A tale of two pumps: Blue light and abscisic acid alter Arabidopsis leaf hydraulics via bundle sheath cell H+-ATPases.

The bundle sheath cell (BSC) layer tightly enveloping the xylem throughout the leaf is recognized as a major signal-perceiving "valve" in series with stomata, regulating leaf hydraulic conductance (Kleaf) and thereby radial water flow via the transpiring leaf. The BSC blue light (BL) signaling pathway increases Kleaf and the underlying BSC water permeability. Here, we explored the hypothesis that BSCs also harbor a Kleaf-downregulating signaling pathway related to the stress phytohormone abscisic acid (ABA). We employed fluorescence imaging of xylem sap in detached leaves and BSC protoplasts from different genotypes of Arabidopsis (Arabidopsis thaliana) plants, using pH and membrane potential probes to monitor physiological responses to ABA and BL in combination with pharmacological agents. We found that BL-enhanced Kleaf required elevated BSC cytosolic Ca2+. ABA inhibited BL-activated xylem-sap-acidifying BSC H+-ATPase AHA2 (Arabidopsis H+-ATPase 2), resulting in depolarized BSCs and alkalinized xylem sap. ABA also stimulated BSC vacuolar H+-ATPase (VHA), which alkalinized the BSC cytosol. Each pump stimulation, AHA2 by BL and VHA by ABA (under BL), also required Ca2+. ABA stimulated VHA in the dark depending on Ca2+, but only in an alkaline external medium. Taken together with earlier findings on the pH sensitivity of BSC osmotic water permeability (i.e. aquaporin activity), our results suggest a Ca2+-dependent and pH-mediated causative link between the BL- and ABA-regulated activities of two BSC H+-ATPases and Kleaf.

Out of the blue: Phototropins of the leaf vascular bundle sheath mediate the regulation of leaf hydraulic conductance by blue light.

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.

Potassium transporter OsHAK18 mediates potassium and sodium circulation and sugar translocation in rice.

High-affinity potassium (K+) transporter (HAK)/K+ uptake permease (KUP)/K+ transporter (KT) have been identified in all genome-sequenced terrestrial plants. They play an important role in K+ acquisition and translocation and in enhancing salt tolerance. Here, we report that plasma membrane-located OsHAK18 functions in K+ and sodium (Na+) circulation and sugar translocation in rice (Oryza sativa). OsHAK18 was expressed mainly, though not exclusively, in vascular tissues and particularly in the phloem. Knockout (KO) of OsHAK18 reduced K+ concentration in phloem sap and roots but increased K+ accumulation in the shoot of both 'Nipponbare' and 'Zhonghua11' cultivars, while overexpression (OX) of OsHAK18 driven by its endogenous promoter increased K+ concentration in phloem sap and roots and promoted Na+ retrieval from the shoot to the root under salt stress. Split-root experimental analysis of rubidium (Rb+) uptake and circulation indicated that OsHAK18-OX promoted Rb+ translocation from the shoot to the root. In addition, OsHAK18-KO increased while OsHAK18-OX reduced soluble sugar content in the shoot and oppositely affected the sugar concentration in the phloem and its content in the root. Moreover, OsHAK18-OX dramatically increased grain yield and physiological K+ utilization efficiency. Our results suggest that-unlike other OsHAKs analyzed heretofore-OsHAK18 is critical for K+ and Na+ recirculation from the shoot to the root and enhances the source-to-sink translocation of photo-assimilates.

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.

Differential gene expression and transport functionality in the bundle sheath versus mesophyll - a potential role in leaf mineral homeostasis.

Under fluctuating ambient conditions, the ability of plants to maintain hydromineral homeostasis requires the tight control of long distance transport. This includes the control of radial transport within leaves, from veins to mesophyll. The bundle sheath is a structure that tightly wraps around leaf vasculature. It has been suggested to act as a selective barrier in the context of radial transport. This suggestion is based on recent physiological transport assays of bundle sheath cells (BSCs), as well as the anatomy of these cells.We hypothesized that the unique transport functionality of BSCs is apparent in their transcriptome. To test this, we compared the transcriptomes of individually hand-picked protoplasts of GFP-labeled BSCs and non-labeled mesophyll cells (MCs) from the leaves of Arabidopsis thaliana. Of the 90 genes differentially expressed between BSCs and MCs, 45% are membrane related and 20% transport related, a prominent example being the proton pump AHA2. Electrophysiological assays showed that the major AKT2-like membrane K+ conductances of BSCs and MCs had different voltage dependency ranges. Taken together, these differences may cause simultaneous but oppositely directed transmembrane K+ fluxes in BSCs and MCs, in otherwise similar conditions.

