José M. Pardo

Ion Homeostasis in NaCl Stress Environments

Homeostasis can be defined as the tendency of a cell or an organism to maintain internal steady state, even in response to any environmental perturbation or stimulus tending to disturb normality, because of the coordinate responses of its constituent components. Typically, ions constantly flux in and out of cells in a controlled fashion with net flux adjusted to accommodate cellular requirements, thus creating an ionic homeostasis. When plant cells are exposed to salinity, mediated by high NaCl concentrations, kinetic steady states of ion transport for Na+ and Cland other ions, such as K+ and Ca2+, are disturbed (Binzel et al., 1988). High apoplastic levels of Na+ and Clalter aqueous and ionic thermodynamic equilibria, resulting in hyperosmotic stress, ionic imbalance, and toxicity. Thus, it is vital for the plant to re-establish cellular ion homeostasis for metabolic functioning and growth, that is, to adapt to the saline environment. Comparisons of what have been interpreted to be adaptive responses among various species lead to the conclusion that some salt-tolerant plants have evolved specialized complex mechanisms that allow adaptation to saline stress conditions. In fact, these unique mechanisms, such as salt glands, exist in few plant species and cannot be presumed to be ubiquitously functional for salt adaptation of all plants. However, intrinsically cellular-based mechanisms appear to be common to all genotypes and are a requisite for salt tolerance. Of paramount importance are those mechanisms that function to regulate ion homeostasis while mediating osmotic adjustment through the accumulation and intracellular compartmentation of ions that are predominant in the external environment. In this update we will focus principally on Na+ homeostasis in sodic environments; however, we also include discussions of H+, K+, Ca2+, and Clbecause of the interrelationship of these ions with Na+ homeostasis. Ion transport processes across the plasma membrane and the tonoplast will be emphasized because these are presumed to be most essential for the control of intracellular Na+ uptake and vacuolar compartmentation.

The Putative Plasma Membrane Na+/H+ Antiporter SOS1 Controls Long-Distance Na+ Transport in Plants

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na+/H+ antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na+ transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na+ transporters, SOS1 was able to reduce Na+ accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1–green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter–β-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na+ than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na+ than did the wild type. There also was greater Na+ content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na+ transport from root to shoot. We present a model in which SOS1 functions in retrieving Na+ from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na+ into the xylem.

Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis

The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

Conservation of the Salt Overly Sensitive Pathway in Rice

The salt tolerance of rice (Oryza sativa) correlates with the ability to exclude Na+ from the shoot and to maintain a low cellular Na+/K+ ratio. We have identified a rice plasma membrane Na+/H+ exchanger that, on the basis of genetic and biochemical criteria, is the functional homolog of the Arabidopsis (Arabidopsis thaliana) salt overly sensitive 1 (SOS1) protein. The rice transporter, denoted by OsSOS1, demonstrated a capacity for Na+/H+ exchange in plasma membrane vesicles of yeast (Saccharomyces cerevisiae) cells and reduced their net cellular Na+ content. The Arabidopsis protein kinase complex SOS2/SOS3, which positively controls the activity of AtSOS1, phosphorylated OsSOS1 and stimulated its activity in vivo and in vitro. Moreover, OsSOS1 suppressed the salt sensitivity of a sos1-1 mutant of Arabidopsis. These results represent the first molecular and biochemical characterization of a Na+ efflux protein from monocots. Putative rice homologs of the Arabidopsis protein kinase SOS2 and its Ca2+-dependent activator SOS3 were identified also. OsCIPK24 and OsCBL4 acted coordinately to activate OsSOS1 in yeast cells and they could be exchanged with their Arabidopsis counterpart to form heterologous protein kinase modules that activated both OsSOS1 and AtSOS1 and suppressed the salt sensitivity of sos2 and sos3 mutants of Arabidopsis. These results demonstrate that the SOS salt tolerance pathway operates in cereals and evidences a high degree of structural conservation among the SOS proteins from dicots and monocots.

SCABP8/CBL10, a Putative Calcium Sensor, Interacts with the Protein Kinase SOS2 to Protect Arabidopsis Shoots from Salt Stress

The SOS (for Salt Overly Sensitive) pathway plays essential roles in conferring salt tolerance in Arabidopsis thaliana. Under salt stress, the calcium sensor SOS3 activates the kinase SOS2 that positively regulates SOS1, a plasma membrane sodium/proton antiporter. We show that SOS3 acts primarily in roots under salt stress. By contrast, the SOS3 homolog SOS3-LIKE CALCIUM BINDING PROTEIN8 (SCABP8)/CALCINEURIN B-LIKE10 functions mainly in the shoot response to salt toxicity. While root growth is reduced in sos3 mutants in the presence of NaCl, the salt sensitivity of scabp8 is more prominent in shoot tissues. SCABP8 is further shown to bind calcium, interact with SOS2 both in vitro and in vivo, recruit SOS2 to the plasma membrane, enhance SOS2 activity in a calcium-dependent manner, and activate SOS1 in yeast. In addition, sos3 scabp8 and sos2 scabp8 display a phenotype similar to sos2, which is more sensitive to salt than either sos3 or scabp8 alone. Overexpression of SCABP8 in sos3 partially rescues the sos3 salt-sensitive phenotype. However, overexpression of SOS3 fails to complement scabp8. These results suggest that SCABP8 and SOS3 are only partially redundant in their function, and each plays additional and unique roles in the plant salt stress response.

