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The understanding on how Na+ is sensed is still very limited in most cellular systems. In theory, Na+ can be sensed either before or after entering the cell, or both. Extracellular Na+ may be sensed by a membrane receptor, whereas intracellular Na+ may be sensed either by membrane proteins or by any of the several Na+-sensitive enzymes in the cytoplasm (Zhu 2003). Even though the molecular identity of Na+ sensors remains elusive, the plasma-membrane Na+/H+ antiporter SOS1 (SALT OVERLY SENSITIVE1) is a probable candidate (Shi et al 2000). The transport activity of SOS1 is essential for Na+ efflux from Arabidopsis cells (Qiu et al. 2002; Quintero et al. 2002), but, additionally, its unusually long cytoplasmic tail is thought to be involved in the Na+ sensing (reviewed by Zhu 2003).
The AtSOS1 transporter (Figure 1) is a good example of a membrane transporter protein with atypical dual functions of solute transport and sensing, a phenomenon that has been increasingly observed (Conde et al. 2010). The SOS1 gene encodes a transmembrane protein with significant identities to plasma membrane Na+/ H+ antiporters from bacteria and fungi, and NaCl stress strongly up-regulates a usually constant level of gene transcription (Shi et al. 2000). Undifferentiated callus cultures regenerated from transgenic plants were also more salt-tolerant, a fact correlated with reduced Na+ content in the transgenic cells (Shi et al. 2003). When expressed in a yeast mutant devoid of endogenous Na+ transporters, SOS1 was able to reduce Na+ accumulation and improve salt stress tolerance of the mutant cells (Shi et al., 2002). The SOS pathway was found out when three salt-overly-sensitive mutants (sos1, sos2 and sos3) were characterized in a genetic screen designed to identify components of the cellular mechanisms that contribute to salt tolerance in Arabidopsis. SOS2 protein is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase and SOS3 encodes a Ca2+ sensor protein that shares significant sequence similarity with the calcineurin B subunit from yeast and neuronal calcium sensors from animals (Liu and Zhu, 1998; Liu et al. 2000; reviewed by Silva and Gerós 2009).
SOS1 has been demonstrated to be a target of the SOS pathway, whose signaling is controlled by SOS2/SOS3. SOS1 transcription up-regulated in response to salt stress but this positive regulation does not occur in sos3 or sos2 mutant plants (Shi et al. 2000). SOS1 cation transporter, the SOS2 protein kinase, and its associated Ca2+ binding/sensor myristoylated SOS3 indeed make up a functional module, where SOS1 is the phosphorylation substrate for the SOS2/ SOS3 kinase complex (Quintero et al. 2002).
In plant cells, Ca2+ acts as a second messenger connecting a wide range of extracellular stimuli with various intracellular responses (Snedden and Fromm, 1998, 2001; DeFalco, Bender and Snedden, 2010). Several major classes of Ca2+ sensors have been characterized in plants. These classes are calmodulin, calcium-dependent protein kinase (CDPKs), and calcineurin B-like proteins (CBLs) (Yang and Poovaiah 2003). Several lines of evidence suggest that all these three classes of Ca2+ sensors are involved in stress signal transduction (Snedden and Fromm 2001; Luan et al. 2002; Zhu 2000). The involvement of Ca2+ signaling in response to osmotic and ionic stress is well documented. Salt stress originates a fast and transient increase in cytosolic Ca2+ that in turn triggers many signal transduction pathways, such as the previously referred SOS, involved in ion channel activity, the regulation of enzymatic activity, and gene transcription. This results in a wide variety of cellular responses (Snedden and Fromm 1998, 2001) and mediates salt adaptation, all leading to ion homeostasis (Bressan et al. 1998; Liu and Zhu 1998; Serrano et al. 1999; Serrano and Rodriguez-Navarro 2001). The involvement of Ca2+ in the re-establishment of cellular homeostasis has to be tightly regulated, as the spatial and temporal dynamics of the Ca2+ signal encodes the response to different osmotic stresses (Knight and Knight, 2001). For instance, in response to salt, osmotic and low temperature stresses, the alterations in cytosolic Ca2+ levels were cell-type specific in Arabidopsis root cells (Kiegle et al. 2000; reviewed by Bartels and Sunkar 2005).
