Plasma Membrane Of Guard Cells Research Articles

Roles of ion channels and transporters in guard cell signal transduction

Stomatal complexes consist of pairs of guard cells and the pore they enclose. Reversible changes in guard cell volume alter the aperture of the pore and provide the major regulatory mechanism for control of gas exchange between the plant and the environment. Stomatal movement is facilitated by the activity of ion channels and ion transporters found in the plasma membrane and vacuolar membrane of guard cells. Progress in recent years has elucidated the molecular identities of many guard cell transport proteins, and described their modulation by various cellular signal transduction components during stomatal opening and closure prompted by environmental and endogenous stimuli.

Blue light inhibits guard cell plasma membrane anion channels in a phototropin‐dependent manner

Guard cells respond to light through two independent signalling pathways. The first pathway is initiated by photosynthetically active radiation and has been associated with changes in the intercellular CO(2) concentration, leading to inhibition of plasma membrane anion channels. The second response is blue-light-specific and so far has been restricted to the activation of plasma membrane H(+)-ATPases. In a search for interactions of both signalling pathways, guard cells of Vicia faba and Arabidopsis thaliana were studied in intact plants. Vicia faba guard cells recorded in CO(2)-free air responded to blue light with a transient outward plasma membrane current that had an average peak value of 17 pA. In line with previous reports, changes in the current-voltage relation of the plasma membrane indicate that this outward current is based on the activation of H(+)-ATPases. However, when V. faba guard cells were blue-light-stimulated in air with 700 microl l(-1) CO(2), the outward current increased to 56 pA. The increase in current was linked to inhibition of S-type anion channels. Blue light also inhibited plasma membrane anion channels in A. thaliana guard cells, but not in the phot1 phot2 double mutant. These results show that blue light inhibits plasma membrane anion channels through a pathway involving phototropins, in addition to the stimulation of guard cell plasma membrane H(+)-ATPases.

Osmo-sensitive and stretch-activated calcium-permeable channels in Vicia faba guard cells are regulated by actin dynamics.

In responses to a number of environmental stimuli, changes of cytoplasmic [Ca(2+)](cyt) in stomatal guard cells play important roles in regulation of stomatal movements. In this study, the osmo-sensitive and stretch-activated (SA) Ca(2+) channels in the plasma membrane of Vicia faba guard cells are identified, and their regulation by osmotic changes and actin dynamics are characterized. The identified Ca(2+) channels were activated under hypotonic conditions at both whole-cell and single-channel levels. The channels were also activated by a stretch force directly applied to the membrane patches. The channel-mediated inward currents observed under hypotonic conditions or in the presence of a stretch force were blocked by the Ca(2+) channel inhibitor Gd(3+). Disruption of actin filaments activated SA Ca(2+) channels, whereas stabilization of actin filaments blocked the channel activation induced by stretch or hypotonic treatment, indicating that actin dynamics may mediate the stretch activation of these channels. In addition, [Ca(2+)](cyt) imaging demonstrated that both the hypotonic treatment and disruption of actin filaments induced significant Ca(2+) elevation in guard cell protoplasts, which is consistent with our electrophysiological results. It is concluded that stomatal guard cells may utilize SA Ca(2+) channels as osmo sensors, by which swelling of guard cells causes elevation of [Ca(2+)](cyt) and consequently inhibits overswelling of guard cells. This SA Ca(2+) channel-mediated negative feedback mechanism may coordinate with previously hypothesized positive feedback mechanisms and regulate stomatal movement in response to environmental changes.

