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Potassium is the most abundant cation in living cells, while Na+ is less abundant and may be toxic. Although the toxic levels of Na differ, even among taxonomically close species (Benito et al., 2009), it is widely accepted that the substitution of Na+ for a significant part of the cytoplasmic K+ produces toxicity. In contrast to this toxicity, all living cells take up Na+ and require Na+-efflux systems that control the Na+ content, which is especially important in environments in which Na+ is much more abundant than K+. Flowering plants apparently have a single Na+ efflux system, SOS1, which is essential because the Na+ content of soils may be high (Munns & Tester, 2008). Two genes encoding SOS1-like proteins exist in Arabidopsis, namely AtSOS1 (Shi et al., 2000) and AtNHX8 (An et al., 2007), and in Cymodocea nodosa, namely CnSOS1A and CnSOS1B (Garciadeblás et al., 2007b), but apparently only one gene encoding an SOS1-like protein occurs in rice (Oryza sativa) (Martínez-Atienza et al., 2007). Bryophytes are different from flowering plants in that in addition to SOS1 transporters they have Na+ efflux ATPases (Benito & Rodríguez-Navarro, 2003) that are required when the external pH is alkaline (Fraile-Escanciano et al., 2009).
The SOS1 gene of Arabidopsis was initially identified as a genetic locus required for salt tolerance (Wu et al., 1996) and the gene was cloned a few years later (Shi et al., 2000). Consistent with the notion of the absence of other Na+ efflux systems, Arabidopsis sos1 mutant plants are extremely sensitive to the presence of Na+ and show defective root-to-shoot Na+ translocation and partitioning (Shi et al., 2002). Since the cloning of the AtSOS1 gene, significant progress has been made in understanding the function of the SOS1 system, especially regarding its transcriptional and functional regulation by the SOS2 Ser–Thr kinase and the SOS3 Ca2+ sensor, which belongs to the SOS3-like Ca2+ sensor/binding proteins (SCaBPs)/calcineurin B-like (CBL) protein family (Qiu et al., 2002; Zhu, 2003; Kolukisaoglu et al., 2004; Batistic & Kudla, 2009; Li et al., 2009). It is also known that the SOS1 messenger RNA (mRNA) in Arabidopsis is unstable under normal growth conditions but is stabilized under stress conditions through the action of reactive oxygen species (Chung et al., 2008). In addition to the SOS1 from Arabidopsis, the SOS1 transporters from C. nodosa (Garciadeblás et al., 2007b), rice (Martínez-Atienza et al., 2007), wheat (Triticum aestivum) (Xu et al., 2008), Populus (Wu et al., 2007), tomato (Solanum lycopersicum) (Olías et al., 2009) and Thellungiella (Oh et al., 2009) have been characterized.
Although progress has been made in understanding the functional regulation of SOS1 transporters, knowledge of their inherent functional mechanism is still incomplete. As a member of the bacterial NhaP Na+/H+ antiporter group in the CAP1 (cation proton antiporter 1) family of transporters, SOS1 can be expected to mediate Na+ efflux by exchanging Na+ and H+ (Shi et al., 2000; Brett et al., 2005; Pardo et al., 2006). However, although Arabidopsis sos1 mutant plants are extremely sensitive to the presence of Na+, defective Na+ efflux in sos1 plant cells has not been reported. Furthermore, sos1 mutants do not have a higher Na+ content than wild-type plants (Ding & Zhu, 1997; Zhu et al., 1998), which would be expected if SOS1 dominated Na+ efflux in roots. Indeed, in a Thsos1-RNAi (RNA interference) line of Thellungiella, the Na+ content of roots was higher than in the control wild-type line (Oh et al., 2009) but the reasons for this are not clear because the differences are small and the motilities of Na+ from shoots to roots in wild-type and Thsos1-RNAi plants are different (Oh et al., 2009). Indirect support for SOS1-mediated Na+ efflux might be the decreased Na+ efflux (Elphick et al., 2001) and increased Na+ content (Liu & Zhu, 1997; Zhu et al., 1998) exhibited by an Arabidopsis sos3 mutant. However, because SOS3 participates in complex signaling networks (Zhu, 2003; Kolukisaoglu et al., 2004; Batistic & Kudla, 2009; Li et al., 2009) the decreased Na+ efflux of the sos3 mutant might be an indirect effect, occurring, for example, through an increased accumulation of Na+ into the vacuole. In the absence of physical evidence of SOS1-mediated Na+ efflux in plant cells this function has been inferred from SOS1 functional expression in Na+-efflux defective yeast mutants (Quintero et al., 2002; Garciadeblás et al., 2007b; Martínez-Atienza et al., 2007; Xu et al., 2008; Oh et al., 2009; Olías et al., 2009) or bacterial mutants (Garciadeblás et al., 2007b; Wu et al., 2007), and also from Na+-dependent H+ movements in plasma membrane vesicles from plant roots, when the wild-type and sos1 plants were compared (Qiu et al., 2002, 2003; Vera-Estrella et al., 2005; Olías et al., 2009).
