Selective transport capacity for K+ over Na+ is ... - CSIRO Publishing

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Functional Plant Biology, 2012, 39, 1047–1057 http://dx.doi.org/10.1071/FP12174

Selective transport capacity for K+ over Na+ is linked to the expression levels of PtSOS1 in halophyte Puccinellia tenuiflora Qiang Guo A,B, Pei Wang A,B, Qing Ma A, Jin-Lin Zhang A, Ai-Ke Bao A and Suo-Min Wang A,C A

State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, PR China. B These authors have contributed equally to this work. C Corresponding author. Email: [email protected]

Abstract. The plasma membrane Na+/H+ antiporter (SOS1) was shown to be a Na+ efflux protein and also involved in K+ uptake and transport. PtSOS1 was characterised from Puccinellia tenuiflora (Griseb.) Scribn. et Merr., a monocotyledonous halophyte that has a high selectivity for K+ over Na+ by roots under salt stress. To assess the contribution of PtSOS1 to the selectivity for K+ over Na+, the expression levels of PtSOS1 and Na+, K+ accumulations in P. tenuiflora exposed to different concentrations of NaCl, KCl or NaCl plus KCl were analysed. Results showed that the expression levels of PtSOS1 in roots increased significantly with the increase of external NaCl (25–150 mM), accompanied by an increase of selective transport (ST) capacity for K+ over Na+ by roots. Transcription levels of PtSOS1 in roots and ST values increased under 0.1–1 mM KCl, then declined sharply under 5–10 mM KCl. Under 150 mM NaCl, PtSOS1 expression levels in roots and ST values at 0.1 mM KCl was significantly lower than that at 5 mM KCl with the prolonging of treatment time. A significant positive correlation was found between root PtSOS1 expression levels and ST values under various concentrations of NaCl, KCl or 150 mM NaCl plus 0.1 or 5 mM KCl treatments. Therefore, it is proposed that PtSOS1 is the major component of selective transport capacity for K+ over Na+ and hence, salt tolerance of P. tenuiflora. Finally, we hypothesise a function model of SOS1 in regulating K+ and Na+ transport system in the membrane of xylem parenchyma cells by sustaining the membrane integrity; it also appears that this model could reasonably explain the phenomenon of Na+ retrieval from the xylem when plants are exposed to severe salt stress. Additional keywords: K+, Na+, plasma membrane Na+/H+ antiporter, salt tolerance. Received 15 June 2012, accepted 19 August 2012, published online 24 September 2012

Introduction Salinity is a major constraint to crop productivity because it reduces yield and limits expansion of agriculture onto uncultivated land (Flowers and Yeo 1995; Zhang et al. 2010). Most crops, including graminaceous crops, are very sensitive to salt: sodium disequilibrium is the primary consequence of ionic stress and often leads to adverse effects on nutrient K+ acquisition, water uptake, enzyme activities, photosynthesis and metabolism (Niu et al. 1995; Tester and Davenport 2003; Bao et al. 2009). However, halophytes have evolved various mechanisms to overcome salt stress through long-term natural selection (Flowers and Colmer 2008; Wu et al. 2011; Ma et al. 2012). Puccinellia tenuiflora (Griseb.) Scribn. et Merr. is a monocotyledonous halophyte found in saline marshes in north China and is used as forage as well as for soil improvement. Previous studies showed that the endodermal apoplastic barrier in P. tenuiflora roots restricted Na+ uptake while the uptake of K+ is maintained (Peng et al. 2004). Further studies suggest that 92–95% of unidirectional Na+ influx in P. tenuiflora roots under Journal compilation CSIRO 2012

salinity condition is pumped out to the external solution, resulting in a strong selectivity for K+ over Na+ and contributing to salt tolerance of P. tenuiflora (Wang et al. 2009). Although the above studies have implicated a high selectivity for K+ over Na+ as a key factor in salt tolerance of P. tenuiflora, the molecular mechanism of selectivity for K+ over Na+ remains unknown, especially the mechanism of selective transport for K+ over Na+ in regulating salt tolerance. The plasma membrane Na+/H+ antiporter encoded by SOS1 is thought to be involved in Na+ efflux and hence, salt tolerance (Shi et al. 2000; Martinez-Atienza et al. 2007; Wu et al. 2007; Xu et al. 2008; Maughan et al. 2009; Olías et al. 2009; Takahashi et al. 2009; Chen et al. 2010; Cosentino et al. 2010). Previous studies have shown that sos1 mutant of Arabidopsis was extremely sensitive to NaCl stress (Zhu et al. 1998). AtSOS1 expression was observed at the epidermal cells of the root tip, implying its role in extruding Na+ to the growth medium (Shi et al. 2002). Further studies suggested that overexpression of AtSOS1 can significantly improve salt tolerance in Arabidopsis by limiting Na+ accumulation in plant cells (Shi et al. 2003). www.publish.csiro.au/journals/fpb