The major physiological role played by Ca2+-ATPases, also designated as Ca2+ pump, is to restore and keep homeostasis by pumping Ca2+ out of the cytosol to end a signaling occurrence, and is critical in all eukaryotic cells and not only during environmental stress conditions (Sze et al. 2000). Both animal and plant cells use two distinct types of Ca2+-ATPases, the type IIA and the type IIB. The expression of genes encoding the type IIA Ca2+ -ATPase in tomato, soybean, and tobacco was demonstrated to be triggered by salt stress (Wimmers, Ewing and Bennett, 1992; Chung et al. 2000). The consequence of up-regulating the Ca2+ pump in response to salinity is thought to provide an adaptive response. The soybean Ca2+-ATPase1 was up-regulated by NaCl but not by KCl and mannitol, indicating that specific Ca2+ signals trigger the enhancement in the gene expression (Chung et al. 2000). The Arabidopsis Ca2+-ATPase isoform 4 (ACA4), a calmodulin-regulated Ca2+-ATPase has also been reported to be part of the Ca2+-dependent signal transduction pathway associated to salt stress (Geisler et al. 2000a). According to the authors, Arabidopsis seedlings treated with increasing concentrations of NaCl for 24 h demonstrated a dose-dependent increase in ACA4 gene expression, and, additionally, when N-terminal truncated ACA4 was heterologously expressed in yeast, it conferred increased salt tolerance to its host (Geisler et al. 2000b; reviewed by Bartels and Sunkar 2005).
Role of Ca2+ in cold sensing
In cold stress conditions, Ca2+ also plays a vital role as messenger in a low temperature signal transduction pathway. Modifications in cytosolic Ca2+ levels is a primary step in a temperature sensing mechanism, enabling the plant to tolerate further cold more effectively. In both Arabidopsis (Knight, Trewavas and Knight 1996; Polisenski and Braam 1996) and alfalfa (Monroy and Dhindsa 1995) cytoplasmic Ca2+ levels increase rapidly in response to low temperature, largely due to an influx of Ca2+ from extracellular stores. Calcium is responsible for an increased expression of several cold-induced genes including the CRT/DRE controlled COR6 and KIN1 genes of Arabidopsis (Monroy et al. 1993; Knight, Trewavas and Knight 1996; Monroy and Dhindsa 1995). For instance, Ca2+ chelators such as BAPTA and Ca2+ channel blockers such as La3+ inhibited the cold-induced influx of Ca2+ and resulted in a lowered expression of the cold-inducible Cas15 gene, impairing the capacity of alfalfa to acclimate to a cold environment. Interestingly, Cas15 expression can be induced at the high temperature of 25 Â°C by treating the cells with A23187, a Ca2+ ionophore that causes a rapid influx of this divalent cation (Monroy and Dhindsa 1995, reviewed by Mahajan and Tuteja 2005).
Besides the ability to sense salt, osmotic and low temperature stresses, plants have sensing mechanisms capable to detect high temperature stress. Even though the existence of a "plant thermometer" has not been recognized, it is suggested that changes in membrane fluidity plays a key role in sensing and influencing gene expression not only under freezing, but also high temperatures. Therefore, sensors are probably located in microdomains of membranes, which are capable of detecting physical phase transition, eventually leading to conformational changes and/or phosphorylation/dephosphorylation events when temperature changes (Plieth 1999). According to this, a model for temperature sensing and regulation of heat shock responses should integrate detectable membrane modifications.
The expression and activity of heat stress-responsive transcription factors (HSFs) is probably altered by changes in the proportion of saturated and unsaturated fatty acids when the temperature threshold responsible for the induction of a heat shock response is attained. Moreover, stiffness of the thylakoid membranes is suggested to invoke altered expression profiles of heat shock genes, HSPs (heat shock proteins) being significantly up-regulated; this suggests that the temperature sensing mechanism may be located on the thylakoid membrane (Horváth et al. 1998). The prospect for a role of the thylakoid membrane as a high-temperature sensor is physiologically crucial, because it is highly susceptible to temperature increases, due to its highly unsaturated constitution, and the presence of photosystems, which are very susceptible to temperature alterations (Sung et al. 2003; reviewed by Wahid et al. 2007).
