The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier

Malate plays a central role in plant metabolism. It is an intermediate in the Krebs and glyoxylate cycles, it is the store for CO2 in C4 and crassulacean acid metabolism plants, it protects plants from aluminum toxicity, it is essential for maintaining the osmotic pressure and charge balance, and it is therefore involved in regulation of stomatal aperture. To fulfil many of these roles, malate has to be accumulated within the large central vacuole. Many unsuccessful efforts have been made in the past to identify the vacuolar malate transporter; here, we describe the identification of the vacuolar malate transporter [A. thaliana tonoplast dicarboxylate transporter (AttDT)]. This transporter exhibits highest sequence similarity to the human sodium/dicarboxylate cotransporter. Independent T-DNA [portion of the Ti (tumor-inducing) plasmid that is transferred to plant cells] Arabidopsis mutants exhibit substantially reduced levels of leaf malate, but respire exogenously applied [14C]malate faster than the WT. An AttDT-GFP fusion protein was localized to vacuole. Vacuoles isolated from Arabidopsis WT leaves exhibited carbonylcyanide m-chlorophenylhydrazone and citrate inhibitable malate transport, which was not stimulated by sodium. Vacuoles isolated from mutant plants import [14C]-malate at strongly reduced rates, confirming that this protein is the vacuolar malate transporter.     

Divergent functions of VTI12 and VTI11 in trafficking to storage and lytic vacuoles in Arabidopsis

The protein storage vacuole (PSV) is a plant-specific organelle that accumulates reserve proteins, one of the main agricultural products obtained from crops. Despite the importance of this process, the cellular machinery required for transport and accumulation of storage proteins remains largely unknown. Interfering with transport to PSVs has been shown to result in secretion of cargo. Therefore, secretion of a suitable marker could be used as an assay to identify mutants in this pathway. CLV3, a negative regulator of shoot stem cell proliferation, is an extracellular ligand that is rendered inactive when targeted to vacuoles. We devised an assay where trafficking mutants secrete engineered vacuolar CLV3 and show reduced meristems, a phenotype easily detected by visual inspection of plants. We tested this scheme in plants expressing VAC2, a fusion of CLV3 to the vacuolar sorting signal from the storage protein barley lectin. In this way, we determined that trafficking of VAC2 requires the SNARE VTI12 but not its close homologue, the conditionally redundant VTI11 protein. Furthermore, a vti12 mutant is specifically altered in transport of storage proteins, whereas a vti11 mutant is affected in transport of a lytic vacuole marker. These results demonstrate the specialization of VTI12 and VTI11 in mediating trafficking to storage and lytic vacuoles, respectively. Moreover, they validate the VAC2 secretion assay as a simple method to isolate genes that mediate trafficking to the PSV.     

Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation

The productivity of higher plants as a major source of food and energy is linked to their ability to buffer changes in the concentrations of essential and toxic ions. Transport across the tonoplast is energized by two proton pumps, the vacuolar H⁺-ATPase (V-ATPase) and the vacuolar H⁺-pyrophosphatase (V-PPase); however, their functional relation and relative contributions to ion storage and detoxification are unclear. We have identified an Arabidopsis mutant in which energization of vacuolar transport solely relies on the activity of the V-PPase. The vha-a2 vha-a3 double mutant, which lacks the two tonoplast-localized isoforms of the membrane-integral V-ATPase subunit VHA-a, is viable but shows day-length-dependent growth retardation. Nitrate content is reduced whereas nitrate assimilation is increased in the vha-a2 vha-a3 mutant, indicating that vacuolar nitrate storage represents a major growth-limiting factor. Zinc is an essential micronutrient that is toxic at excess concentrations and is detoxified via a vacuolar Zn²⁺/H⁺-antiport system. Accordingly, the double mutant shows reduced zinc tolerance. In the same way the vacuolar Na⁺/H⁺-antiport system is assumed to be an important component of the system that removes sodium from the cytosol. Unexpectedly, salt tolerance and accumulation are not affected in the vha-a2 vha-a3 double mutant. In contrast, reduction of V-ATPase activity in the trans-Golgi network/early endosome (TGN/EE) leads to increased salt sensitivity. Taken together, our results show that during gametophyte and embryo development V-PPase activity at the tonoplast is sufficient whereas tonoplast V-ATPase activity is limiting for nutrient storage but not for sodium tolerance during vegetative and reproductive growth.     