In addition to the experimental difficulties entailed in studying SOS1-mediated Na+ efflux, the functional understanding of SOS1 is complicated by other activities of SOS1, either direct or indirect, some of which are revealed by the sos1 mutant of Arabidopsis. In this mutant, high-affinity K+ uptake and K+ content are reduced (Wu et al., 1996; Zhu et al., 1998), the Na+ uptake rate in plants treated with low concentrations of K+ is slower than in wild-type plants (Ding & Zhu, 1997), the K+ efflux produced by the addition of Na+ is higher (Shabala et al., 2005), and membrane traffic and vacuolar functions are affected (Oh et al., 2010). Some of these defects might result from a defective pH homeostasis (Oh et al., 2010). In line with the notion of its functional complexity, SOS1 mediates or stimulates K+ uptake in Escherichia coli (Garciadeblás et al., 2007b). Populus euphratica SOS1 also improves the K+/Na+ ratio in E. coli (Wu et al., 2007) but the flux that is primarily enhanced – K+ influx or Na+ efflux – has not been determined.
To overcome the difficulties that complex vascular plants impose on investigating the cellular functions of plant transporters, the moss Physcomitrella patens has been proposed as a useful tool (Garciadeblás et al., 2007a). This plant is anatomically simple and has an SOS1 gene that encodes a typical SOS1 transporter (Benito & Rodríguez-Navarro, 2003). P. patens also has ENA ATPases that mediate Na+ efflux, and disruption mutants of the ENA genes are available (Fraile-Escanciano et al., 2009). Therefore, we undertook a study of the SOS1 transporter of P. patens (PpSOS1) and report data which strongly support the hypothesis that PpSOS1 mediates Na+ efflux and that the involved mechanism is an electroneutral Na+/H+ exchange, as previously proposed for AtSOS1 (Qiu et al., 2003). PpΔena1/PpΔsos1 double-mutant plants showed excessive accumulation of Na+ at all external pH values, and both the single PpΔsos1 mutant and the double-mutant plants died at relatively low Na+ concentrations. The system encoded by a second SOS1 gene in P. patens, PpSOS1B, is apparently not involved in plasma-membrane Na+ efflux.
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The molecular cloning of the PpSOS1 gene and the construction of Δsos1 and Δena1/Δsos1 mutants of P. patens offers new opportunities for studying plant SOS1 systems at the molecular level. Our findings indicate that the SOS1 transport system is conserved from bryophytes to flowering plants, including the clubmoss S. moellendorffii, and sequence analysis of the proteins, including the complex C-terminus, does not reveal any significant difference between the most distant members (Fig. 1b). The Synechocystis NhaP and the plant SOS1 antiporters share an ancestral precursor (Hamada et al., 2001) but the shorter C-terminus of SynNhaP suggests that regulatory functions of the C-terminus of land-plant SOS1 transporters do not exist in cyanobacteria/NhaP antiporters. A feature of the regulation of AtSOS1 is its activation by AtSOS2 and AtSOS3 (Qiu et al., 2002; Zhu, 2003; Kolukisaoglu et al., 2004; Batistic & Kudla, 2009; Li et al., 2009) and we found that suppression of the Na+ sensitivity of an Na+ efflux yeast mutant by PpSOS1 was enhanced by the co-expression of AtSOS2 and AtSOS3. This heterologous activation of PpSOS1, which is similar to previous descriptions for the SOS1 transporters of Cymodocea (Garciadeblás et al., 2007b), rice (Martínez-Atienza et al., 2007), tomato (Olías et al., 2009) and Thellungiella (Oh et al., 2009), suggests that the regulation of PpSOS1 and AtSOS1 is similar and highly conserved in land-plant evolution, in that the components of the Arabidopsis pathway recognize and modify the activity of the P. patens transporter.
The function of the second SOS1 transporter, PpSOS1B, in P. patens was not examined in this study. Although the transcripts of PpSOS1 and PpSOS1B are abundant and show a parallel regulation, PpSOS1B may localize to endomembranes (Fig. 2), where it could not mediate cellular Na+ loss. Consistent with this notion, the suppression of Na+ efflux in the PpΔena1/PpΔsos1 line, in comparison with the PpΔena1/PpSOS1 line at pH 4.5, indicates that PpSOS1B does not cooperate with PpSOS1 in Na+ efflux.
It has been previously shown that PpENA1 mediates Na+ efflux (Fraile-Escanciano et al., 2009). Therefore, the function of PpSOS1 was established by comparing Na+ uptake and loss in PpΔena1 and PpΔena1/PpΔsos1 lines. In these comparisons we found a progressive reduction of Na+ accumulation and rapid loss of Na+ at pH 4.5 in PpΔena1 vs PpΔena1/PpΔsos1 plants (Figs 5,6). These results reveal that both PpENA1 and PpSOS1 reduce Na+ accumulation at pH 4.5 with reference to PpΔena1/PpΔsos1 plants, and that PpSOS1 was more effective than PpENA1. Therefore, the disruption of the SOS1 gene in PpΔena1 plants increased Na+ accumulation substantially. By contrast, at pH 9.0 the presence or the absence of the SOS1 gene did not show any effect, which also occurred when the PpENA1 ATPase was active. These results support the fact that Na+ efflux mediated by PpSOS1 was driven by the ΔpH, which is consistent with the notion that PpSOS1 mediates electroneutral Na+/H+ exchange, as proposed for AtSOS1 (Qiu et al., 2003).
