measuring-osmotic-water-permeability-coefficient-pf-spherical-cells

Measuring the Osmotic Water Permeability Coefficient (P<sub>f</sub>) 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 ...

The Hebrew University of Jerusalem

In plants, KT/HAK/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. We determined its cellular and tissue localization, and analyzed its functions in rice using both OsHAK5 knockout mutants and overexpression lines at three genetic backgrounds. GUS reporter driven by 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 was 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 [K]/[Na] ratio in the shoots and salt stress tolerance (shoot growth), while knockout of OsHAK5 decreased [K]/[Na] 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 role of OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels.

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.

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

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.

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

To prepare reagents for a study of the interactions of prolactin (PRL) and growth hormone (GH) receptors (Rs) with suppressor of cytokine signaling (SOCS) proteins in living cells by fluorescence resonance energy transfer methodology, the respective proteins were tagged with cyan (CFP) or yellow (YFP) fluorescent protein. Constructs encoding ovine (o)PRLR-YFP, oPRLR-CFP, oGHR-YFP, and oGHR-CFP tagged downstream of the receptor DNA were prepared in the plasmid pcDNA plasmid and tested for biological activity in HEK 293T cells transiently cotransfected with those constructs and the reporter gene encoding luciferase. All four constructs were biologically active and as potent as their untagged counterparts. Cells transfected with those proteins exhibited fluorescence in the cytoplasm and the membrane. Constructs encoding DNA tagged with YFP or CFP upstream of SOCS1, SOCS2, SOCS3, and SOCS6 were prepared in pECFP-C1 and pEYFP-C1 plasmids. The biological activities of SOCS1 and SOCS3 tagged at their amino termini were assayed by their ability to inhibit placental lactogen (PL)- or GH-induced activation of JAK2/STAT5-mediated luciferase transcription in HEK 293T cells; the activity of SOCS2 was assayed by its ability to abolish SOCS1-induced inhibition. The tagged proteins exhibited biological activity that was equal to or even more potent than their untagged counterparts. The biological activities of CFP-SOCS2 and YFP-SOCS2 were also assayed using GST-GHR binding assay. Their interaction with the cytosolic domain of GHR was equivalent to their respective untagged counterparts. The biological activity of the construct encoding SOCS6 was not tested because of lack of a suitable assay. Cells transfected with eight of these tagged constructs expressed the fluorescent proteins in both the nucleus and cytosol; the tagged SOCS2 was localized mostly in the latter compartment.

Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent proteins.

Alder (Alnus glutinosa) and more than 200 angiosperms that encompass 24 genera are collectively called actinorhizal plants. These plants form a symbiotic relationship with the nitrogen-fixing actinomycete Frankia strain HFPArI3. The plants provide the bacteria with carbon sources in exchange for fixed nitrogen, but this metabolite exchange in actinorhizal nodules has not been well defined. We isolated an alder cDNA from a nodule cDNA library by differential screening with nodule versus root cDNA and found that it encoded a transporter of the PTR (peptide transporter) family, AgDCAT1. AgDCAT1 mRNA was detected only in the nodules and not in other plant organs. Immunolocalization analysis showed that AgDCAT1 protein is localized at the symbiotic interface. The AgDCAT1 substrate was determined by its heterologous expression in two systems. Xenopus laevis oocytes injected with AgDCAT1 cRNA showed an outward current when perfused with malate or succinate, and AgDCAT1 was able to complement a dicarboxylate uptake-deficient Escherichia coli mutant. Using the E. coli system, AgDCAT1 was shown to be a dicarboxylate transporter with a K(m) of 70 microm for malate. It also transported succinate, fumarate, and oxaloacetate. To our knowledge, AgDCAT1 is the first dicarboxylate transporter to be isolated from the nodules of symbiotic plants, and we suggest that it may supply the intracellular bacteria with dicarboxylates as carbon sources.

A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family.

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.

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

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.

Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea 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.

Osmoregulation of leaf motor cells.

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.

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

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.

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

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.

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

Basal signaling regulates plant growth and development.

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.

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

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.

Article	Year
The role of OsHAK5 in potassium acquisition and transport from roots to shoots in rice at low potassium supply levels. Plant physiology\| PubMed ID: 25157029	2014
Diurnal and circadian regulation of putative potassium channels in a leaf moving organ. Plant physiology\| PubMed ID: 11842166	2002
Plasma membrane aquaporins in the motor cells of Samanea saman: diurnal and circadian regulation. The Plant cell\| PubMed ID: 11910017	2002
Preparation and expression of biologically active prolactin and growth hormone receptors and suppressor of cytokine signaling proteins 1, 2, 3, and 6 tagged with cyan and yellow fluorescent ... Protein expression and purification\| PubMed ID: 12182826	2002
A nodule-specific dicarboxylate transporter from alder is a member of the peptide transporter family. Plant physiology\| PubMed ID: 15001700	2004
Dynamic changes in the osmotic water permeability of protoplast plasma membrane. Plant physiology\| PubMed ID: 15310831	2004
Phosphorylation of SPICK2, an AKT2 channel homologue from Samanea motor cells. Journal of experimental botany\| PubMed ID: 16968880	2006
Osmoregulation of leaf motor cells. FEBS letters\| PubMed ID: 17434488	2007
Expression of the novel wheat gene TM20 confers enhanced cadmium tolerance to bakers' yeast. The Journal of biological chemistry\| PubMed ID: 18411273	2008
Phosphatidylinositol (4,5)bisphosphate inhibits K+-efflux channel activity in NT1 tobacco cultured cells. Plant physiology\| PubMed ID: 19052153	2009
Phosphatidylinositol 4,5-bisphosphate regulates plant K(+) channels. Biochemical Society transactions\| PubMed ID: 20298247	2010
Basal signaling regulates plant growth and development. Plant physiology\| PubMed ID: 20921159	2010
The Arabidopsis-related halophyte Thellungiella halophila: boron tolerance via boron complexation with metabolites? Plant, cell & environment\| PubMed ID: 21999349	2012
Is the leaf bundle sheath a "smart flux valve" for K+ nutrition? Journal of plant physiology\| PubMed ID: 24629888	2014

Nava Moran

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