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.

The role of a potassium transporter OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels.

In plants, K transporter (KT)/high affinity K transporter (HAK)/K uptake permease (KUP) is the largest potassium (K) transporter family; however, few of the members have had their physiological functions characterized in planta. Here, we studied OsHAK5 of the KT/HAK/KUP family in rice (Oryza sativa). We determined its cellular and tissue localization and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines in three genetic backgrounds. A ß-glucuronidase reporter driven by the OsHAK5 native promoter indicated OsHAK5 expression in various tissue organs from root to seed, abundantly in root epidermis and stele, the vascular tissues, and mesophyll cells. Net K influx rate in roots and K transport from roots to aerial parts were severely impaired by OsHAK5 knockout but increased by OsHAK5 overexpression in 0.1 and 0.3 mm K external solution. The contribution of OsHAK5 to K mobilization within the rice plant was confirmed further by the change of K concentration in the xylem sap and K distribution in the transgenic lines when K was removed completely from the external solution. Overexpression of OsHAK5 increased the K-sodium concentration ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased the K-sodium concentration ratio in the shoots, resulting in sensitivity to salt stress. Taken together, these results demonstrate that OsHAK5 plays a major role in K acquisition by roots faced with low external K and in K upward transport from roots to shoots in K-deficient rice plants.

The Arabidopsis-related halophyte Thellungiella halophila: boron tolerance via boron complexation with metabolites?

Tolerance to boron (B) is still not completely understood. We tested here the hypothesis that Thellungiella halophila, an Arabidopsis thaliana-related 'extremophile' plant, with abundance of B in its natural environment, is tolerant to B, and examined the potential mechanisms of this tolerance. With 1-10 mm B applied ([B](ext)) to Thellungiella and Arabidopsis grown in hydroponics, the steady-state accumulated B concentration ([B](int)) in the root was below [B](ext), and was similar in both, suggesting both extrude B actively. Whether grown in soil or hydroponically, the shoot [B](int) was higher in Arabidopsis than in Thellungiella, suggesting more effective net B exclusion by Thellungiella root. Arabidopsis exhibited toxicity symptoms including reduced shoot fresh weight (FW), but Thellungiella was not affected, even at similar levels of shoot-accumulated [B](int) (about 10 to 40 mm B in 'shoot water'), suggesting additional B tolerance mechanism in Thellungiella shoot. At [B](ext) = 5 mm, the summed shoot concentration of the potentially B-binding polyhydroxyl metabolites (malic acid, fructose, glucose, sucrose and citric acid) in Arabidopsis was below [B](int) , but in Thellungiella it was over twofold higher than [B](int) , and therefore likely to allow appreciable 1:2 boron-metabolite complexation in the shoot. This, we suggest, is an important component of Thellungiella B tolerance mechanism.

Phosphatidylinositol 4,5-bisphosphate regulates plant K(+) channels.

Phosphoinositides play an important role in both abiotic and biotic signalling in plants. The signalling cascade may include the production of second messengers by hydrolysis of PtdIns(4,5)P(2). However, increasingly, PtdIns(4,5)P(2) itself is shown to mediate signalling by regulating target proteins. The present mini-review summarizes the experimentally demonstrated effects of PtdIns(4,5)P(2) on plant K(+) channels and examines their structure for candidate sites of direct PtdIns(4,5)P(2)-protein interaction.

The Potassium Transporter OsHAK5 Alters Rice Architecture via ATP-Dependent Transmembrane Auxin Fluxes.