An Arabidopsis syntaxin homologue isolated by functional complementation of a yeast pep12 mutant.

The syntaxin family of integral membrane proteins are thought to function as receptors for transport vesicles, with different isoforms of this family localized to various membranes throughout the cell. The yeast Pep12 protein is a syntaxin homologue which may function in the trafficking of vesicles from the trans-Golgi network to the vacuole. We have isolated an Arabidopsis thaliana cDNA by functional complementation of a yeast pep12 mutant. The Arabidopsis cDNA (aPEP12) potentially encodes a 31-kDa protein which is homologous to yeast Pep12 and to other members of the syntaxin family, indicating that this protein may function in the docking or fusion of transport vesicles with the vacuolar membrane in plant cells. Northern blot analysis indicates that the mRNA is expressed in all tissues examined, although at a very low level in leaves. The mRNA is found in all cell types in roots and leaves, as shown by in situ hybridization experiments. The existence of plant homologues of proteins of the syntaxin family indicates that the basic vesicle docking and fusion machinery may be conserved in plants as it is in yeast and mammals.     

The role of phospholipase D in Glut-4 translocation

Insulin-stimulated Glut-4 translocation is regulated through a complex pathway. Increasing attention is being paid to the role undertaken in this process by Phospholipase D, a signal transduction-activated enzyme that generates the lipid second-messenger phosphatidic acid. Phospholipase D facilitates Glut-4 translocation at potentially multiple steps in its outward movement. Current investigation is centered on Phospholipase D promotion of Glut-4-containing membrane vesicle trafficking and vesicle fusion into the plasma membrane, in part through activation of atypical protein kinase C isoforms.     

From the Cover: Feature Article: Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.     

Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting

All eukaryotic cells present at the cell surface a specific set of plasma membrane proteins that modulate responses to internal and external cues and whose activity is also regulated by protein degradation. We characterized the lytic vacuole-dependent degradation of membrane proteins in Arabidopsis thaliana by means of in vivo visualization of vacuolar targeting combined with quantitative protein analysis. We show that the vacuolar targeting pathway is used by multiple cargos including PIN-FORMED (PIN) efflux carriers for the phytohormone auxin. In vivo visualization of PIN2 vacuolar targeting revealed its differential degradation in response to environmental signals, such as gravity. In contrast to polar PIN delivery to the basal plasma membrane, which depends on the vesicle trafficking regulator ARF-GEF GNOM, PIN sorting to the lytic vacuolar pathway requires additional brefeldin A-sensitive ARF-GEF activity. Furthermore, we identified putative retromer components SORTING NEXIN1 (SNX1) and VACUOLAR PROTEIN SORTING29 (VPS29) as important factors in this pathway and propose that the retromer complex acts to retrieve PIN proteins from a late/pre-vacuolar compartment back to the recycling pathways. Our data suggest that ARF GEF- and retromer-dependent processes regulate PIN sorting to the vacuole in an antagonistic manner and illustrate instrumentalization of this mechanism for fine-tuning the auxin fluxes during gravitropic response.     

The two-pore channel TPK1 gene encodes the vacuolar K+ conductance and plays a role in K+ homeostasis

The Arabidopsis thaliana genome contains five genes that encode two pore K+ (TPK) channels. The most abundantly expressed isoform of this family, TPK1, is expressed at the tonoplast where it mediates K+ -selective currents between cytoplasmic and vacuolar compartments. TPK1 open probability depends on both cytoplasmic Ca2+ and cytoplasmic pH but not on the tonoplast membrane voltage. The channel shows intrinsic rectification and can be blocked by Ba2+, tetraethylammonium, and quinine. TPK1 current was found in all shoot cell types and shows all of the hallmarks of the previously described vacuolar K (VK) tonoplast channel characterized in guard cells. Characterization of TPK1 loss-of-function mutants and TPK1-overexpressing plants shows that TPK1 has a role in intracellular K+ homeostasis affecting seedling growth at high and low ambient K+ levels. In stomata, TPK1 function is consistent with vacuolar K+ release, and removal of this channel leads to slower stomatal closure kinetics. During germination, TPK1 contributes to the radicle development through vacuolar K+ deposition to provide expansion growth or in the redistribution of essential minerals.     