4. The role of solute transport and compartmentation in cellular homeostasis during stress
One of the most important parts in the complex and remarkable ability to tolerate an environmental stress such as salinity and drought, always intimately connected, is played by the wide variety of modifications in ion transport inside and outside the cell.
4.1 Na+ homeostasis
Sodium is deleterious to many organisms, except for halotolerant ones such as halobacteria and halophytes, which possess specific mechanisms that maintain low concentrations of intracellular Na+. In halophytes, the accumulation of Na+ in the cytoplasm is prevented by inhibiting its influx across the plasma membrane and instead by promoting its efflux or sequestration into the vacuole (Hasegawa et al. 2000). The activity of most enzymes is negatively affected by high salt concentrations due to perturbation of the hydrophobic-electrostatic balance between the forces maintaining protein structure. However, toxic effects on cells occur even at moderate salt concentrations of about 100 mM, unveiling specific salt toxicity targets (Serrano 1996). Apoplastic enzymes from halophytes have been shown in vitro to be remarkably salt-insensitive, coping with NaCl concentrations up to 500 mM (Thiyagarajah, Fry and Yeo, 1996).
As previously mentioned, Na+ toxicity arises not only due to toxic effects of Na+ in the cytosol, but also because of the impairment of K+ homeostasis, probably due to competition of Na+ for K+ binding sites. Ion transporters have long been known to play a key role in ion homeostasis (Hasegawa et al. 2000; Blumwald, Aharon and Apse 2000; Apse and Blumwald 2002; Zhu 2003). Under salt/drought stress, to avoid excessive Na+ accumulation in the cytosol and reach ion homeostasis, plant cells exhibit three major mechanisms: restriction of Na+ permeation and uptake catalyzed Na+ transporters; sequestration of Na+ into the vacuole; and efflux of excess sodium, with symplastic Na+ being transported back to the the apoplast through plasma membrane Na+/H+ antiporters (reviewed by Bartels and Sunkar 2005).
Sodium enters plant cells through the high-affinity K+ transporter HKT1 (Rus et al. 2001, Máser et al. 2002) and through non-selective cation channels benefiting from the significant negative membrane potential across the plasma membrane (Amtmann and Sanders 1999). Also, in several plant species such as rice, Na+ leakage into the transpiration stream via the apoplast is responsible for a vast part of Na+ entry into plants (Yeo et al. 1999). Sodium currents that are mediated by non-selective cation channels are also partially sensitive to calcium signaling, as demonstrated by the inhibition of Na+ uptake by roots caused by Ca2+ (Tester and Davenport 2003). It remains, however, yet to be fully comprehended if the regulation of the activity of non-selective cation channels by Ca2+ is direct or indirect via intracellular signaling cascades (Zhu 2003).
The Arabidopsis AtHKT1 protein mediates Na+ influx when heterologously expressed in yeast and Xenopus oocytes (Uozumi et al. 2000). AtHKT1 is in fact the best-characterized member of class-1 HKTs in A. thaliana, and its mediation of Na+ transport is actually well established (Møller et al. 2009) although its main role is currently believed to be in regulating Na+ transport between root and shoot (Máser et al. 2002; Hauser and Horie 2010; Kronzucker and Britto 2011). Mutation in AtHKT1 suppresses the hypersensitivity of sos3 mutants (Rus et al. 2001) suggesting that the wild-type SOS3 and other components of the SOS regulatory pathway may inhibit the activity of AtHKT1 as a Na+ influx transporter (Bartels and Sunkar 2005).