Plant HAK/KUP/KT family members function as plasma membrane (PM) H+/K+ symporters and may modulate chemiosmotically-driven polar auxin transport (PAT). Here, we show that inactivation of OsHAK5, a rice K+ transporter gene, decreased rootward and shootward PAT, tiller number, and the length of both lateral roots and root hairs, while OsHAK5 overexpression increased PAT, tiller number, and root hair length, irrespective of the K+ supply. Inhibitors of ATP-binding-cassette type-B transporters, NPA and BUM, abolished the OsHAK5-overexpression effect on PAT. The mechanistic basis of these changes included the OsHAK5-mediated decrease of transmembrane potential (depolarization), increase of extracellular pH, and increase of PM-ATPase activity. These findings highlight the dual roles of OsHAK5 in altering cellular chemiosmotic gradients (generated continuously by PM H+-ATPase) and regulating ATP-dependent auxin transport. Both functions may underlie the prominent effect of OsHAK5 on rice architecture, which may be exploited in the future to increase crop yield via genetic manipulations.

Osmoregulation of leaf motor cells.

"Osmotic Motors"--the best-documented explanation for plant leaf movements--frequently reside in specialized motor leaf organs, pulvini. The movements result from dissimilar volume and turgor changes in two oppositely positioned parts of the pulvinus. This Osmotic Motor is powered by a plasma membrane proton ATPase, which drives KCl fluxes and, consequently, water, across the pulvinus into swelling cells and out of shrinking cells. Light signals and signals from the endogenous biological clock converge on the channels through which these fluxes occur. These channels and their regulatory pathways in the pulvinus are the topic of this review.

Is the leaf bundle sheath a "smart flux valve" for K+ nutrition?

Evidence has started to accumulate that the bundle sheath regulates the passage of water, minerals and metabolites between the mesophyll and the conducting vessels of xylem and phloem within the leaf veins which it envelops. Although potassium (K(+)) nutrition has been studied for several decades, and much is known about the uptake and recirculation of K(+) within the plant, the potential regulatory role of bundle sheath with regard to K(+) fluxes has just begun to be addressed. Here we have collected some facts and ideas about these processes.

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).

Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells.

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed "cytosolic" Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: "Low PIs" had depressed levels of these PIs, and "High PIs" had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 microM) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5-4 microM), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells.

Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast.

Cadmium causes the generation of reactive oxygen species, which in turn causes cell damage. We isolated a novel gene from a wheat root cDNA library, which conferred Cd(II)-specific tolerance when expressed in yeast (Saccharomyces cerevisiae). The gene, which we called TaTM20, for Triticum aestivum transmembrane 20, encodes a putative hydrophobic polypeptide of 889 amino acids, containing 20 transmembrane domains arranged as a 5-fold internal repeating unit of 4 transmembrane domains each. Expression of TaTM20 in yeast cells stimulated Cd(II) efflux resulting in a decrease in the content of yeast intracellular cadmium. TaTM20-induced Cd(II) tolerance was maintained in yeast even under conditions of reduced GSH. These results demonstrate that TaTM20 enhances Cd(II) tolerance in yeast through the stimulation of Cd(II) efflux from the cell, partially independent of GSH. Treatment of wheat seedlings with Cd(II) induced their expression of TaTM20, decreasing subsequent root Cd(II) accumulation and suggesting a possible role for TaTM20 in Cd(II) tolerance in wheat.

Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells.

SPICK2, a homologue of the weakly-inward-rectifying Shaker-like Arabidopsis K channel, AKT2, is a candidate K+-influx channel participating in light- and clock-regulated leaf movements of the legume, Samanea saman. Light and the biological clock regulate the in situ K+-influx channel activity differentially in extensor and flexor halves of the pulvinus (the S. saman leaf motor organ), and also-though differently-the transcript level of SPICK2 in the pulvinus. This disparity between the in situ channel activity versus its candidate transcript, along with the sequence analysis of SPICK2, suggest an in situ regulation of the activity of SPICK2, possibly by phosphorylation and/or by interaction with cAMP. Consistent with this (i) the activity of the voltage-dependent K+-selective fraction of the inward current in extensor and flexor cells was affected differentially in whole-cell patch-clamp assays promoting phosphorylation (using the protein phosphatase inhibitor okadaic acid); (ii) several proteins in isolated plasma membrane-enriched vesicles of the motor cells underwent phosphorylation without an added kinase in conditions similar to patch-clamp; and (iii) the SPICK2 protein was phosphorylated in vitro by the catalytic subunit of the broad-range cAMP-dependent protein kinase. All of these results are consistent with the notion that SPICK2 is the K+-influx channel, and is regulated in vivo directly by phosphorylation.

