AtHKT1;1 and AtHAK5 mediate low-affinity Na + uptake in Arabidopsis thaliana under mild salt stress

Transcription

1 DOI /s ORIGINAL PAPER AtHKT1;1 and AtHAK5 mediate low-affinity Na + uptake in Arabidopsis thaliana under mild salt stress Qian Wang Chao Guan Pei Wang Mao-Lin Lv Qing Ma Guo-Qiang Wu Ai-Ke Bao Jin-Lin Zhang Suo-Min Wang Received: 23 April 2014 / Accepted: 4 August 2014 Ó Springer Science+Business Media Dordrecht 2014 Abstract Salinity is a serious problem for agricultural production worldwide. Reducing Na? influx is one of the key steps for controlling Na? accumulation in plants and improving salt tolerance of crop plants. Researches on a number of species are now converging on HKT-type and KUP/HAK/KT type proteins, both of them are probable candidates of Na? uptake into the root. To assess the contribution of AtHKT1;1 and AtHAK5 to low-affinity Na? uptake in Arabidopsis thaliana, the 22 Na? influx in A. thaliana wild type (WT) and hkt1;1 mutant (athkt1;1) with or without inhibitors (10 mm TEA? or 5 mm NH? 4 ) were investigated, in addition, the expression levels of AtH- KT1;1 and AtHAK5 in plants exposed to different concentrations of NaCl, KCl or KCl plus NaCl were analyzed. Results showed that TEA? or NH? 4 have no significant influence on 22 Na? influx in WT, but reduced 22 Na? influx by 42 and 46 %, respectively, in athkt1;1. Under 25 mm NaCl, 0.01 mm K? facilitated higher net Na? uptake rate in both WT and athkt1;1 than 2.5 mm K?. In addition, 0.01 mm K? down-regulated AtHKT1;1 and up-regulated Qian Wang and Chao Guan have contributed equally to this work. Electronic supplementary material The online version of this article (doi: /s ) contains supplementary material, which is available to authorized users. Q. Wang C. Guan P. Wang M.-L. Lv Q. Ma A.-K. Bao J.-L. Zhang S.-M. Wang (&) State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou , People s Republic of China smwang@lzu.edu.cn G.-Q. Wu School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou , People s Republic of China AtHAK5 in WT roots compared with 2.5 mm K?, and more interestingly, the transcript of AtHAK5 in athkt1;1 roots was always higher than that in WT roots during 48 h of 2.5 mm K? plus 25 mm NaCl, and it increased continuously during 48 h of 0.01 mm K? plus 25 mm NaCl. Therefore, it is proposed that AtHKT1;1 and AtHAK5 mediate low-affinity Na? uptake, and both of them are regulated by external K? concentrations. AtHKT1;1 might mediate low-affinity Na? uptake under 2.5 mm K?, while 0.01 mm K? might activate AtHAK5 and facilitate lowaffinity Na? uptake in WT. When AtHKT1;1 lost its function, AtHAK5 might mediate low-affinity Na? uptake instead of AtHKT1;1 and this function becomes more important under low K? condition. Keywords Mild salt stress Low-affinity Na? uptake 22 Na? influx Net Na? uptake rate AtHKT1;1 AtHAK5 Abbreviations AKT Arabidopsis K? transporter ANOVA Analysis of variance Ct Threshold cycle HAK High-affinity K? transporter HKT High-affinity K? transporter KIRC K? inward rectifying channel KORC K? outward rectifying channel KT K? transporter KUP K? uptake transporter NHX Tonoplast Na? /H? antiporter PCR Polymerase chain reaction RFW Root fresh weight SE Standard error SOS1 Plasma membrane Na? /H? antiporter TEA Tetraethylammonium WT Wild type

2 Introduction Salinity is one of adverse environments that limits production of crop plants worldwide (Zhu 2001; Flowers 2004). High [NaCl] ext disturbs intracellular ion homeostasis, leads to cell membrane dysfunction and attenuation of metabolic activity, which cause growth inhibition and lead ultimately to plant death (Rains and Epstein 1967; Hasegawa et al. 2000). In order to cope with salt stress, multifarious adaptation strategies have evolved in plants, including the extrusion of Na? from the root, compartmentalization of Na? into the vacuole and reducing Na? influx into the root (Tester