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Recently, RNAi-based silencing of ThSOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis and apoplastic sodium influx into the stele and hence, the shoot, suggesting that ThSOS1 are important in mediating Na+ efflux from root (Oh et al. 2009). Moreover, the preferential expression of AtSOS1 in the cells adjacent to vascular tissues suggested a role of SOS1 in controlling long-distance Na+ transport in Arabidopsis (Shi et al. 2002). Similar studies showed that tomato (Lycopersicon esculentum Mill.) have the ability to retain Na+ in stem, thus, preventing Na+ from reaching the photosynthetic tissues, which is largely dependent on the function of SlSOS1 (Olías et al. 2009). In addition to above functions, SOS1 may be involved in root K+ uptake and maintain pH homeostasis in plants. In the Arabidopsis sos1 mutant, high affinity K+ uptake and K+ content were reduced (Wu et al. 1996; Zhu et al. 1998), K+ efflux triggered by the addition of Na+ is higher (Shabala et al. 2005) and membrane traffic and vacuolar functions were affected (Oh et al. 2010). Some of these defects may result from a defective pH homeostasis (Guo et al. 2009; Oh et al. 2010). In line with the notion of its functional complexity, elevated cytoplasmic Na+ levels resulting from loss of SOS1 function impaired K+ permeability in root cells and compromised K+ nutrition during salinity stress (Qi and Spalding 2004). SOS1 in Cymodocea nodosa (Ucria) Asch. mediated K+ uptake in Escherichia coli (Garciadeblás et al. 2007). Taken together, SOS1 plays important roles in maintaining ions homeostasis in planta under salt stress (Zhu 2003). However, whether SOS1 is involved in regulating the selective transport for K+ over Na+ in P. tenuiflora has not been explored. To test the contribution of SOS1 to selective transport for K+ over Na+ in P. tenuiflora, in this study, the PtSOS1 gene was isolated and characterised and Na+ and K+ accumulations in P. tenuiflora exposed to various NaCl, KCl and NaCl plus different KCl interaction treatments were investigated. Our results indicated that PtSOS1 was induced and regulated by NaCl or KCl and there was a significant positive correlation between the transcription levels of PtSOS1 and ST values in roots of P. tenuiflora. Materials and methods Plant growth conditions and treatments Seeds of Puccinellia tenuiflora (Griseb.) Scribn. et Merr. were germinated for 7 days at 25C on filter paper wetted with distilled water in rectangular dishes (60 30 cm). When the plumule emerged, seedlings were cultured in similar dishes with modified Hoagland nutrient solution containing 5 mM KNO3, 1 mM NH4H2PO4, 0.5 mM Ca(NO3)2, 0.5 mM MgSO4, 60 mM Fe-citrate, 92 mM H3BO3, 18 mM MnCl2 4H2O, 1.6 mM ZnSO47H2O, 0.6 mM CuSO45H2O and 0.7 mM (NH4)6Mo7O244H2O. Once plants had three leaves, they were transferred into black painted containers and watered daily with the same modified Hoagland nutrient solution. Solutions were renewed every 3 days. All the plants were grown in a room where the temperature was 25/18C (light/dark), the daily photoperiod was 16/8 h (light/dark); the flux density was 600 mmol m–2 s–1 and the RH was ~65%. Fourweek-old plants were used for following treatments: (i) plants