The ATP Binding Cassette Transporter AtMRP5 Modulates Anion and Calcium Channel Activities in Arabidopsis Guard Cells

Stomatal guard cells control CO(2) uptake and water loss between plants and the atmosphere. Stomatal closure in response to the drought stress hormone, abscisic acid (ABA), results from anion and K(+) release from guard cells. Previous studies have shown that cytosolic Ca(2+) elevation and ABA activate S-type anion channels in the plasma membrane of guard cells, leading to stomatal closure. However, membrane-bound regulators of abscisic acid signaling and guard cell anion channels remain unknown. Here we show that the ATP binding cassette (ABC) protein AtMRP5 is localized to the plasma membrane. Mutation in the AtMRP5 ABC protein impairs abscisic acid and cytosolic Ca(2+) activation of slow (S-type) anion channels in the plasma membrane of guard cells. Interestingly, atmrp5 insertion mutant guard cells also show impairment in abscisic acid activation of Ca(2+)-permeable channel currents in the plasma membrane of guard cells. These data provide evidence that the AtMRP5 ABC transporter is a central regulator of guard cell ion channel during abscisic acid and Ca(2+) signal transduction in guard cells.

Guard cells in albino leaf patches do not respond to photosynthetically active radiation, but are sensitive to blue light, CO2 and abscisic acid

Stomatal openings can be stimulated by light through two signalling pathways. The first pathway is blue light specific and involves phototropins, while the second pathway mediates a response to photosynthetically active radiation (PAR). This second pathway was studied with the use of albino Vicia faba plants and variegated leaves of Chlorophytum comosum. Treatment of V. faba with norflurazon (Nf) inhibits the synthesis of carotenoids and leads to albino leaves with guard cells that lack functional green chloroplasts. Guard cells in albino leaf patches of C. comosum, however, do contain photosynthetically active chloroplasts. Stomata in albino leaf patches of both plants did not respond to red light, although blue light could still induce stomatal opening. This shows that the response to PAR is not functioning in albino leaf patches, even though guard cells of C. comosum harbour chloroplasts. Stomata of Nf-treated plants still responded to CO2 and abscisic acid (ABA). The size of Nf-treated guard cells was increased, but impalement studies with double-barrelled microelectrodes revealed no changes in ion-transport properties at the plasma membrane of guard cells. Blue light could hyperpolarize albino guard cells by triggering outward currents with peak values of 37 pA in albino plants and 51 pA in green control cells. Because of the inhibition of carotenoid biosynthesis, Nf-treated V. faba plants contained only 4% of the ABA content found in green control plants. The ABA dose dependence of anion channel activation in guard cells was shifted in these plants, causing a reduced response to 10 microM ABA. These data show that despite the dramatic changes in physiology caused by Nf, the gross responsiveness of guard cells to blue light, CO2 and ABA remains unaltered. Stomata in albino leaf patches, however, do not respond to PAR, but require photosynthetically active mesophyll cells for this response.

H2O2 intensifies CN(-)-induced apoptosis in pea leaves.

H2O2 intensifies CN(-)-induced apoptosis in stoma guard cells and to lesser degree in basic epidermal cells in peels of the lower epidermis isolated from pea leaves. The maximum effect of H2O2 on guard cells was observed at 10(-4) M. By switching on non-cyclic electron transfer in chloroplasts menadione and methyl viologen intensified H2O2 generation in the light, but prevented the CN--induced apoptosis in guard cells. The light stimulation of CN- effect on guard cell apoptosis cannot be caused by disturbance of the ribulose-1,5-bisphosphate carboxylase function and associated OH* generation in chloroplasts with participation of free transition metals in the Fenton or Haber-Weiss type reactions as well as with participation of the FeS clusters of the electron acceptor side of Photosystem I. Menadione and methyl viologen did not suppress the CN(-)-induced apoptosis in epidermal cells that, unlike guard cells, contain mitochondria only, but not chloroplasts. Quinacrine and diphenylene iodonium, inhibitors of NAD(P)H oxidase of cell plasma membrane, had no effect on the respiration and photosynthetic O2 evolution by leaf slices, but prevented the CN(-)-induced guard cell death. The data suggest that NAD(P)H oxidase of guard cell plasma membrane is a source of reactive oxygen species (ROS) needed for execution of CN(-)-induced programmed cell death. Chloroplasts and mitochondria were inefficient as ROS sources in the programmed death of guard cells. When ROS generation is insufficient, exogenous H2O2 exhibits a stimulating effect on programmed cell death. H2O2 decreased the inhibitory effects of DCMU and DNP-INT on the CN(-)-induced apoptosis of guard cells. Quinacrine, DCMU, and DNP-INT had no effect on CN(-)-induced death of epidermal cells.