and Davenport 2003; Zhang et al. 2010; Kronzucker and Britto 2011). It has been demonstrated that the extrusion of Na? out of cell and compartmentalization of Na? into the vacuole are mediated by SOS1 (Shi et al. 2000; Xu et al. 2008; Chen et al. 2010) and NHX (Yamaguchi and Blumwald 2005; Apse and Blumwald 2007; Wu et al. 2011), respectively. The pathways by which plants take up Na? are still unclear. It is evident that Na? can be transported into the cell through K? carriers, although the mechanisms for Na? influx across the plasma membranes have not yet been established (Blumwald et al. 2000; Munns and Tester 2008). Transport systems with not only high-affinity for K? but also lowaffinity for Na? include KIRCs, KORCs, HKTs, and KUP/ HAK/KT family (Maathuis and Amtmann 1999; Blumwald et al. 2000; Schachtman 2000). AtHKT1;1 isolated from Arabidopsis thaliana appears to function as a Na? -selective uniporter that controls Na? uptake when studied in heterologous expression systems (Uozumi et al. 2000). In planta, Rus et al. (2001) demonstrated that athkt1;1 (under a sos3 mutant background) had lower total tissues Na? accumulation than WT, suggesting a potential role of AtHKT1;1 in Na? absorption by root. However, Berthomieu et al. (2003) found that loss-offunction of AtHKT1;1 did not reduce Na? influx, but changed Na? distribution (the more Na? in the shoot and the less in phloem), indicating that the characteristic of AtHKT1;1 was loading Na? into the phloem sap in the shoot and unloading Na? in the root to perform Na? recirculation from shoot to root. Sunarpi et al. (2005) pointed out that AtHKT1;1 was localized to the plasma membrane of xylem parenchyma cells in the shoot, and found that athkt1;1 had less phloem Na? accumulation and more xylem Na? accumulation than WT in the shoot, therefore, proposed that AtHKT1;1 protected leaf from Na? stress by retrieval of Na? from the xylem and recirculation of Na? to root via the phloem. Davenport et al. (2007) elucidated that AtHKT1;1 contributed to control both root accumulation of Na? and retrieval of Na? from the xylem, but did not involve in influx in the root or recirculation in the phloem. However, Zhang et al. (2008) found that the soil bacterium Bacillus subtilis GB03- exposed plants had less AtHKT1;1 transcript in the root and accumulated less Na? either in the root or in a whole plant under 100 mm NaCl, implying that AtHKT1;1 involved in Na? uptake in the root. Therefore, whether AtHKT1;1 mediates Na? entry in A. thaliana roots remains to be further researched. K? uptake transporters from bacteria named KUP (Schleyer and Bakker 1993) and high-affinity K? transporters from fungi named HAK (Bañuelos et al. 1995; Haro et al. 1999) comprise a family of K? transporters that are also present in plants. The expression levels of their genes are strongly induced by K? starvation (Fu and Luan 1998; Rubio et al. 2000; Bañuelos et al. 2002; Ahn et al. 2004; Martínez-Cordero et al. 2004; Gierth et al. 2005). However, other researchers also implied that several members of this family were linked to permeate Na? from soil into root. Takahashi et al. (2007) proved that the expression of PhaHAK5 was induced by salt stress in reed (Phragmites australis) and it was more remarkable in salt-sensitive species, further study showed that PhaHAK5 could absorb Na? in yeast heterologous system. Benito et al. (2012) confirmed that HAK transporters from moss (Physcomitrella patens) and yeast (Yarrowia lipolytica) mediated Na? uptake. Zhang et al. (2013) indicated that a KUP/HAK/KT type transporter might mediate low-affinity Na? uptake in Suaeda maritima. However, whether KUP/HAK/KT family involves in low-affinity Na? uptake in A. thaliana is still undiscovered. In the present work, 22 Na? influx, net Na? and K? uptake rate and the expression patterns of AtHKT1;1 and AtHAK5 were investigated in both WT and athkt1;1. Finally, we