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were treated with the modified Hoagland nutrient solution supplemented with 0, 25, 50, 100 and 150 mM NaCl for 48 h, respectively; (ii) plants were grown at the modified Hoagland nutrient solution deprived of KNO3 for 7 days and 5 mM KNO3 was substituted by 2.5 mM NH4NO3. Different concentrations of K+ treatments were supplemented by adding 0, 0.1, 0.5, 1, 5 and 10 mM KCl for 48 h, respectively; (iii) after the treatment of depriving of K+ as described above, plants were irrigated with Hoagland nutrient solution modified to contain 0.1 or 5 mM final KCl plus 25 or 150 mM NaCl, then plants were harvested at 0, 6, 12, 24, 48, 72 and 96 h after treatments respectively. Cloning of PtSOS1 Total RNA was extracted from roots of P. tenuiflora seedlings exposed to 150 mM NaCl for 48 h using the method described by Chomczynski and Sacchi (1987) with minor modifications. First-strand cDNA was synthesised from 4 mg of total RNA using an Oligo (dT)18 primer and MMLV-RTase Sangon (Shanghai, China). The partial cDNA fragment was amplified by PCR using degenerate primers P1 and P2 (see Table S1, available as Supplementary Material to this paper). PCR amplification was programmed at 94C for 2 min; 30 cycles of 94C for 30 s, 55C for 50 s and 72C for 1 min; and a final extension at 72C for 10 min. PCR products were purified from agarose gels, ligated into the pUCm-T vector (BBI, Shanghai, China) and sequenced by Sangon. The 50 - and 30 -ends of PtSOS1 were obtained with the kit of RNA ligase mediated rapid amplification of 50 - and 30 -cDNA Ends (RLM-RACE, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and specific primers P3, P4, P5 and P6 respectively (Table S1). These fragments were assembled to obtain the full-length of the PtSOS1 cDNA. Sequence analysis Sequence BLAST search was performed on the NCBI platform (http://www.ncbi.nlm.nih.gov/BLAST, accessed 11 July 2009). The cDNA sequence was analysed by the DNASTAR 7.1 software (DNASTAR Inc., Madison, WI, USA). The molecular mass and isoelectric point of the deduced protein was predicted on the ExPASy protromics server (http://www. expasy.org, accessed 11 July 2009). Sequence multi-alignment analysis was performed with DNAMAN6.0 software (Lynnon Biosoft, Vaudreuil, Quebec, Canada). The hydrophobicity values were calculated by the program TMPRED available at http:// www.ch.embnet.orgy/software/TMPREDform.html (accessed 11 July 2009). The specific primers were designed with Primer 5.0 software (Premier Biosoft International, Palo Alto, CA, USA). Phylogenetic analysis To investigate the phylogenetic relationship of PtSOS1 with other Na+/H+ antiporters, multiple sequence alignment was performed with the Clustal X software (Thompson et al. 1997) and the phylogenetic tree was constructed with MEGA 4.1 software (Tamura et al. 2007). Evolutional distances were computed using neighbour-joining method (Saitou and Nei 1987).

PtSOS1 is a key determinant of ST in Puccinellia tenuiflora

Semiquantitative RT–PCR Total RNA was extracted with a Trizol Kit (Sangon) following the manufacturer’s instructions. First-strand cDNA was synthesised from 4 mg of total RNA with MMLV-reverse transcriptase by Sangon. Semiquantitative RT–PCR was performed with the primer pairs P7 and P8 (Table S1), which yielded a RT–PCR product of 553 bp (Table S1). ACTIN was used as the internal control in the semiquantitative RT–PCR. The specific primers of ACTIN that amplified a 598-bp fragment are A1 and A2 (Table S1) designed according to the cDNA sequence of ACTIN from P. tenuiflora (GenBank accession number FJ545641). The PCR was performed as follows: 94C for 2 min; 30 cycles of 94C for 30 s, 53C for 40 s (PtSOS1)/ 56C for 50 s (ACTIN), 72C for 50 s; and a final extension at 72C for 10 min. PCR products were separated on 1.2% (w/v) agarose gels containing ethidium bromide and visualised by AlphaImager (ver. 4.0.1) for subsequent analysis. The ratios of the quantity of mRNA for PtSOS1 to that for ACTIN were calculated and the results reflect the relative expression level. Experiments were repeated at least three times to obtain similar results. Determination of Na+ and K+ concentration At the end of the treatments, plant roots were washed twice for 8 min in ice-cold 20 mM CaCl2 to exchange cell wall-bound Na+ and shoots rinsed in deionised water to remove surface salts (Wang et al. 2007). Plants were separated into shoots and roots then dried in an oven at 80C for 3 days. Na+ and K+ were extracted from dried plant tissue in 100 mM acetic acid at 90C for 2 h. Ion analysis was performed using a flame spectrophotometer (2655–00; Cole Parmer Instrument Co., Chicago, IL, USA).


residues with estimated molecular mass of 126 kDa and a theoretical isoelectric point of 6.7. The cDNA sequence of PtSOS1 was registered at GenBank under accession number GQ452778. Multiple sequence alignment revealed PtSOS1 shared high amino acid sequence identity (88%) with TaSOS1. Hydrophobicity plot analysis by the TMpred program showed PtSOS1 had predicted 11 transmembrane domains and Pfamdirected (http://www.sanger.ac.uk/Software/Pfam/, accessed 11 July 2009) sequence analysis identified a cyclic nucleotide binding domain (738–828 amino acids) located in the long C-terminal tail (see Fig. S1, available as Supplementary Material to this paper), as previously reported for other plant plasma membrane SOS1 (Shi et al. 2000; Oh et al. 2007; Maughan et al. 2009). Moreover, phylogenetic analysis showed that PtSOS1 was grouped with other plasma membrane Na+/H+ antiporters (SOS1), but not with tonoplast Na+/H+ antiporters (NHX1) (Fig. S2). Further analysis indicated that PtSOS1 formed a clade with the most closely related the monocotyledons SOS1 homologue (e.g. LpSOS1 and TaSOS1), but was distinct from the cluster of dicotyledon SOS1 such as AtSOS1 (Fig. S2). These results suggested that putative PtSOS1 encodes a plasma membrane Na+/H+ antiporter. Expression of PtSOS1, Na+ and K+ accumulation and ST values in P. tenuiflora treated with NaCl Compared with control, the addition of 25–150 mM NaCl significantly increased the transcription levels of PtSOS1 and Na+ concentrations in both shoots and roots; the increase in the roots was higher than that in the shoots (Figs 1, 2a). NaCl concentration (mM)