Protein Tyrosine Phosphatases Mediate the Signaling Pathway of Stomatal Closure of Vicia faba L.

Abstract: The regulation of stomatal movement is one of the most important signaling networks in plants. The H+‐ATPase at the plasma membrane of guard cells plays a critical role in the stomata opening, while there are some conflicting results regarding the effectiveness of the plasma membrane H+‐ATPase inhibitor, vanadate, in inhibiting stomata opening. We observed that 2 mmol/L vanadate hardly inhibited light‐stimulated stomata opening in epidermal peels of Vicia faba L., but significantly inhibited dark‐ and ABA‐induced stomatal closure. These results cannot be explained with the previous findings that H+‐ATPase was inhibited by vanadate. In view of the fact that vanadate is an inhibitor of protein tyrosine phosphatases (PTPases), we investigated whether the stomatal movement regulated by vanadate is through the regulation of PTPase. As expected, phenylarsine oxide (PAO), a specific inhibitor of PTPase, has very similar effects and even more effective than vanadate. Typical PTPase activity was found in guard cells of V. faba; moreover, the phosphatase activity could be inhibited by both vanadate and PAO. These results not only provide a novel explanation for conflicting results about vanadate modulating stomatal movement, but also provide further evidence for the involvement of PTPases in modulating signal transduction of stomatal movement.(Managing editor: Ping HE)

Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility.

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.

The poplar K+ channel KPT1 is associated with K+ uptake during stomatal opening and bud development.

To gain insights into the performance of poplar guard cells, we have measured stomatal conductance and aperture, guard cell K+ content and K+-channel activity of the guard cell plasma membrane in intact poplar leaves. In contrast to Arabidopsis, broad bean and tobacco grown under same conditions, poplar stomata operated just in the dynamic range - any change in conductance altered the rate of photosynthesis. In response to light, CO2 and abscisic acid (ABA), the stomatal opening velocity was two to five times faster than that measured for Arabidopsis thaliana, Nicotiana tabacum and Vicia faba. When stomata opened, the K+ content of guard cells increased almost twofold, indicating that the very fast stomatal opening in this species is mediated via potassium uptake. Following impalement of single guard cells embedded in their natural environment of intact leaves with triple-barrelled microelectrodes, time-dependent inward and outward-rectifying K+-channel-mediated currents of large amplitude were recorded. To analyse the molecular nature of genes encoding guard cell K+-uptake channels, we cloned K+-transporter Populustremula (KPT)1 and functionally expressed this potassium channel in a K+-uptake-deficient Escherichia coli mutant. In addition to guard cells, this K+-transporter gene was expressed in buds, where the KPT1 gene activity strongly correlated with bud break. Thus, KPT1 represents one of only few poplar genes associated with bud flush.