Dynamic changes in the osmotic water permeability of protoplast plasma membrane.

The osmotic water permeability coefficient (P(f)) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The best-fitting model had three free parameters: initial P(f), P(f) rate-of-change (slope(P(f))), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P(f) during the delay was < or =1 microm s(-1). During the swelling period following the delay, P(f) changed dynamically: within the first 15 s P(f) either (1) increased gradually to approximately 8 microm s(-1) (in the majority population of low-initial-P(f) cells) or (2) increased abruptly to 10 to 20 microm s(-1) and then decreased gradually to 3 to 6 microm s(-1) (in the minority population of high-initial-P(f) cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P(f) values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P(f) in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P(f) increase, thereby confirming the hypothesis that P(f) dynamics, delay included, reflected the varying activity of aquaporins.

Plasma membrane aquaporins in the motor cells of Samanea saman: diurnal and circadian regulation.

Leaf-moving organs, remarkable for the rhythmic volume changes of their motor cells, served as a model system in which to study the regulation of membrane water fluxes. Two plasma membrane intrinsic protein homolog genes, SsAQP1 and SsAQP2, were cloned from these organs and characterized as aquaporins in Xenopus laevis oocytes. Osmotic water permeability (P(f)) was 10 times higher in SsAQP2-expressing oocytes than in SsAQP1-expressing oocytes. SsAQP1 was found to be glycerol permeable, and SsAQP2 was inhibited by 0.5 mM HgCl(2) and by 1 mM phloretin. The aquaporin mRNA levels differed in their spatial distribution in the leaf and were regulated diurnally in phase with leaflet movements. Additionally, SsAQP2 transcription was under circadian control. The P(f) of motor cell protoplasts was regulated diurnally as well: the morning and/or evening P(f) increases were inhibited by 50 microM HgCl(2), by 2 mM cycloheximide, and by 250 microM phloretin to the noon P(f) level. Our results link SsAQP2 to the physiological function of rhythmic cell volume changes.

Diurnal and circadian regulation of putative potassium channels in a leaf moving organ.

In a search for potassium channels involved in light- and clock-regulated leaf movements, we cloned four putative K channel genes from the leaf-moving organs, pulvini, of the legume Samanea saman. The S. saman SPOCK1 is homologous to KCO1, an Arabidopsis two-pore-domain K channel, the S. saman SPORK1 is similar to SKOR and GORK, Arabidopsis outward-rectifying Shaker-like K channels, and the S. saman SPICK1 and SPICK2 are homologous to AKT2, a weakly-inward-rectifying Shaker-like Arabidopsis K channel. All four S. saman sequences possess the universal K-channel-specific pore signature, TXXTXGYG, strongly suggesting a role in transmembrane K(+) transport. The four S. saman genes had different expression patterns within four leaf parts: "extensor" and "flexor" (the motor tissues), the leaf blades (mainly mesophyll), and the vascular bundle ("rachis"). Based on northern blot analysis, their transcript level was correlated with the rhythmic leaf movements: (a) all four genes were regulated diurnally (Spick2, Spork1, and Spock1 in extensor and flexor, Spick1 in extensor and rachis); (b) Spork1 and Spock1 rhythms were inverted upon the inversion of the day-night cycle; and (c) in extensor and/or flexor, the expression of Spork1, Spick1, and Spick2 was also under a circadian control. These findings parallel the circadian rhythm shown to govern the resting membrane K(+) permeability in extensor and flexor protoplasts and the susceptibility of this permeability to light stimulation (Kim et al., 1993). Thus, Samanea pulvinar motor cells are the first described system combining light and circadian regulation of K channels at the level of transcript and membrane transport.