Sorting inhibitors (Sortins): Chemical compounds to study vacuolar sorting in Arabidopsis

Chemical genomics is an interdisciplinary approach that unites the power of chemical screens and genomics strategies to dissect biological processes such as endomembrane trafficking. We have taken advantage of the evolutionary conservation between plants and Saccharomyces cerevisiae to identify such chemicals. Using S. cerevisiae, we screened a library of diverse chemical structures for compounds that induce the secretion of carboxypeptidase Y, which is normally targeted to the vacuole. Among 4,800 chemicals screened, 14 compounds, termed sorting inhibitors (Sortins), were identified that stimulated secretion in yeast. In Arabidopsis seedlings, application of Sortin1 and -2 led to reversible defects in vacuole biogenesis and root development. Sortin1 was found to redirect the vacuolar destination of plant carboxypeptidase Y and other proteins in Arabidopsis suspension cells and cause these proteins to be secreted. Sortin1 treatment of whole Arabidopsis seedlings also resulted in carboxypeptidase Y secretion, indicating that the drug has a similar mode of action in cells and intact plants. We have demonstrated that screening of a simple eukaryote, in which vacuolar biogenesis is not essential, can be a powerful tool to find chemicals that interfere with vacuolar delivery of proteins in plants, where vacuole biogenesis is essential. Our studies were done by using a sublethal dose of Sortin1, demonstrating the powerful ability of the chemical to control the induced phenotype in a manner that would be difficult to achieve using conventional genetics.     

Vacuolar Na+/H+ antiporter cation selectivity is regulated by calmodulin from within the vacuole in a Ca2+- and pH-dependent manner

The selective movement of ions between intracellular compartments is fundamental for eukaryotes. Arabidopsis thaliana Na(+)/H(+) exchanger 1 (AtNHX1), the most abundant vacuolar Na(+)/H(+) antiporter in A. thaliana, has important roles affecting the maintenance of cellular pH, ion homeostasis, and the regulation of protein trafficking. Previously, we have shown that the AtNHX1 C-terminal hydrophilic region localized in the vacuolar lumen plays an important role in regulating the antiporter's activity. Here, we have identified A. thaliana calmodulin-like protein 15 (AtCaM15), which interacts with the AtNHX1 C terminus. When expressed in yeast, AtCaM15 is localized in the vacuolar lumen. The transient expression of AtCaM15 in Arabidopsis leaf protoplasts showed that AtCaM15 is present in the central vacuole. The binding of AtCaM15 to AtNHX1 was Ca(2+)- and pH-dependent and decreased with increasing pH values. Our results also show that the binding of AtCaM15 to AtNHX1 modified the Na(+)/K(+) selectivity of the antiporter, decreasing its Na(+)/H(+) exchange activity. Taken together, the presence of a vacuolar calmodulin-like protein acting on the vacuolar-localized AtNHX1 C terminus in a Ca(2+)- pH-dependent manner suggests the presence of signaling entities acting within the vacuole.     