Sensing Extracellular Calcium

In plants, various physiological processes are regulated by extracellular calcium (Ca 2+ o ). For instance, Ca 2+ o triggers increases and oscillations in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) of guard cells and thereby promotes stomatal closure. However, the mechanisms whereby plants sense and respond to changes in Ca 2+ o remain unclear. Han et al. used a functional screening approach, in which Arabidopsis cDNA was expressed in human embryonic kidney (HEK 293) cells, and the intracellular response to changes in Ca 2+ o was monitored with the Ca 2+ indicator Fura-2, to clone a protein that mediated Ca 2+ o -induced [Ca 2+ ] i increase (CICI). The protein, which the authors named CAS, was predicted to have a single transmembrane domain with an extracellular N terminus that did not resemble any functional domains known. The authors used a combination of 45 Ca blotting of purified N and C termini, equilibrium dialysis, and time-course analysis of CAS activation and deactivation to determine that the N terminus showed low-affinity, high-capacity Ca 2+ binding. Northern analysis indicated that CAS was mostly localized in shoots, and experiments with gene reporters, as well as immunolocalization, indicated that it was localized in the plasma membrane of guard cells. Reducing CAS expression with antisense disrupted CICI signaling in guard cells and inhibited bolting (rapid upward growth at the initiation of seed production) in response to Ca 2+ deficiency. Thus, CAS appears to represent a novel Ca 2+ o sensor with physiological functions relevant to stomata closure and plant development. S. Han, R. Tang, L. K. Anderson, T. E. Woerner, Z.-M. Pei, A cell surface receptor mediates extracellular Ca 2+ sensing in guard cells. Nature 425 , 196-200 (2003). [Online Journal]

Nitric oxide regulates K+ and Cl- channels in guard cells through a subset of abscisic acid-evoked signaling pathways.

Abscisic acid (ABA) triggers a complex sequence of signaling events that lead to concerted modulation of ion channels at the plasma membrane of guard cells and solute efflux to drive stomatal closure in plant leaves. Recent work has indicated that nitric oxide (NO) and its synthesis are a prerequisite for ABA signal transduction in Arabidopsis and Vicia guard cells. Its mechanism(s) of action is not well defined in guard cells and, generally, in higher plants. Here we show directly that NO selectively regulates Ca2+-sensitive ion channels of Vicia guard cells by promoting Ca2+ release from intracellular stores to raise cytosolic-free [Ca2+]. NO-sensitive Ca2+ release was blocked by antagonists of guanylate cyclase and cyclic ADP ribose-dependent endomembrane Ca2+ channels, implying an action mediated via a cGMP-dependent cascade. NO did not recapitulate ABA-evoked control of plasma membrane Ca2+ channels and Ca2+-insensitive K+ channels, and NO scavengers failed to block the activation of these K+ channels evoked by ABA. These results place NO action firmly within one branch of the Ca2+-signaling pathways engaged by ABA and define the boundaries of parallel signaling events in the control of guard cell movements.

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells.

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

Modulation of guard cell plasma membrane potassium currents by methyl jasmonate.

Previous studies have suggested a role for jasmonates in the promotion of stomatal closure ([Raghavendra and Reddy, 1987][1]; [Gehring et al., 1997][2]). This report describes whole-cell patch-clamp experiments that demonstrate that methyl jasmonate (Me-JA) has concentration-dependent effects on

Biochemical evidence for the requirement of 14-3-3 protein binding in activation of the guard-cell plasma membrane H+-ATPase by blue light.

Blue light (BL) activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus with concomitant binding of 14-3-3 protein to the terminus in stomatal guard cells. However, the binding site and role of 14-3-3 protein in this physiological response have not been elucidated. We investigated the above using synthetic phosphopeptides designed from the C-terminus of Vicia H(+)-ATPase (isoform 1; VHA1). The presence of KGLDIDTIQQHYphospho-T(950)V peptide (P-950) prevented binding of 14-3-3 protein to the phosphorylated H(+)-ATPase. Dephosphorylated P-950 and other phosphopeptides, including typical phosphorylation sites in the C-terminus, had no effect on the binding. Incubation of BL-activated plasma membrane H(+)-ATPase with P-950 dissociated the 14-3-3 protein from the H(+)-ATPase without affecting phosphorylation levels and decreased the H(+)-ATPase activity. By contrast, incubation of P-950 with the activated H(+)-ATPase from fusicoccin-treated guard-cell protoplasts neither dissociated the 14-3-3 protein nor decreased the H(+)-ATPase activity. These results indicate that BL induces phosphorylation on threonine residue (Thr(950)) in the C-terminus of H(+)-ATPase, and that the binding of 14-3-3 to this site is required for the activation of H(+)-ATPase in stomatal guard cells.