At first sight, the efflux of Na+ in individual cells is not logical in multicellular organisms like plants, as the extrusion of Na+ could negatively impact the surrounding cells (Zhu 2003). Thus, Na+ efflux needs to be considered in specific tissues and in a whole-plant context. In Arabidopsis, Na+ efflux is catalyzed by the plasmamembrane Na+/H+ antiporter encoded by the previously mentioned SOS1 gene (Shi et al. 2000, 2002; Qiu et al. 2002; Quintero et al. 2002). This transmembrane protein is an electroneutral Na+/H+ exchanger that is specific for Na+ being unable to transport Li2+ or K+ (Qiu et al. 2002, 2003). Activity of the SOS1 promoter is detected ubiquitously in virtually all tissues, but its greatest activity occurs in root epidermal cells, particularly at the root tip, and in cells bordering the vascular tissue throughout the plant (Shi et al. 2002). This SOS1 expression pattern, together with the results of ion analysis in sos1 mutant plants, suggests several roles for SOS1: Na+ efflux into the root medium; time gaining for Na+ storage in the vacuole by slowing down Na+ accumulation in the cytoplasm; and the control over long-distance Na+ transport between roots and leaves by loading and unloading of Na+ into and from the xylem and phloem. The function of SOS1 in long distance transport is important for coordination between transpirational Na+ flow and the vacuolar sequestration of Na+ in leaves. As previously mentioned, the transcript level of SOS1 is up-regulated at a transcriptional level by salt stress (Shi et al. 2000, 2003). Indeed, increased expression of SOS1 results in improved ion homeostasis and salt tolerance in transgenic Arabidopsis (Shi et al. 2003; Zhu 2003).
4.2. K+ homeostasis
A high cytosolic K+/Na+ ratio is important for the normal functioning of cellular metabolism and, of course, plant growth and productivity. Under normal conditions of nutrient availability, about 80% of K+ intake by plants happens through the action of two major systems, KUP/HAK/KT and AKT (Kronzucker and Britto 2011). Respectively, they catalyze high- and low-affinity uptake (Hirsch et al. 1998; Rubio et al. 2008; Szczerba, Britto and Kronzucker, 2009). Back-up uptake mechanisms such as the non-characterized cation and K+ transporting families CHX and KEA respectively, may provide additional K+ influx capacity at higher external K+ concentrations (Pardo et al. 2006; Pyo et al. 2010). Both KUP/HAK/KT and AKT potassium uptake systems are severely inhibited by Na+ (Fu and Luan 1998; Britto et al. 2010). In theory, under salt stress, Na+ competes with K+ for influx into roots. The transcript amounts of several K+ transporter genes are either down- or up-regulated in salinity, so that plants can keep the uptake of K+ under salt stress. For example, in the common ice plant, salt-stress significantly increases the expression of KMT1, a member of the AKT/KAT family, and of several HAK/KUP genes, whereas, on the other hand, transcript levels of MKT1, also part of the AKT/KAT family, are down-regulated (Su et al. 2001, 2002). Also, gene expression of the Arabidopsis root K+-transporter AtKC1 is up-regulated by salt (Pilot et al. 2003).
The activity of K+ channels are known to be regulated by protein kinases (Li, Lee and Assmann, 1998) and phosphatases (Cherel et al. 2002), and if these proteins are influenced or somehow regulated directly or indirectly by salt stress is yet to be clarified. In Eucalyptus camaldulensis, two Na+-K+ co-transporter HKT1 homologs display intrinsic osmosensing capabilities when expressed in Xenopus oocytes (Liu et al. 2001). Their Na+- and K+-transport activities are enhanced by a downshift in extracellular osmolarity.
As previously referred to, the A. thaliana plasma membrane SOS1 protein is a specific Na+/H+ antiporter responsible for Na+ efflux and regulates its distribution between root and shoot. However, surprisingly, it also interacts with K+ influx mechanisms by roots, suggesting an influence of the SOS signaling pathway in K+ homeostasis. Concordantly, Arabidopsis sos mutant plants display a growth impairment under K+-limiting conditions (Zhu, Liu and Xiong, 1998). The involvement of the SOS pathway in K+ uptake and homeostasis is possibly indirect, because in theory, the severe inhibition of Na+ efflux in sos mutant plants can lead to an accumulation of cytoplasmic Na+ that is repressive to K+-uptake transporters like AKT1. Under K+-limiting conditions, inhibitory levels of cytoplasmic Na+ may emerge in sos mutant plants, even when grown in media without extra NaCl addition (Zhu et al. 2003).
Additionally, two CHX isoforms, AtCHX17 and AtCHX23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively (Cellier et al. 2004; Song et al. 2004; Pardo et al. 2006). In parallel with the recently discovered KEA family of K+ transporters, the CHX family still needs further characterization studies, to fully understand its role in K+ homeostasis.