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 β-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.

We present the first evidence for a fast activation of the nuclear protein poly(ADP-ribose) polymerase (PARP) by signals evoked in the cell membrane, constituting a novel mode of signaling to the cell nucleus. PARP, an abundant, highly conserved, chromatin-bound protein found only in eukaryotes, exclusively catalyzes polyADP-ribosylation of DNA-binding proteins, thereby modulating their activity. Activation of PARP, reportedly induced by formation of DNA breaks, is involved in DNA transcription, replication, and repair. Our findings demonstrate an alternative mechanism: a fast activation of PARP, evoked by inositol 1,4,5,-trisphosphate–Ca2+ mobilization, that does not involve DNA breaks. These findings identify PARP as a novel downstream target of phospholipase C, and unveil a novel fast signal–induced modification of DNA-binding proteins by polyADP-ribosylation.

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.

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 bestfitting model had three free parameters: initial P f , P f rate-of-change (slope Pf ), 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 #1 mm s 21 . 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 mm s 21 (in the majority population of low-initial-P f cells) or (2) increased abruptly to 10 to 20 mm s 21 and then decreased gradually to 3 to 6 mm s 21 (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.The regulation of plant aquaporins is becoming a focus of research in an increasing number of laboratories Tyerman et al., 2002). However, the reports on the regulation of the water permeability of plant cells are still relatively few, very likely reflecting the technical difficulties inherent in such measurements.In order to quantify the permeability of a plant cell to water, one of the approaches consists of isolating protoplasts and monitoring the initial rate of change of their volume upon an osmotic challenge. If the osmotic potential of the external solution is changed instantaneously, the osmotic water permeability (termed P f or P os ) can be deduced from the initial rate of volume relaxation (e.g. Zhang et al., 1990;Verkman, 2000). There are at least two problems with this approach: (1) even when an instantaneous change of solution is possible, a systematic error is introduced, causing an underestimate of P f because, already during the initial phase of protoplast swelling, the volume, the surface area, and the internal concentration of solutes do not remain constant; and (2) instantaneous bath perfusion has technical-and physiological-limitations: unlike animal cells, isolated plant cell protoplasts (the terms protoplast and cell will be used here interchangeably) do not stick well to the chamber floor and defeat attempts of rapid (let alone, instantaneous) solution flushes. Very fast external solution exchange has been achieved by immobilizing the protoplast with a suction micropipette (e.g. Ramah...

''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.

ABSTRACILeaflet movements in Samanea saman are driven by the shrinking and swelling of cells in opposing (extensor and flexor) regions of the motor organ (pulvinus). Changes in cell volume, in turn, depend upon large changes in motor cell content of K+, Cl and other ions. We performed patch-clamp experiments on extensor and flexor protoplasts, to determine whether their plasma membranes contain channels capable of carrying the large K currents that flow during leaflet movement. Recordings in the "whole-cell" mode reveal depolarization-activated K+ currents in extensor and flexor cells that increase slowly (t½ = ca. 2 seconds) and remain active for minutes. Recordings from excised patches reveal a single channel conductance of ca. 20 picosiemens in both cell types. The magnitude of the K+ currents is adequate to account quantitatively for K+ loss, previously measured in vivo during cell shrinkage. The K+ channel blockers tetraethylammonium (5 millimolar) or quinine (1 millimolar) blocked channel opening and decreased light-and dark-promoted movements of excised leaflets. These results provide evidence for the role of potassium channels in leaflet movement.Leaflet movements in nyctinastic (night closure) plants often involve significant changes in the volume and up to severalfold variation in the ionic content of motor cells in the pulvinus (reviewed in Ref. 21). These variations may occur in response to light, darkness, and an endogenous biological clock. Cells in the extensor region of the pulvinus take up K+ and Cl-as they swell during leaflet opening, and lose both ions as they shrink during leaflet closure, while cells in the opposing (flexor) region behave in the reverse manner (12,22,23,25,30,32).We undertook this study to examine a possible role for K+ channels in leaflet movement-related K+ fluxes and changes in cell volume in the nyctinastic legume Samanea saman. K+ channels have already been described in giant algae (1, 5) and in protoplasts isolated from wheat mesophyll cells (13,14), Vicia faba guard cells (27,29) Protoplast Isolation. Terminal secondary pulvini from the fourth to ninth leaf (counting from the apex) were harvested 2 to 3 h after the beginning of the light period in the growth chamber, or 2 to 3 h after sunrise in the greenhouse. Protoplasts were prepared by enzymic digestion of slices of extensor or flexor tissue (pooled separately), as described in (7) but with the following modifications. (a) The osmotic pressure of the pre-digestion solution was raised in two steps to 680 mosmol to ensure plasmolysis. (b) Pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan) was added to the digestion solution (which contained cellulase, pectinase and Driselase) to a final concentration of 0.2% (w/v). (c) A second purification step was added, as follows: the cells collected from the Ficoll interface were layered again on a sucrose gradient (2), spun at 60 to IOOg for 5 min, and collected and kept on ice for up to 24 h for patch-clamp experiments.Forty to fifty protoplasts of each type, flexor and ...

The transcriptome analysis of leaf bundle sheath cells compared with mesophyll cells, supported by physiological assays, suggests a potential role of the bundle sheath in radial leaf transport.

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 m 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.Some plants and microorganisms engage in reciprocal symbiosis for the purpose of exchanging nutrients. For example, in nitrogen-fixing nodules, the intracellular bacteria supply the host plant with combined nitrogen and are in turn provided with carbon sources (Mylona et al., 1995). There are two types of nodule symbioses between nitrogen-fixing soil bacteria and higher plants, namely, the symbiosis between legumes and rhizobia, and the actinorhizal symbiosis between actinomycetes of the genus Frankia and a diverse group of angiosperms collectively called actinorhizal plants. In both cases, nutrient exchange between the host and its microsymbiont is controlled by the plant plasma membrane-derived interface enclosing the microsymbiont. In most legume symbioses, where the bacteria are taken up into the plant cells in a complete endocytotic process (Verma, 1992), this interface is the peribacteroid membrane (PBM). In primitive legume symbioses (de Faria et al., 1987) and the actinorhizal symbioses (Mylona et al., 1995), the interface is reported to be the invaginated and incompletely enclosed plasma membrane of the infected cell.The nutrient exchange between the symbiotic partners requires transporters of the carbon sources and trace elements that flow from the plant to the microsymbiont along with the transporters of the products of bacterial nitrogen fixation that flow from the microsymbiont to the plant (Pawlowski and Bisseling, 1996). Soybean (Glycine max) nodules, which represent legume symbioses, show evidence of the physiological or biochemical activities of transporters of ammonium, which i...