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Statistical analyses All the data are presented as means with standard errors. Statistical analyses, one-way ANOVA and Duncan’s multiple range tests were performed with statistical software (ver.13.0, SPSS Inc., Chicago, IL, USA). Results Isolation and characterisation of PtSOS1 A fragment of 925 bp was initially isolated with degenerated primers P1 and P2 by the RT–PCR (Table S1). Nucleotide blast search showed that the isolated cDNA fragment shared high sequence homology (84–98%) with many known SOS1 from other plants (e.g. Oryza sativa, Phragmites australis, Triticum aestivum and Lolium perenne), indicating that partial potential SOS1 was isolated from P. tenuiflora. After obtaining 50 RACE and 30 RACE products, a full-length cDNA designated as PtSOS1 was obtained, which was 3811 bp long and contained an open reading frame (ORF) of 3432 bp encoding 1143 amino acid

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Calculation of ST value Selective transport capacity for K+ over Na+ (ST) were estimated according to the following equation as described by Wang et al. (2005): ST = (K+/Na+ in shoots)/(K+/Na+ in roots). The higher ST value indicates the stronger net capacity of selection for transport of K+ over Na+ from root to shoot (Wang et al. 2005, 2009).

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NaCl concentration (mM) Fig. 1. Expression of PtSOS1 in Puccinellia tenuiflora under different concentrations of NaCl (0, 25, 50, 100 and 150 mM) for 48 h. (a) Semiquantitative RT–PCR analysis of PtSOS1 in shoot and root. (b) The relative expression level of PtSOS1 in shoot and root (related to ACTIN). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Data are means s.e. (n = 3).

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Fig. 2. Tissue Na+ and K+ concentration in Puccinellia tenuiflora exposed to 0, 25, 50, 100 and 150 mM NaCl for 48 h. (a) Na+, (b) K+ concentration in shoot and root and (c) selective transport (ST) capacity for K+ over Na+, ST = (K+/Na+ in shoots)/(K+/Na+ in roots), the higher ST value indicates the stronger net capacity of selection for transport of K+ over Na+ from root to shoot (according to Wang et al. 2005). Ten plants were pooled in each replicate (n = 8). (d) Relationship between ST values and relative PtSOS1 expression level in root under 0 (*), 25 (¤), 50 (*), 100 (&) and 150 (&) mM NaCl treatments for 48 h (n = 3–8). Data are means s.e. Different letters indicate significant difference at P < 0.05 (Duncan’s test).

Expression of PtSOS1, Na+, K+ accumulation and ST values in P. tenuiflora treated with KCl Transcription levels of PtSOS1 in both shoots and roots increased rapidly from 0 to 1 mM and peaked at 1 mM KCl, then declined sharply from 1 to 10 mM KCl (Fig. 3), indicating that the expression of PtSOS1 was induced and regulated by external KCl. Under 0–10 mM external KCl treatments, Na+ concentration in roots was significant higher than that in shoots, but any significant differences of Na+ concentration were observed neither in shoots nor in roots among different concentrations of KCl treatments (Fig. 4a). K+ concentration in shoots was higher than that in roots under 0–0.5 mM KCl; but no significant difference was found between shoots and roots under 1–10 mM KCl (Fig. 4b). ST values were increased with the increase of external KCl concentrations from 0 to 1 mM and peaked at 1 mM KCl, then exhibited declining trends (Fig. 4c). Moreover, there

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The K+ concentration in root was significantly reduced under 100 and 150 mM NaCl; but no significant differences were observed in shoots among different concentrations of NaCl treatments (Fig. 2b). With the increase of external NaCl concentrations, ST values increased gradually and peaked at 150 mM NaCl (Fig. 2c) and there was a significant positive correlation between ST values and expression levels of PtSOS1 in roots of P. tenuiflora exposed to external 0–150 mM NaCl concentrations (Fig. 2d).