Protein phosphorylation activates the guard cell Ca2+ channel and is a prerequisite for gating by abscisic acid.

Protein phosphorylation and cytosolic-free [Ca2+] ([Ca2+]i) contribute to signalling cascades evoked by the water-stress hormone abscisic acid (ABA) that lead to stomatal closure in higher-plant leaves. ABA activates an inward-rectifying Ca2+ channel at the plasma membrane of stomatal guard cells, promoting Ca2+ entry by shifting the voltage-sensitivity of the channels. Because many of these effects could be mediated by kinase/phosphatase action at the membrane, we examined a role for protein (de-)phosphorylation in plasma membrane patches from Vicia guard cells. Ca2+ channel activity decayed rapidly in excised patches, and recovered on adding ATP (K1/2, 1.3 +/- 0.7 mm) but not the non-hydrolyzable analog ATPgammaS. ABA activation of the channel required the presence of ATP and like ABA, the 1/2 A-type protein phosphatase antagonists okadaic acid (OA) and calyculin A (CA) enhanced Ca2+ channel activity by increasing the open probability and number of active channels. Neither ATP nor the antagonists affected the mean open lifetime of the channel, suggesting an action through changes in closed lifetime distributions. Like ABA, OA and CA shifted the voltage-sensitivities of the Ca2+ current and [Ca2+]i increases in intact guard cells towards positive voltages. OA and CA also augmented the [Ca2+]i rise evoked by hyperpolarization and delayed its recovery. These results demonstrate a membrane-delimited interaction between 1/2 A-type protein phosphatase(s) and the Ca2+ channel or associated proteins, and they are consistent with a role for protein (de-)phosphorylation in ABA signalling mediated directly through Ca2+ channel gating that leads to [Ca2+]i increases in the guard cells.

Requirements for activation of the signal-transduction network that leads to regulatory phosphorylation of leaf guard-cell phospho enolpyruvate carboxylase during fusicoccin-stimulated stomatal opening

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K + or other osmolytes. K + accumulation is stoichiometric with H + extrusion, which is compensated for by phospho enolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [ 32P]orthophosphate ( 32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H +-ATPase. [ 32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In −FC controls, stomatal size, guard-cell malate, and [ 32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by ∼80% when K + uptake was associated with Cl − uptake and was essentially abolished when stomatal opening was sucrose—rather than K +—dependent. Finally, alkalinization by NH 4 + in the presence of K + did not cause PEPC phosphorylation (as it does in C 4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H +-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H +-ATPase influence the phosphorylation state of this PEPC isoform.

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells.

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.

Identification of K(+) channels in the plasma membrane of maize subsidiary cells.

The stomatal complex of Zea mays consists of two guard cells with the pore in between them and two flanking subsidiary cells. Both guard cells and subsidiary cells are important elements for stoma physiology because a well-coordinated transmembrane shuttle transport of potassium and chloride ions occurs between these cells during stomatal movement. To shed light upon the corresponding transport systems from subsidiary cells, subsidiary cell protoplasts were enzymatically isolated and in turn, analyzed with the patch-clamp technique. Thereby, two K(+)-selective channel types were identified in the plasma membrane of subsidiary cells. With regard to their voltage-dependent gating behavior, they may act as hyperpolarization-dependent K(+) uptake and depolarization-activated K(+) release channels during stomatal movement. Interestingly, the K(+) channels from subsidiary cells and guard cells similarly responded to membrane voltage as well as to changes in the K(+) gradient. Further, the inward- and outward-rectifying K(+) current amplitude decreased upon a rise in the intracellular free Ca(2+) level from 2 nM to the micro M-range. The results indicate that the plasma membrane of subsidiary cells and guard cells has to be inversely polarized in order to achieve the anti-parallel direction of K(+) fluxes between these cell types during stomatal movement.

