation and mature zones. Two genes, ZmPIP1;5 and ZmPIP2;5, showed the greatest increase in expression (up to 11- and 17-fold, respectively) in the mature zone, where they accounted for 50% of the total expressed ZmPIPs. The immunocytochemical localization of ZmPIP2;1 and ZmPIP2;5 in the exodermis and endodermis indicated that they are involved in root radial water movement. In addition, we detected a polar localization of ZmPIP2;5 to the external periclinal side of epidermal cells in root apices, suggesting an important role in water uptake from the root surface. Finally, protoplast swelling assays showed that root cells display a variable, but globally low, osmotic water permeability coefficient (Pf < 10 microm/s). However, the presence of a population of cells with a higher Pf (up to 26 microm/s) in mature zone of the root might be correlated with the increased expression of several ZmPIP genes.

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.

Summary• Anisohydric plants are thought to be more drought tolerant than isohydric plants. However, the molecular mechanism determining whether the plant water potential during the day remains constant or not regardless of the evaporative demand (isohydric vs anisohydric plant) is not known.• Here, it was hypothesized that aquaporins take part in this molecular mechanism determining the plant isohydric threshold. Using computational mining a key tonoplast aquaporin, tonoplast intrinsic protein 2;2 (SlTIP2;2), was selected within the large multifunctional gene family of tomato (Solanum lycopersicum) aquaporins based on its induction in response to abiotic stresses. SlTIP2;2-transformed plants (TOM-SlTIP2;2) were compared with controls in physiological assays at cellular and whole-plant levels.• Constitutive expression of SlTIP2;2 increased the osmotic water permeability of the cell and whole-plant transpiration. Under drought, these plants transpired more and for longer periods than control plants, reaching a lower relative water content, a behavior characterizing anisohydric plants. In 3-yr consecutive commercial glasshouse trials, TOM-SlTIP2;2 showed significant increases in fruit yield, harvest index and plant mass relative to the control under both normal and water-stress conditions.• In conclusion, it is proposed that the regulation mechanism controlling tonoplast water permeability might have a role in determining the whole-plant ishohydric threshold, and thus its abiotic stress tolerance.