A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking

The homotypic fusion of yeast vacuoles occurs in an ordered cascade of priming, docking, and fusion. The linkage between these steps has so far remained unclear. We now report that Vam7p (the vacuolar SNAP-23/25 homolog) signals from the cis-SNARE complex to Ypt7p (the vacuolar Rab/Ypt) to initiate the docking process. After Vam7p has been released from the cis-SNARE complex by Sec18p-mediated priming, it is still required for Ypt7p-dependent docking and it needs Ypt7p to remain on the vacuole. Thus, after priming, Vam7p is released from the vacuole altogether if Ypt7p has been extracted by Gdi1p or inactivated by antibody but is not released if docking is blocked simply by vacuole dilution; it is therefore Ypt7p function, and not docking per se, that retains Vam7p. In accord with this finding, cells deleted for the gene encoding Ypt7 have normal amounts of Vam7p but have little Vam7p on their isolated vacuoles. Interaction of Vam7p and Ypt7p is further indicated by two-hybrid analysis [Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000) Nature (London) 403, 623-627] and by the effect of Vam7p on the association of the Rab/Ypt-effector HOPS complex (homotypic fusion and vacuole protein sorting; Vam2p and Vam6p plus four vacuole protein sorting class C proteins) with Ypt7p. Vam7p provides a functional link between the priming step, which releases it from the cis-SNARE complex, and docking.     

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.     

SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana

Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Additionally, transgenic plants with CaMV35S promoter::SRK2C-GFP displayed higher overall drought tolerance than control plants. Whereas stomatal regulation in 35S::SRK2C-GFP plants was not altered, microarray analysis revealed that their drought tolerance coincided with up-regulation of many stress-responsive genes, for example, RD29A, COR15A, and DREB1A/CBF3. From these results, we concluded that SRK2C is capable of mediating signals initiated during drought stress, resulting in appropriate gene expression. Our present study reveals new insights around signal output from osmotic-stress-activated SnRK2 protein kinase as well as supporting feasibility of manipulating SnRK2 toward improving plant osmotic-stress tolerance.     

Minireview: aquaporin 2 trafficking

In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.     

Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion

Vacuole fusion occurs in three stages: priming, in which Sec18p mediates Sec17p release, LMA1 (low M(r) activity 1) relocation, and cis-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex disassembly; docking, mediated by Ypt7p and trans-SNARE association; and fusion of docked vacuoles. Ca(2+) and calmodulin regulate late stages of the reaction. We now show that the vacuole proton gradient, generated by the vacuolar proton ATPase, is needed for trans-SNARE complex formation during docking and hence for the subsequent LMA1 release. Though neither the vacuolar Pmc1p Ca(2+)-ATPase nor the Vcx1p Ca(2+)/H(+) exchanger are needed for the fusion reaction, they participate in Ca(2+) and Delta mu(H)(+) homeostasis. Fusion itself does not require the maintenance of trans-SNARE pairs.     

Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake

Stomatal movements rely on alterations in guard cell turgor. This requires massive K⁺ bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K⁺ into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K⁺/H⁺ exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K⁺ and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K⁺ accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K⁺ is a requirement for stomatal opening and a critical component in the overall K⁺ homeostasis essential for stomatal closure, and suggest that vacuolar K⁺ fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.The rapid accumulation and release of K⁺ and of organic and inorganic anions by guard cells controls the opening and closing of stomata and thereby gas exchange and transpiration of plants. The intracellular events that underlie stomatal opening start with plasma membrane hyperpolarization caused by the activation of H⁺-ATPases, which induces K⁺ uptake through voltage-gated inwardly rectifying K⁺_in channels (1). Potassium uptake is accompanied by the electrophoretic entry of the counterions chloride, nitrate, and sulfate, and by the synthesis of malate. These osmolytes, together with sucrose accumulation, increase the turgor in guard cells and thereby drive stomatal opening. Stomatal closure is initiated by activation of the plasma membrane localized chloride and nitrate efflux channels SLAC1 and SLAH3 that are regulated by the SnRK2 protein kinase OST1 and the Ca²⁺-dependent protein kinases CPK21 and 23 (2, 3). CPK6 also activates SLAC1 and coordinately inhibits rectifying K⁺_in channels to hinder stomatal opening (4, 5). Sulfate and organic acids exit the guard cell through R-type anion channels. The accompanying reduction in guard cell turgor results in stomatal closure (1).Despite the established role of plasma membrane transport in guard cell function and stomatal movement, ion influx into the cytosol represents only a transit step to the vacuole, as more than 90% of the solutes released from guard cells originate from vacuoles (6). In contrast to the plasma membrane, knowledge of the transport processes occurring in intracellular compartments of guard cells during stomatal movements is less advanced (7). Only recently, AtALMT9 has been shown to act as a malate-induced chloride channel at the tonoplast that is required for stomatal opening (8). Vacuoles govern turgor-driven changes in guard cell volumes by increases and decreases in vacuolar volume during stomatal opening and closure, respectively, by more than 40% (9, 10). Monitoring the dynamic changes in guard cell vacuolar structures revealed an intense remodeling during stomatal movements (11, 12). Pharmacological and genetic approaches indicated that dynamic changes of the vacuole are crucial for achieving the full amplitude of stomatal movement (12–14). However, so far, no specific tonoplast transport proteins or processes have been functionally linked to vacuolar dynamics during guard cell movements.Cation channel activities mediating K⁺ release and stomatal closure have been characterized at the tonoplast, including fast vacuolar, slow vacuolar, and K⁺-selective vacuolar cation channels (7, 15). Genetic inactivation of K⁺-release channels leads to slower stomatal closure kinetics (7, 16). By contrast, the transporters responsible for the uptake of K⁺ into vacuoles against the vacuolar membrane potential that drive the stomatal aperture have remained unknown. We have recently reported that the tonoplast-localized K⁺,Na⁺/H⁺ exchangers NHX1 and NHX2 from Arabidopsis are involved in the accumulation of K⁺ into the vacuole of plant cells, thereby increasing their osmotic potential and driving the uptake of water that generates the turgor pressure necessary for cell expansion and growth (17). The involvement of K⁺,Na⁺/H⁺ exchangers in the regulation of plant transpiration was also proposed, as the nhx1 nhx2 mutant exhibited enhanced transpirational water loss compared with WT when subjected to osmotic stress. Here, to resolve whether active K⁺ uptake at the tonoplast directly regulates stomatal activity by mediating K⁺ accumulation in the vacuole of guard cells, we analyzed the stomatal movements of nhx1 nhx2 double mutant lines by using a range of physiological, molecular, and imaging-based approaches. Moreover, we have developed a noninvasive, fluorescence ratiometric method to measure vacuolar pH (pHv) in guard cells by using the H⁺-sensitive and cell-permeant dye Oregon green and epidermal peels. Our data establish that (i) the capacity for K⁺ accumulation into guard cell vacuoles is essential for stomatal activity by facilitating not only stomatal aperture but also closure, (ii) K⁺/H⁺ exchange at the guard cell tonoplast mediates the luminal pHv shifts associated to stomata opening, and (iii) the dynamic morphological changes that guard cell vacuoles undergo during stomatal movements are brought about by the uptake of K⁺ into the vacuole.     

The plasma membrane Na+/H+ antiporter SOS1 interacts with RCD1 and functions in oxidative stress tolerance in Arabidopsis

The adverse effects of high salt on plants include Na(+) toxicity and hyperosmotic and oxidative stresses. The plasma membrane-localized Na(+)/H(+) antiporter SOS1 functions in the extrusion of toxic Na(+) from cells and is essential for plant salt tolerance. We report here that, under salt or oxidative stress, SOS1 interacts through its predicted cytoplasmic tail with RCD1, a regulator of oxidative-stress responses. Without stress treatment, RCD1 is localized in the nucleus. Under high salt or oxidative stress, RCD1 is found not only in the nucleus but also in the cytoplasm. Like rcd1 mutants, sos1 mutant plants show an altered sensitivity to oxidative stresses. The rcd1mutation causes a decrease in salt tolerance and enhances the salt-stress sensitivity of sos1 mutant plants. Several genes related to oxidative-stress tolerance were found to be regulated by both RCD1 and SOS1. These results reveal a previously uncharacterized function of a plasma membrane Na(+)/H(+) antiporter in oxidative-stress tolerance and shed light on the cross-talk between the ion-homeostasis and oxidative-stress detoxification pathways involved in plant salt tolerance.