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Fig. 4. Tissue Na+ and K+ concentration in Puccinellia tenuiflora exposed to 0, 0.1, 0.5, 1, 5 and 10 mM KCl for 48 h. (a) Na+, (b) K+ concentration in shoot and root and (c) selective transport (ST) capacity for K+ over Na+ (see legend to Fig. 2 for details of ST). Ten plants were pooled in each replicate (n = 8). (d) Relationship between ST values and relative PtSOS1 expression level in root under 0 (&), 0.1 (~), 0.5 (*), 1 (&), 5 (*) and 10 (~) mM KCl treatments for 48 h (n = 3–8). Data are means s.e. Different letters indicate significant difference at P < 0.05 (Duncan’s test).

was a significant positive correlation between ST values and expression levels of PtSOS1 in roots of P. tenuiflora treated with 0–10 mM KCl (Fig. 4d). Expression of PtSOS1, Na+, K+ accumulation and ST values in P. tenuiflora under 25 mM NaCl plus 0.1 or 5 mM KCl treatments Under 25 mM NaCl plus 0.1 or 5 mM KCl treatments, transcription levels of PtSOS1 increased gradually in both roots and shoots with increasing treatment time, but the magnitude of the former was remarkably higher than that of the latter; however, the transcription abundance of PtSOS1 in neither shoots nor in roots was significantly different between 0.1 and 5 mM KCl treatments (Fig. 5), indicating that the expression of PtSOS1 was not regulated by external K+ treatments under moderate salt stress. Under 25 mM NaCl, Na+ concentrations in shoots and roots increased with prolonging of treatment time under 0.1 and 5 mM KCl treatments (Fig. 6a), while K+ concentrations declined (Fig. 6b). ST values increased from 6 to 24 h, then decreased after 24 h (Fig. 6c). However, no significant differences in Na+, K+ concentrations or ST values were found between 0.1 and 5 mM KCl treatments (Fig. 6a–c). Further analysis indicated that there was no significant correlation between ST values and expression levels of PtSOS1 in roots (Fig. 6d).

Expression of PtSOS1, Na+, K+ accumulation and ST values in P. tenuiflora under 150 mM NaCl plus 0.1 or 5 mM KCl treatments Under 150 mM NaCl plus 0.1 or 5 mM KCl, transcription levels of PtSOS1 in both shoots and roots increased gradually with prolonging of treatment time. There was no significant difference between 0.1 and 5 mM KCl treatments before 24 h, but after 24 h of treatments the magnitude of the latter was significantly higher than that of the former (Fig. 7a, b). Under 150 mM NaCl, Na+ concentrations in shoots increased with increasing time of treatment time under both 0.1 and 5 mM KCl treatments, but the increasing degree of the former was greater than that of the latter (Fig. 8a). Na+ concentrations in roots also exhibited increasing trends, but the extent under 0.1 mM KCl treatment was greater than that under 5 mM KCl from 6 to 24 h of treatments; thereafter, no significant differences were found between 0.1 and 5 mM KCl treatments (Fig. 8a). With increasing treatment time, K+ concentrations in shoots was relatively stable under both 0.1 and 5 mM KCl treatments, suggesting that P. tenuiflora has a strong ability to maintain K+ concentrations in shoot under 150 mM NaCl plus 0.1 mM KCl treatment. K+ concentrations in roots decreased with increasing treatment time and it was significantly lower under 0.1 mM KCl than under 5 mM KCl after 24 h (Fig. 8b). In addition, no significant differences were observed in ST values between 0.1 and 5 mM KCl treatments from 6 to 24 h; thereafter, the

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magnitude of the latter was higher than that of the former (Fig. 8c). Based on above results, a significant positive correlation was found between ST values and expression levels of PtSOS1 in roots (Fig. 8d).

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Structural and evolutionary analysis of PtSOS1 in P. tenuiflora

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Structural analysis showed that the PtSOS1 has 11 transmembrane domains in its N-terminal portion and a long cytoplasmic tail at the carboxyl terminus (Fig. S1). Previous studies showed that the partial truncation of Nha1p C-terminus improved the tolerance of yeast cells to Na+, Li+ and Rb+ or decreased Na+ and Li+ export activity, suggesting that the long cytoplasmic tail functions as a regulatory domain (Kinclová et al. 2001). In plants, the long cytosolic tail in SOS1 was suggested to interact with various regulatory proteins such as RCD1, a regulator of oxidative stress responses (Katiyar-Agarwal et al. 2006). Furthermore, a highly conserved cyclic nucleotide binding domain was found in the C-terminal region of SOS1 in plant species (Shi et al. 2000; Oh et al. 2007; Maughan et al. 2009). In this study, we found that the C-terminal region of PtSOS1 also contains a cyclic nucleotide binding domain (Fig. S1). Donaldson et al. (2004) found that cyclic GMP (cGMP) could regulate Ca2+ signal pathway. Furthermore, a membrane-permeable cGMP homologue could induce the expression of AtSOS1 and further

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Treatment time (h) Fig. 5. Time courses of PtSOS1 expression in Puccinellia tenuiflora under 25 mM NaCl plus 0.1 or 5 mM KCl over a 96-h period. (a) Semiquantitative RT–PCR analysis of PtSOS1 in shoot and root. (b) The relative expression level of PtSOS1 in shoot and root (related to ACTIN). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Data are means s.e. (n = 3).