Our previous (Fraile-Escanciano et al., 2009) and present results demonstrate that PpENA1 and PpSOS1 are complementary systems. PpENA1 is relevant exclusively at high pH values, where PpSOS1 is inactive, but is unnecessary at low pH values where PpSOS1 is active. The model, an Na+-ATPase for high pH values and an electroneutral Na+/H+ antiporter for low pH values, has been previously proposed in fungi (Benito et al., 2009) and, interestingly, it does not apply to vascular plants. Whilst Na+-ATPases exist in mosses and liverworts, they are not present in clubmosses (Fraile-Escanciano et al., 2009) or in flowering plants (Garciadeblas et al., 2001), indicating the evolutionary loss of this system in the tracheophytes.
The most important consequence of the elimination of the PpSOS1 system is the enhanced Na+ sensitivity of the resulting PpΔsos1 mutant, which suffered Na+-triggered cell death. Consistent with the partial redundancy of the SOS1 and ENA1 functions, the PpΔena1/PpΔsos1 line was hypersensitive to Na+. The Na+-triggered cell death of the PpΔsos1 and PpΔena1/PpΔsos1 lines is consistent with previous reports that link salt toxicity with programmed cell death in plants (Shabala, 2009, and references therein) and in sos1 Arabidopsis mutants (Huh et al., 2002). Furthermore, the cell injuries (Fig. 7) were very similar to those produced by cell-free culture filtrates from Erwinia carotovora or by treatments with Botrytis cinerea spores (Ponce-de-León et al., 2007). Because the deletions of PpSOS1 and PpENA1 affect both Na+ efflux and K+ uptake we have not clearly determined whether cell death was produced exclusively by the high amounts of Na+ or by the interaction of several effects. Furthermore, the multiple effects of the sos1 mutation on membrane traffic and vacuolar functions in Arabidopsis (Oh et al., 2010) strongly suggest that the cell death observed in the PpΔsos1 and PpΔena1/PpΔsos1 lines may not occur exclusively as a result of the increase in Na+ content.
Our analysis of K+ and Rb+ uptake in K+-starved plants of the PpΔsos1 and PpΔena1/PpΔsos1 lines revealed that high-affinity K+ uptake is impaired in these mutants, as it is in the sos1 mutant of Arabidopsis (Wu et al., 1996). In our experiments, plants were prepared in a growth medium that was practically Na+ free (< 10 μM) and the absence or presence of 5 mM Na+ during the uptake tests did not affect the results. Under these conditions we can rule out that Na+ is involved in the impairment of K+ uptake, as proposed for Arabidopsis (Qi & Spalding, 2004). The direct participation of PpENA1 or PpSOS1 on high-affinity K+ uptake can also be ruled out because in the experimental conditions of Fig. 8 high-affinity Rb+ uptake is mediated by PpHAK1 (Garciadeblás et al., 2007a). Therefore, the relevant question is how PpSOS1 enhances K+ uptake without involving Na+. The first observation that sheds light on this question is that the PpΔena1 mutation did not produce any effect on Rb+ influx, but substantially increased the negative effect of the PpΔsos1 mutation (Fig. 8). Therefore, PpSOS1 and PpENA1 are complementary systems for maintaining a high Rb+ influx, as described for Na+ efflux at acidic pH values. Although PpSOS1 and PpENA1 may fulfil the same function of Na+ efflux in the plasma membrane using different driving forces –ΔpH and ATP, respectively – the impairment of K+(Rb+) influx by the ena1 and sos1 mutations may not be caused by the loss of the plasma membrane functions of PpENA1 and PpSOS1. Further research is necessary to establish the roles of these systems in organelle functions (Benito et al., 2009; Oh et al., 2010).
In summary, our results strongly support the fact that SOS1 mediates Na+ efflux in plant cells and confirm that SOS1 controls K+ uptake. This opens a line of research for functional studies of SOS1 transporters from different plant species by expressing them in the PpΔsos1/PpΔena1 line. The substitution of P. patens for yeast cells provides the substantial technical advantage of using a homologous system (Bassham & Raikhel, 2000). Future lines of research include analysis of the activation of SOS1 transporters by the SOS2–SOS3 pathway in P. patens given the activation of PpSOS1 by AtSOS2–AtSOS3 in yeast cells, and amenability of P. patens for targeted mutagenesis of the corresponding members of the CBL and CIPK (CBL-interacting protein kinases) gene families identified in the P. patens genome (Batistic & Kudla, 2009).