hypothesized a function model of AtHKT1;1 and AtHAK5 in mediating Na? uptake in A. thaliana. AtHKT1;1 and AtHAK5 could coordinately mediated lowaffinity Na? uptake in WT roots under mild salt stress (25 mm NaCl), which was affected by external K? concentrations. AtHAK5 might also participate in low-affinity Na? uptake in athkt1;1 roots under mild salt stress, which was more remarkable at 0.01 mm K? concentrations. Materials and methods Plant materials and growth conditions Seeds of Arabidopsis thaliana wild type (WT) and hkt1;1 mutant (athkt1;1) (the seeds are kindly provided by Prof. Mark Tester) were sterilized for 3 min with 75 % (v/v) ethanol and 5 % (v/v) bleacher, respectively, and rinsed 5 times with distilled water, soaked in distilled water at 4 C

3 for 2 days. Then seeds were sown on substrate containing 1.2 % (w/v) agar and 0.5 % (w/v) sucrose dissolved in modified Hoagland nutrient solution (2 mm KNO 3, 0.5 mm KH 2 PO 4, 0.5 mm MgSO 4 7H 2 O, 0.25 mm Ca(NO 3 ) 2 4H 2 O, 1.25 mm CaCl 2 2H 2 O, 0.06 mm Fe-citrate, 50 lm H 3 BO 3, 10 lm MnCl 2 4H 2 O, 1.6 lm ZnSO 4 7H 2 O, 0.6 lm CuSO 4 5H 2 O, 0.05 lm Na 2 MoO 4-2H 2 O) for sterile culture (ph was adjusted to 5.7 by 1 M Tris). After 3 weeks, all the seedlings were transferred to black painted containers containing above Hoagland nutrient solution. Solutions were renewed every 4 days. All the seedlings were grown in the same chamber. The environmental conditions were as follows: 22 ± 2 C/ 20 ± 2 C (day/night), 200 lmol m -2 s -1 photon flux density with 8/16 h for the day/night cycle, 60 ± 5% relative humidity. 22 Na? influx experiments Four-week-old plants were transferred to modified Hoagland nutrient solution supplemented with 25 mm NaCl for 24 h. Then the seedlings were used to evaluate 22 Na? influx according to the method described by Essah et al. (2003). Roots were pretreated for 10 min in nutrient solution containing inhibitors (10 mm TEA-Cl or 5 mm NH 4 Cl) and 25 mm unlabeled NaCl, with a change of solution after 5 min, and then transferred to above corresponding solution (10 ml) labeled with kbq L -1 of 22 Na? as uptake solution. After 2 min, roots were removed from the uptake solution, blotted, and transferred to 200 ml of ice-cold NaCl (25 mm plus 20 mm CaCl 2 ) for two successive rinses of 2 min and then a further rinse of 3 min. Finally, the roots were blotted gently, weighed, and transferred to glass vials containing 2.5 ml of Optiphase Hisafe (Fisher Chemicals, Loughborough) and 22 Na? uptake was determined using a scintillation counter (LS 6000 IC, Reckman Coulter). 22 Na? influx was calculated as: counts/specific activity/time/root fresh weight (RFW) and expressed as nmol/g RFW/min. Net Na? and K? uptake rate measurements Six-week-old plants were used for the following treatments, respectively: (1) plants were grown at the modified Hoagland nutrient solution for 7 days, then they were treated with modified Hoagland nutrient solutions supplemented with additional 25 mm NaCl for 96 h. (2) plants were subjected to 0.01 mm K? for 7 days (the modified Hoagland nutrient solution deprived of KNO 3 and KH 2- PO 4, 2 mm KNO 3 was substituted by 2 mm HNO 3, 0.5 mm KH 2 PO 4 was substituted by 0.5 mm H 3 PO 4, and 0.01 mm K? was provided by 0.01 mm KCl, ph was adjusted to 5.7 by 1 M Tris), then they were treated with above modified Hoagland nutrient solutions containing 0.01 mm K? supplemented with additional 25 mm NaCl for 96 h. Net uptake rate of Na? and K? were calculated according to the following equation described by Wang et al. (2007, 2009): Using the control data as an example, as (Na? content in whole plant of control Na? content in whole plant before NaCl treatments)/(fresh weight of control root 9 treatment time) and expressed as nmol/ grfw/min. Real-time quantitative PCR analysis Six-week-old plants were used for the following treatments, respectively: (1) plants were grown at the modified Hoagland nutrient solution for 7 days, then they were treated with modified Hoagland nutrient solutions with 25 mm NaCl, and roots were harvested at 0, 3, 6, 12, 24 and 48 h, respectively. (2) plants were subjected to 0.01 mm K? for 7 days (the modified Hoagland nutrient solution deprived of KNO 3 and KH 2 PO 4, 2 mmkno 3 was substituted by 2 mm HNO 3, 0.5 mmkh 2 PO 4 was substituted by 0.5 mm H 3 PO 4, and 0.01 mm K? was provided by 0.01 mmkcl, ph was adjusted to 5.7 by 1 mm Tris), then they were treated with above modified Hoagland nutrient solutions containing 0.01 mm K? with 25 mm NaCl, and roots were harvested at 0, 3, 6, 12, 24 and 48 h, respectively. Total RNA were extracted with the RNAprep pure plant Kit (TianGen, Biotech Co., Ltd, Beijing, China) following the manufacturer s instructions. First strand cdna was synthesized with MMLV-reverse transcriptase (Takara, Biotech Co., Ltd, Dalian, China). The reverse transcribed cdna samples were used for real-time quantitative PCR, which was performed on a thermal cycler (ABI PRISM 7500, USA). A specific fragment (116 bp) of AtHKT1;1 was amplified with a pair of primers P1 and P2 (Table S1). Another specific fragment (107 bp) of AtHAK5 was amplified with a pair of primers P3 and P4 (Table S1). ACTIN was used for RNA normalization, the specific primers of ACTIN that amplified a 170 bp fragment were A1 and A2 (Table S1). SYBR Green PCR master mix (Takara, Biotech Co., Ltd, Dalian, China) was used for 20 ll PCR reactions as follow: 95 C for 30 s, and 40 cycles of 95 C for 5 s and 60 C for 34 s. Each sample was assayed three times. The relative expression levels of all the samples were calculated and analyzed (ABI PRISM 7500 sequence detection system), the Ct value of target genes and ACTIN in different samples were obtained after quantitative real-time PCR reaction. In brief, the normalizer ACTIN Ct value was subtracted from the gene of interest Ct (target gene) to produce the dct value of the sample. The dct value of the calibrator (the sample with the 0 h dct value in our experiment) was subtracted from every other sample to produce the ddct value. Two to the

4 ddct power (2 ddct ) was taken for every sample as the relative expression levels (Livak and Schmittgen 2001). Statistical analysis Results of 22 Na? influx, net Na? and K? uptake rate and genes expression levels are presented as means with SE and data analysis was performed by ANOVA using SPSS statistical software (Ver. 13.0, SPSS Inc., Chicago, IL, USA). Duncan s multiple range test was used to detect a difference between means at a significance level of P \ Results TEA? or NH 4? inhibits 22 Na? influx of athkt1;1 under mild salt stress TEA? inhibits K? uptake through most of K? channels (such as AKT) and some other K? transporters (such as KUP/HAK/KT) (Hedrich and Schroeder 1989; Tester 1990; Fu and Luan 1998). HAK5 as an important member of KUP/HAK/KT family has specific sensibility to NH 4? (Santa-María et al. 1997, 2000; Martínez-Cordero et al. 2004, 2005; Nieves-Cordones et al. 2007). Therefore, we firstly analyzed the effect of TEA? or NH 4? on 22 Na? influx in WT and athkt1;1. Results showed that there was no significant difference on 22 Na? influx between WT and athkt1;1 under 25 mm NaCl without TEA? or NH 4? (control condition) (Fig. 1). TEA? or NH 4? significantly reduced 22 Na? influx of athkt1;1 by 42 and 46 %, respectively, while had no effect on that of WT (Fig. 1), suggesting a Na? uptake pathway that was insensitive to TEA? or NH 4? existed in WT, whereas another one that was sensitive to TEA? or NH 4? existed in athkt1;1 under mild salt stress. Net Na? uptake rate is enhanced under low K? condition Neither external K? concentrations nor loss-of-function of AtHKT1;1 had significant influence on net Na? uptake rate without salt stress (Supplemental Fig. S1a). Under 25 mm NaCl, the