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Fig. 6. Time courses of Na+ and K+ concentration in Puccinellia tenuiflora under 25 mM NaCl plus 0.1 or 5 mM KCl over a 96-h period. (a) Na+, (b) K+ concentration in shoot and root and (c) selective transport (ST) capacity for K+ over Na+ (see legend to Fig. 2 for details of ST). Ten plants were pooled in each replicate (n = 8). (d) Relationship between ST values and relative PtSOS1 expression level in root under 25 mM NaCl plus 0.1 or 5 mM KCl treatments for 6–96 h. Data are means s.e. (n = 3–8).



reduce Na+ influx and increase K+ uptake to enhance salt tolerance of Arabidopsis (Maathuis and Sanders 2001; Maathuis 2006). In addition, the Na+/H+ antiporters were divided into two distinct subgroups corresponding to plasma membrane Na+/H+ antiporters (SOS1) and tonoplast Na+/H+ antiporters (NHX1) (Brett et al. 2005). Phylogenetic analysis showed that PtSOS1 was evolutionarily closer to (I) SOS1 isoforms, such as LpSOS1 and TaSOS1, rather than to (II) NHX1 isoforms, such as OsNHX1 and AtNHX1 (Fig. S2). Therefore, it is suggested that PtSOS1 encodes a plasma membrane Na+/H+ antiporter.

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Treatment time (h) Fig. 7. Time courses of PtSOS1 expression in Puccinellia tenuiflora under 150 mM NaCl plus 0.1 or 5 mM KCl over a 96 h period. (a) Semiquantitative RT–PCR analysis of PtSOS1 in shoot and root. (b) The relative expression level of PtSOS1 in shoot and root (related to ACTIN). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Data are means s.e. (n = 3).

The Na+/H+ antiporter SOS1 is the only Na+ efflux protein at the plasma membrane of plants characterised so far (Shi et al. 2002, 2003). It has been shown that salt upregulates SOS1 activities and enhances Na+ exclusion from the cytosol to the extracellular space (Qiu et al. 2002; Quintero et al. 2002). This is due to the increase of the transcription level of SOS1 gene (Shi et al. 2002, 2003). The detailed profiling of SOS1 gene transcripts under a range of conditions covering a wide spectrum of stress doses and exposure times before species-specific regulation of genes that are potentially important for salt tolerance can be assessed (Amtmann 2009). Shi et al. (2000) found that in both roots and 2.4


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Fig. 8. Time courses of Na+ and K+ concentration in Puccinellia tenuiflora under 150 mM NaCl plus 0.1 or 5 mM KCl over a 96-h period. (a) Na+, (b) K+ concentration in shoot and root and (c) selective transport (ST) capacity for K+ over Na+ (see legend to Fig. 2 for details of ST). Ten plants were pooled in each replicate (n = 8). (d) Relationship between ST values and relative PtSOS1 expression level in root under 150 mM NaCl plus 0.1 or 5 mM KCl treatments for 6–96 h. Data are means s.e. (n = 3–8).

