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.

The Role of Aquaporins in pH-Dependent Germination of Rhizopus delemar Spores.

Rhizopus delemar and associated species attack a wide range of fruit and vegetables after harvest. Host nutrients and acidic pH are required for optimal germination of R. delemar, and we studied how this process is triggered. Glucose induced spore swelling in an acidic environment, expressed by an up to 3-fold increase in spore diameter, whereas spore diameter was smaller in a neutral environment. When suspended in an acidic environment, the spores started to float, indicating a change in their density. Treatment of the spores with HgCl2, an aquaporin blocker, prevented floating and inhibited spore swelling and germ-tube emergence, indicating the importance of water uptake at the early stages of germination. Two putative candidate aquaporin-encoding genes-RdAQP1 and RdAQP2-were identified in the R. delemar genome. Both presented the conserved NPA motif and six-transmembrane domain topology. Expressing RdAQP1 and RdAQP2 in Arabidopsis protoplasts increased the cells' osmotic water permeability coefficient (Pf) compared to controls, indicating their role as water channels. A decrease in R. delemar aquaporin activity with increasing external pH suggested pH regulation of these proteins. Substitution of two histidine (His) residues, positioned on two loops facing the outer side of the cell, with alanine eliminated the pH sensing resulting in similar Pf values under acidic and basic conditions. Since hydration is critical for spore switching from the resting to activate state, we suggest that pH regulation of the aquaporins can regulate the initial phase of R. delemar spore germination, followed by germ-tube elongation and host-tissue infection.

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.

Do phosphoinositides regulate membrane water permeability of tobacco protoplasts by enhancing the aquaporin pathway?

MAIN CONCLUSION: Enhancing the membrane content of PtdInsP 2 , the already-recognized protein-regulating lipid, increased the osmotic water permeability of tobacco protoplasts, apparently by increasing the abundance of active aquaporins in their membranes. While phosphoinositides are implicated in cell volume changes and are known to regulate some ion channels, their modulation of aquaporins activity has not yet been reported for any organism. To examine this, we compared the osmotic water permeability (P f) of protoplasts isolated from tobacco (Nicotiana tabacum) cultured cells (NT1) with different (genetically lowered or elevated relative to controls) levels of inositol trisphosphate (InsP3) and phosphatidyl inositol [4,5] bisphosphate (PtdInsP2). To achieve this, the cells were transformed with, respectively, the human InsP3 5-phosphatase ('Ptase cells') or human phosphatidylinositol (4) phosphate 5-kinase ('PIPK cells'). The mean P f of the PIPK cells was several-fold higher relative to that of controls and Ptase cells. Three results favor aquaporins over the membrane matrix as underlying this excessive P f: (1) transient expression of the maize aquaporin ZmPIP2;4 in the PIPK cells increased P f by 12-30 µm s(-1), while in the controls only by 3-4 µm s(-1). (2) Cytosol acidification-known to inhibit aquaporins-lowered the P f in the PIPK cells down to control levels. (3) The transcript of at least one aquaporin was elevated in the PIPK cells. Together, the three results demonstrate the differences between the PIPK cells and their controls, and suggest a hitherto unobserved regulation of aquaporins by phosphoinositides, which could occur through direct interaction or indirect phosphoinositides-dependent cellular effects.

Measuring the osmotic water permeability coefficient (Pf) of spherical cells: isolated plant protoplasts as an example.

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P(f)) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the 'PfFit'), to yield P(f).

The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics.

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 CO2, 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 Pf 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 CO2, 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.

Smart pipes: the bundle sheath role as xylem-mesophyll barrier.

Signs of abiotic toxicity often appear first at the margins of leaves and gradually spread toward the midrib. It has been suggested that the bundle sheath tissue surrounding the shoot vascular system acts as a solute transport-regulating barrier that prevents excessive quantities of toxic ions from entering the leaf and pushes them toward the hydathodes. We examined this hypothesis by examining the distribution of toxic boron (B) in mutant Arabidopsis leaves with flooded mesophyll and comparing it with that observed in control leaves that exuded guttation drops. As opposed to the control plants, which showed classical symptoms of B toxicity (necrosis starting at the leaf margins), in the mutants, necrosis was first observed inside the leaf. We will discuss this result and how it supports the hypothesis that the bundle sheath serves as a selective barrier filtering the xylem-to-leaf radial transport flow and pushing toxic solutes toward the hydathodes.

Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA?

The 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.