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis

Intracellular Na+,K+/H+ antiporters (NHXs) play central roles in maintaining ion homeostasis and pH control. Tonoplast-localized proteins NHX1 and NHX2 are critical for active K+ uptake into the vacuole, a process that is required to create osmotic potential for cell expansion and turgor regulation. These proteins are abundantly expressed in guard cells, where they contribute to stomata function. Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function.

Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway

For plants growing in highly saline environments, accumulation of sodium in the cell cytoplasm leads to disruption of metabolic processes and reduced growth. Maintaining low levels of cytoplasmic sodium requires the coordinate regulation of transport proteins on numerous cellular membranes. Our previous studies have linked components of the Salt-Overly-Sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana and demonstrated that the activity of the plasma membrane Na+/H+ exchanger (SOS1) is regulated by SOS2 (a protein kinase) and SOS3 (a calcium-binding protein). Current studies were undertaken to determine if the Na+/H+ exchanger in the vacuolar membrane (tonoplast) of Arabidopsis is also a target for the SOS regulatory pathway. Characterization of tonoplast Na+/H+ exchange demonstrated that it represents activity originating from the AtNHX proteins since it could be inhibited by 5-(N-methyl-N-isobutyl)amiloride and by anti-NHX1 antibodies. Transport activity was selective for sodium (apparent Km = 31 mm) and electroneutral (one sodium ion for each proton). When compared with tonoplast Na+/H+-exchange activity in wild type, activity was significantly higher, greatly reduced, and unchanged in sos1, sos2, and sos3, respectively. Activated SOS2 protein added in vitro increased tonoplast Na+/H+-exchange activity in vesicles isolated from sos2 but did not have any effect on activity in vesicles isolated from wild type, sos1, or sos3. These results demonstrate that (i) the tonoplast Na+/H+ exchanger in Arabidopsis is a target of the SOS regulatory pathway, (ii) there are branches to the SOS pathway, and (iii) there may be coordinate regulation of the exchangers in the tonoplast and plasma membrane.

The plasma membrane Na+/H+ antiporter SOS1 is essential for salt tolerance in tomato and affects the partitioning of Na+ between plant organs

We have identified a plasma membrane Na(+)/H(+) antiporter gene from tomato (Solanum lycopersicum), SlSOS1, and used heterologous expression in yeast to confirm that SlSOS1 was the functional homolog of AtSOS1. Using post-transcriptional gene silencing, we evaluated the role played by SlSOS1 in long-distance Na(+) transport and salt tolerance of tomato. Tomato was used because of its anatomical structure, more complex than that of Arabidopsis, and its agricultural significance. Transgenic tomato plants with reduced expression of SlSOS1 exhibited reduced growth rate compared to wild-type (WT) plants in saline conditions. This sensitivity correlated with higher accumulation of Na(+) in leaves and roots, but lower contents in stems of silenced plants under salt stress. Differential distribution of Na(+) and lower net Na(+) flux were observed in the xylem sap in the suppressed plants. In addition, K(+) concentration was lower in roots of silenced plants than in WT. Our results demonstrate that SlSOS1 antiporter is not only essential in maintaining ion homeostasis under salinity, but also critical for the partitioning of Na(+) between plant organs. The ability of tomato plants to retain Na(+) in the stems, thus preventing Na(+) from reaching the photosynthetic tissues, is largely dependent on the function of SlSOS1.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na(+) in vacuoles. However, all isoforms characterized so far catalyze both Na(+)/H(+) and K(+)/H(+) exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K(+), which in turn affects plant K(+) nutrition and Na(+) tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K(+) vacuolar pools in all growth conditions tested, but no consistent enhancement of Na(+) accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K(+) and to withstand salt-shock. Under K(+)-limiting conditions, greater K(+) compartmentation in the vacuole occurred at the expense of the cytosolic K(+) pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K(+) uptake system, enhanced K(+) uptake by roots, and increased the K(+) content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H(+)-linked K(+) transport that is thought to facilitate active K(+) uptake at the tonoplast, and the partitioning of K(+) between vacuole and cytosol.

A Plant Defense Response Effector Induces Microbial Apoptosis

Osmotin is a tobacco PR-5 protein that has antifungal activity and is implicated in host-plant defense. We show here that osmotin induces apoptosis in Saccharomyces cerevisiae. Induction of apoptosis was correlated with intracellular accumulation of reactive oxygen species and was mediated by RAS2, but not RAS1. Osmotin treatment resulted in suppression of transcription of stress-responsive genes via the RAS2/cAMP pathway. It was therefore concluded that osmotin induced proapoptotic signaling in yeast. The results indicate that the ability of antimicrobial proteins to induce microbial apoptosis could be an important factor in determining a pathogens virulence and could therefore be targeted for the design of new antifungal drugs.