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shoots, the expression of AtSOS1 was upregulated by NaCl stress, but its mRNA in roots was more abundant than that in shoots. A similar expression pattern was observed in OsSOS1 (Martinez-Atienza et al. 2007), TaSOS1 (Xu et al. 2008), PhaNHA1 (Takahashi et al. 2009) and ThSOS1 (Oh et al. 2009) respectively. The present study indicated that with the increase of external NaCl concentrations (25–150 mM), mRNA levels of PtSOS1 showed an increasing trend in both shoots and roots, whereas the mRNA levels in roots at 100 and 150 mM NaCl were more abundant than in shoots (Fig. 1). These results suggest that upregulation of SOS1 expression is consistent with the role of SOS1 in Na+ tolerance (Shi et al. 2000; Oh et al. 2009; Cosentino et al. 2010). We note that compared with the sensitive wheat varieties, the salt-tolerant variety Kharchia 65 showed the highest Na+ efflux activity from roots using MIFE (non-invasive microelectrode ion flux technique), meanwhile, expression level of TaSOS1 in roots is also the highest, suggesting that Na+ efflux from wheat root was regulated by SOS1 gene (Cuin et al. 2011). With the increase of NaCl concentrations, root 22Na+ efflux rate increased gradually in P. tenuiflora (Wang et al. 2009) and the expression of PtSOS1 was also upregulated in roots (Fig. 1). Furthermore, additional NaCl had no impact on Na+ concentration in shoots (Fig. 2a) and K+ concentrations in shoots was unaffected among stress treatments (Fig. 2b). One likely explanation is that 92–95% of unidirectional Na+ influx in P. tenuiflora root was pumped out to the external solution under salt stress, which led to the reduction of Na+ accumulation in plants and further contributed to maintain a higher K+/Na+ ratio (Wang et al. 2009). We note that overexpression of PtSOS1 can significantly reduce Na+ accumulation and increase K+/Na+ ratio in Arabidopsis compared with wild-type in response to NaCl stress (Wang et al. 2011). Recently, our study found that P. tenuiflora infected by the soil microbe Bacillus subtilis (GB03) exhibited increased transcription levels of PtSOS1, K+/Na+ ratio and reduced whole-plant Na+ accumulation compared with control under 200 mM NaCl treatment (JL Zhang JL, PW Paré, unpubl. data). These results suggest that PtSOS1 mediates Na+ efflux from root, which reduces Na+ accumulation and increases K+/Na+ ratio in P. tenuiflora under salt stress. PtSOS1 might play key role in the selective transport for K+ over Na+ in P. tenuiflora Potassium is an essential element for plant growth and plays several important roles in many basic cellular functions, such as osmoregulation, electrical neutralisation of anionic groups, control of cell membrane polarisation and co-transport of sugar (Clarkson and Hanson 1980). It is also crucial in maintaining low levels of protease and endonuclease activity and preventing plant cell damage and death under salt stress (Demidchik et al. 2010). A previous study showed that restricting unidirectional Na+ influx into roots with a strong selectivity for K+ over Na+ and consequently promoting K+ uptake and transport by roots seemed likely to contribute to salt tolerance of P. tenuiflora (Wang et al. 2009). In the present study, a significant positive correlation was found between expression levels of PtSOS1 and ST values in roots of P. tenuiflora exposed to different NaCl (0–150 mM), KCl (0–10 mM) or to 150 mM NaCl

Q. Guo et al.

plus 0.1 or 5 mM KCl treatments (Figs 2d, 4d, 8d), suggesting PtSOS1 could be contributed to maintaining the selective transport for K+ over Na+ by roots in P. tenuiflora. This might be first due to the fact that PtSOS1 is involved in Na+ efflux from root (see above). Second, although several studies have shown that SOS1 is a specific transporter for Na+ and cannot transport K+ (Qiu et al. 2002; Quintero et al. 2002; Shi et al. 2002), the atsos1 mutant was defective in high affinity K+ uptake (Wu et al. 1996) and K+ content was significantly lower than in the wildtype plants under salt stress (Zhu et al. 1998) and overexpression of seagrass (Cymodocea nodosa) CnSOS1 and AtSOS1 in an E. coli strain which is deficient in K+ uptake systems led to the recovery of low-affinity K+ uptake (Garciadeblás et al. 2007), suggesting that the activity of SOS1 is correlated with potassium nutrition (Kronzucker and Britto 2011). Qi and Spalding (2004) found no significant differences in K+ uptake ability of root cells between atsos1 mutant and wild-type plants under Na+ free media with 5 mM NH4+ (in growth medium with millimolar NH4+ concentrations, which blocks non-channel K+ uptake systems, Arabidopsis seedlings rely on the AKT1 channel for physiologically relevant K+ uptake), but the addition of 50 mM NaCl strongly inhibited the K+ uptake ability of rootcell in atsos1 mutants compared with wild-type plants. Therefore, it was thought that SOS1 was necessary for protecting the K+ uptake mediated by AKT1 on which growth depends and the elevated cytoplasmic Na+ levels resulting from loss of SOS1 function impaired K+ uptake ability in root cells and compromised K+ nutrition under salt stress (Qi and Spalding 2004). Meanwhile, recent evidence implicated that the expression of TPK1 (two pore K+ channel) and the corresponding protein activity were drastically declined by salt stress in atsos1 mutant (Oh et al. 2010), indicating that SOS1 participates in and regulates K+ uptake and transport by roots (Pardo et al. 2006). Notably, the downregulation of ThSOS1 by RNA interference decreased the salt tolerance of Thellungiella salsuginea and triggered the fragmentation of xylem parenchyma cells (XPCs) and the destruction of membrane integrity in above cells (Oh et al. 2009). Integrating these findings, we propose a model for explaining the function of SOS1 in the selective transport for K+ over Na+ by roots as follows (Fig. 9). Under salt stress, SOS1 at the plasma membrane of XPCs loads Na+ directly into the xylem or mediates cytoplasmic Na+ efflux to the neighbouring apoplastic spaces and then Na+ diffusing into xylem, which ameliorates cellular Na+ toxicity (Shi et al. 2002). However, Na+ in xylem was delivered rapidly to shoots via transpiration stream, overaccumulated Na+ in shoots would damage the photosynthetic tissues. HKT (high affinity K+ transporter)-like proteins could alleviate this damaging process by mediating Na+ uptake from xylem vessels and the neighbouring apoplastic spaces into XPCs to limit Na+ transport to shoots (Ren et al. 2005; Sunarpi et al. 2005; Byrt et al. 2007). In contrast, the retrieval of Na+ by HKT possibly causes membrane depolarisation of XPCs and triggers K+ penetration into the xylem via membrane depolarisation-induced K+ efflux channels such as SKOR (Stelar K+ outward rectifier) (Wegner and Raschke 1994; Wegner and De Boer 1997; Gaymard et al. 1998), which consequently promotes K+ transport to shoots (Horie et al. 2009; Hauser and Horie 2010; Shabala et al. 2010). However, once excessive, accumulation of Na+ in XPCs would cause cellular Na+ toxicity, resulting in disorder