treatment of 0.01 mm K? significantly increased net Na? uptake rate in both WT (288 %) and athkt1;1 (75 %) compared with 2.5 mm K?. However, net Na? uptake rate between WT and athkt1;1 had no difference under 2.5 or 0.01 mm K? condition (Fig. 2a). Results indicated that Na? uptake by roots was induced by K? deficiency, which seemed to be irrelevant to AtHKT1;1. After treatment of low K? (0.01 mm) for 7 days, K? presented extrusion in both WT and athkt1;1 without salt Fig. 1 Effects of TEA? (10 mm) and NH 4? (5 mm) on root 22 Na? influx of A. thaliana (WT and athkt1;1) under mild slat stress (25 mm NaCl). Four-week-old seedlings were transferred to modified Hoagland nutrient solution supplemented with 25 mm NaCl for 24 h. Then seedlings were transferred to modified Hoagland nutrient solution supplemented with corresponding concentrations of NaCl with or without 10 mm TEA? or 5 mm NH 4? for 10 min before they were transferred into the above corresponding solution labeled with 22 Na?. Five plants were pooled in each replicate (n = 6). Values are mean ± SE and bars indicate SE. Columns with different letters indicate significant differences at P \ 0.05 (Duncan s test) stress (Supplemental Fig. S1b). In addition, 25 mm NaCl inhibited net K? uptake rate of athkt1;1 compared with WT under 2.5 mm K? level (Fig. 2b). Expression of AtHKT1;1 responds to 2.5 or 0.01 mm K? plus 25 mm NaCl in WT roots After treatment of 0.01 mm K? for 7 days, the transcript level of AtHKT1;1 down-regulated notably (0 h) (Fig. 3). Under 2.5 mm K? with 25 mm NaCl, the expression level of AtHKT1;1 was induced by salt, peaked at 6 h. Under 0.01 mm K? with 25 mm NaCl, the mrna level of AtHKT1;1 also was induced by short-term (3 h) of salt stress. Interestingly, the expression level of AtHKT1;1 under 0.01 mm K? was always lower than that under 2.5 mm K? during 48 h of salt stress. Thus, the expression of AtHKT1;1 was restrained by low K? condition, though it was induced by short-term of mild salt stress. Expression of AtHAK5 responds to 2.5 or 0.01 mm K? plus 25 mm NaCl in WT roots With prolonging of salt stress, the transcript abundance of AtHAK5 was increased and peaked at 3 h, then displayed a decreasing trend under both 2.5 and 0.01 mm K? conditions and almost was down-regulated completely to null at 24 and 48 h, respectively (Fig. 4). In addition, the expression level of AtHAK5 under 0.01 mm K? was much higher than that under 2.5 mm K? during 12 h of salt stress (Fig. 4). The results indicated that the expression

5 Fig. 4 Time courses of AtHAK5 expression in WT roots under 2.5 or 0.01 mm K? with 25 mm NaCl. Real-time quantitative PCR analysis of AtHAK5 mrna in 6-week-old plants that treated with 2.5 or 0.01 mm K? for 7 days and then were exposed to 25 mm NaCl over a 48 h period (see legend to Real-time quantitative PCR analysis for details of treatment). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Values are mean ± SE (n = 3) and bars indicate SE Fig. 2 Net Na? a,k? b uptake rate of A. thaliana (WT and athkt1;1) under 2.5 or 0.01 mm K? with 25 mm NaCl. Six-week-old plants treated with 2.5 or 0.01 mm K? for 7 days and then were exposed to 25 mm NaCl for 96 h to calculate net Na? and K? uptake rate (see legend to Net Na? and K? uptake rate measurements for details of treatment). Five plants were pooled in each replicate (n = 6). Values are mean ± SE and bars indicate SE. Columns with different letters indicate significant differences at P \ 0.05 (Duncan s test) level of AtHAK5 could be induced not only by low K? but also by short-term mild salt stress. Expression of AtHAK5 responds to 2.5 or 0.01 mm K? plus 25 mm NaCl in athkt1;1 roots With prolonging of salt stress, the expression of AtHAK5 had no difference between 2.5 and 0.01 mm K? before 3 h (Fig. 5). After that, the expression of AtHAK5 begun to be down-regulated sharply and almost was decreased completely to null