Transporte de iones en vacuolas de plantas superiores en el contexto celular

La vacuola ocupa hasta 95% del volumen celular en las plantas superiores, es un reservorio de solutos y un organelo clave en el control de la concentración de Ca2+ y del pH intracelulares. El papel de la vacuola en la homeostasis iónica celular es también evidente en condiciones ambientales de falta de potasio y estrés salino. La vacuola central está involucrada en la regulación de la turgencia y el volumen celular en células especializadas como las estomáticas, constituyendo el principal componente del motor osmótico celular. Desde hace más de una década, la célula estomática ha ganado un status de modelo celular en plantas superiores, siendo extensamente estudiada mediante técnicas fisiológicas y de biología molecular y celular. A pesar del gran avance en el conocimiento de la transducción de señales en la membrana plasmática, el entendimiento de los mecanismos implicados en el transporte iónico en la membrana vacuolar continúa siendo un gran reto. Al igual que en la membrana plasmática, el transporte iónico en la membrana vacuolar o del tonoplasto, está dirigido y coordinado a través de un complejo sistema integrado por proteínas de transporte que incluye bombas (H+-ATPasa, pirofosfatasa, bomba de Ca2+), transportadores (antiportadores Na+/H+ y Ca2+/H+) y varios canales iónicos. En años recientes, con la aplicación de las técnicas modernas de electrofisiología (patch clamp), numerosos canales iónicos han sido caracterizados funcionalmente, aunque ninguno de ellos ha sido estructuralmente identificado. El trabajo de nuestro grupo se ha enfocado a la caracterización de los canales iónicos vacuolares lentos (SV) y los canales iónicos vacuolares rápidos (FV); estos canales tienen una amplia, si no es que ubicua, distribución en plantas superiores, se les encuentra en células troncales, en células diferenciadas de las hojas o de los tejidos de almacenamiento de las raíces. En este trabajo se discuten las características funcionales de estos canales en cuanto a selectividad iónica, mecanismos de compuerta dependientes de voltaje e influencia de cationes de importancia fisiológica (calcio, magnesio, protones, poliaminas), así como su posible papel fisiológico. Asimismo, se plantean los principales temas que están por resolverse. Particularmente, se analiza la participación de los canales aniónicos en la función de la vacuola como motor osmótico. Se sabe que el tonoplasto presenta una gran permeabilidad para varios aniones, sin embargo, los canales iónicos involucrados no han sido claramente identificados. El otro problema es la identificación de los canales activados por ligandos intracelulares. A pesar de que las células vegetales poseen receptores intracelulares para inositol 1,4,5 trifosfato (IP3) y adenosinadifosfatoribosa cíclica (cADPR), su ubicación celular no está definida. Por el momento no son confiables las evidencias que indican la existencia de canales iónicos vacuolares activados por estos factores.

K+ channels inhibited by hydrogen peroxide mediate abscisic acid signaling in Vicia guard cells.

A number of studies show that environmental stress conditions increase abscisic acid (ABA) and hydrogen peroxide (H2O2) levels in plant cells. Despite this central role of ABA in altering stomatal aperture by regulating guard cell ion transport, little is known concerning the relationship between ABA and H2O2 in signal transduction leading to stomatal movement. Epidermal strip bioassay illustrated that ABA-inhibited stomatal opening and ABA-induced stomatal closure were abolished partly by externally added catalase (CAT) or diphenylene iodonium (DPI), which are a H2O2 scavenger and a NADPH oxidase inhibitor respectively. In contrast, internally added CAT or DPI nearly completely or partly reversed ABA-induced closure in half-stoma. Consistent with these results, whole-cell patch-clamp analysis showed that intracellular application of CAT or DPI partly abolished ABA-inhibited inward K+ current across the plasma membrane of guard cells. H2O2 mimicked ABA to inhibit inward K+ current, an effect which was reversed by the addition of ascorbic acid (Vc) in patch clamping micropipettes. These results suggested that H2O2 mediated ABA-induced stomatal movement by targeting inward K+ channels at plasma membrane.