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osmotic adjustment when Na+ accumulation below the capacity of sequestering Na+ in their vacuoles (Blumwald et al. 2000). A recent study found that salt-tolerant barley varieties were capable of orchestrating raise in both Na+ and K+ concentration in xylem and efficiently sequestered the accumulated Na+ in leaves (Shabala et al. 2010). Under the above conditions, it appears that the transport activities of SOS1 outweigh HKT at the membrane of XPCs and therefore, the direction of Na+ transport is loading. According to the presented model (Fig. 9), we disagree with the hypothesis that SOS1 functions in retrieving Na+ from the xylem stream under severe salt stress (Shi et al. 2002). We suggest that when Na+ in vacuoles of leaves reaches its maximum concentration while plant exposed to severe salt conditions, it seems that the reasonable interpretation about Na+ retrieval from the xylem is that the transport activities of HKT overwhelm SOS1 at the membrane of XPCs and thus, Na+ is unloaded.

Xylem parenchyma cell

Activation by depolarisation?

Acknowledgements This work was supported by the National Natural Science Foundation of China (grant Nos. 31170431 and 31172256) and the PhD Programs Foundation of Ministry of Education of China (grant no. 20090211110001). We are grateful to the anonymous reviewers for their valuable suggestions and comments on the initial version of the manuscript.

Xylem

Apoplast

Fig. 9. Schematic model for the function of SOS1 in regulating K+ and Na+ transport at the membrane of xylem parenchyma cells (XPCs) by sustaining the membrane integrity in plants subjected to salt stress. At the XPCs, SOS1 loads Na+ into xylem (Shi et al. 2002), whereas HKT-like proteins mediate the reverse Na+ flux and unload Na+ from xylem vessels to prevent Na+ overaccumulation in shoots (Ren et al. 2005; Sunarpi et al. 2005; Byrt et al. 2007). Na+ unloading into XPCs possibly depolarises their plasma membrane, which in turn could activate SKOR to load K+ into xylem (Wegner and De Boer 1997; Gaymard et al. 1998; Horie et al. 2009). The opposite Na+ fluxes mediated by SOS1 and HKT must be finely coordinated to Na+ transport, which contribute to sustain the membrane integrity of XPCs, thus, regulating normal HKT and SKOR transport activity at the membrane of XPCs and further maintaining the selective transport capacity for K+ over Na+ by roots.

of or damage to membrane systems, which affects K+ loading into xylem and Na+ unloading from xylem mediating by SKOR and HKT respectively. Therefore, the opposite Na+ fluxes mediated by SOS1 and HKT must be finely co-ordinated to preserve the membrane integrity, which regulates normal HKT and SKOR transport activity at the membrane of XPCs, thereby maintaining the selective transport capacity for K+ over Na+ by roots. Taken together, these suggested PtSOS1 might be a key determinant for maintaining a high selective transport capacity for K+ over Na+ by roots in P. tenuiflora. Our suggested model for the function of SOS1 at xylemparenchyma interface (Fig. 9) is consistent with the model that SOS1 functions in loading Na+ into the xylem under mild salt stress (25 mM NaCl) proposed by Shi et al. (2002). There was no significant correlation between ST values and expression levels of PtSOS1 in roots of P. tenuiflora treated with 25 mM NaCl plus 0.1 or 5 mM KCl (Fig. 6d), suggesting PtSOS1 mediates loading Na+ into xylem and then being transported to shoots for

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