at 48 h under 2.5 mm K? with 25 mm NaCl. On the contrary, the expression of AtHAK5 increased constantly until 48 h under 0.01 mm K? with 25 mm NaCl (Fig. 5). The results showed that loss-of-function of AtHKT1;1 had significant influence on the expression pattern of AtHAK5, and this effect exhibited opposite trend under different K? concentrations. Different expression pattern of AtHAK5 between WT and athkt1;1 in A. thaliana roots Fig. 3 Time courses of AtHKT1;1 expression in WT roots under 2.5 or 0.01 mm K? with 25 mm NaCl. Real-time quantitative PCR analysis of AtHKT1;1 mrna in 6-week-old plants that treated with 2.5 or 0.01 mm K? for 7 days and then were exposed to 25 mm NaCl over a 48 h period (see legend to Real-time quantitative PCR analysis for details of treatment). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Values are mean ± SE (n = 3) and bars indicate SE The expression patterns of AtHAK5 in WT and athkt1;1 roots had been described in Figs. 4 and 5, respectively. Here we compared the transcript level of AtHAK5 in WT roots with that in athkt1;1 roots under 2.5 mm K?, finding that the latter was always higher than the former during 12 h of mild salt stress (Fig. 6a). And likewise, the similar trend was presented under 0.01 mm K?, in addition, with prolonging of salt treatment, the transcript level of AtHAK5

6 Fig. 5 Time courses of AtHAK5 expression in athkt1;1 roots under 2.5 or 0.01 mm K? with 25 mm NaCl. Real-time quantitative PCR analysis of AtHAK5 mrna in 6-week-old plants that treated with 2.5 or 0.01 mm K? for 7 days and then were exposed to 25 mm NaCl over a 48 h period. ACTIN was used as an internal control (see legend to Real-time quantitative PCR analysis for details of treatment). Experiments were repeated at least three times to obtain similar results. Values are mean ± SE (n = 3) and bars indicate SE in athkt1;1 roots up-regulated constantly rather than downregulated gradually in WT roots (Fig. 6b), indicating that functional deficiency of AtHKT1;1 would induce the expression level of AtHAK5. Discussion The hypothesis that K? transport systems also mediate Na? entry derived from studies conducted more than 40 years ago, which established that Na? adversely affects K? acquisition (Rains and Epstein 1967; Maathuis et al. 1996). HKT and KUP/HAK/KT families are two kinds of important high-affinity K? transporters, and are regarded as mediating Na? uptake (Mäser et al. 2002). In the present work, we investigated net Na? uptake rates of both WT and athkt1;1, finding that they had no significant difference with or without 25 mm NaCl (Figs. 2, S1a). There was also no significant difference on 22 Na? influx between WT and athkt1;1 under mild salt stress without TEA?? or NH 4 (Fig. 1). Berthomieu et al. (2003) used excised roots to examine Na? uptake in sas2-1 (loss-of-function of AtH- KT1;1), which was 20 % higher than WT, indicating that AtHKT1;1 did not mediate Na? uptake in A. thaliana roots. However, a contribution of AtHKT1;1 to Na? uptake could not be ruled out, because other transporters maybe take the place of AtHKT1;1 to mediate Na? uptake in sas2-1. Whether AtHKT1;1 is a salt tolerance determinant that controls Na? entry into A. thaliana roots remains to be further elucidated. Fig. 6 Time courses of AtHAK5 expression in both WT and athkt1 roots under 2.5 a or 0.01 b mm K? with 25 mm NaCl. Real-time quantitative PCR analysis of AtHAK5 mrna in 6-week-old plants that treated with 2.5 or 0.01 mm K? for 7 days and then were exposed to 25 mm NaCl over a 48 h period (see legend to Real-time quantitative PCR analysis for details of treatment). ACTIN was used as an internal control. Experiments were repeated at least three times to obtain similar results. Values are mean ± SE (n = 3) and bars indicate SE Previous researches had pointed out that TEA? could restrain the activity of several members of KUP/HAK/KT type transporter (Hedrich and Schroeder 1989; Tester 1990; Fu and Luan 1998), whereas HKT family was insensitive to it (Liu et al. 2001; Garciadeblás et al. 2003). HAK5 as a key member of KUP/HAK/KT had specific sensibility to NH 4? (Spalding et al. 1999; Rubio et al. 2008, 2010). We found that 22 Na? influx was affected by neither TEA? nor NH 4? in WT exposed to 25 mm NaCl (Fig. 1), meanwhile, the expression level of AtHKT1;1 increased steadily within 6 h of salt stress in roots (Fig. 3). Our results manifested that the candidate of low-affinity Na? uptake pathway which is insensitive to TEA? and NH 4? might be AtH- KT1;1 in WT roots. Although the acknowledged function

7 of AtHKT1;1 is involved in Na? unloading from the xylem to surrounding parenchyma cells (Sunarpi et al. 2005; Davenport et al. 2007; Horie et al. 2009) orna? recirculation from shoots to roots (Berthomieu et al. 2003), Rus et al. (2001) had proposed that AtHKT1;1 involved in Na? uptake by roots, and further confirmed by Zhang et al. (2008), founding that reduction of root AtHKT1;1 limited Na? entry into plants. In conclusion, AtHKT1;1 is able to mediate low-affinity Na? uptake in A. thaliana roots. When disruption of AtHKT1;1, we still found that there was high 22 Na? influx in athkt1;1 roots (Fig. 1). Therefore, we assumed that there might be other candidates that involved in low-affinity Na? uptake in athkt1;1 roots. Santa-María et al. (1997) discovered that HvHAK1 not only conferred high-affinity K? uptake to a K? -uptakedeficient yeast mutant, but also transported Na? when this cation was present at millimolar concentrations. Takahashi et al. (2007) reported that the expression of PhaHAK5 was found in salt-sensitive reed, but not in any parts of salttolerant reed; in addition, the K? uptake ability of the yeast strain expressing PhaHAK5 was remarkably lower than that of the yeast strain expressing PhaHAK1 under salt stress, suggesting that PhaHAK5 was one of the routes by which Na? entered cells. Horie et al. (2011) found that OsHAK2 could mediate Na? transport in Escherichia coli cells in the presence of high concentration of extracellular Na?. Most recently, Benito et al. (2012) demonstrated that PpHAK13 was conferred the function of Na? uptake to yeast cell and disruption of the PpHAK13 gene abrogated Na? uptake in P. patens, meanwhile, found that yeast YIHAK1 exhibited the characteristics of Na? uptake. In A. thaliana, it was demonstrated that K? transport by AtKUP1 or AtHAK5 was competitively inhibited by Na? in yeast (Fu and Luan 1998; Rubio et al. 2000). Our results showed that TEA? and NH? 4 decreased 22 Na? influx by 42 and 46 %, respectively, in athkt1;1 roots under mild salt stress (Fig. 1). It was proposed that a low-affinity Na? uptake pathway that might be mediated by a KUP/HAK/KT type transporter, which was sensitive to TEA?, existed in roots of S. maritima when plants exposed to higher external salt concentrations ( mm NaCl) (Wang et al. 2007; Zhang et al. 2013). Simultaneously, the expression level of AtHAK5 in athkt1;1 roots was remarkably higher than that in WT roots when plants exposed to 25 mm NaCl (Fig. 6), indicating AtHAK5 was the candidate of low-affinity Na? uptake pathway that was sensitive to both TEA?? and NH 4 in athkt1;1 roots. Nieves-Cordones et al. (2010) demonstrated that the uptake and accumulation of Na? in T-DNA insertion mutants disrupting AtHAK5 had no remarkable difference compared with WT under 30 mm NaCl, implicating that AtHAK5 did not regulate Na? uptake in A. thaliana. Nevertheless, as we have discussed that AtHAK5 mediated low-affinity Na? uptake in athkt1;1, AtHKT1;1 might mediate low-affinity Na? uptake in athak5. Taken together, our data strongly support the view that both AtHKT1;1 and AtHAK5 might mediate low-affinity Na? uptake in A. thaliana; when any one loses its function, the other one would take place of it to obtain Na? from soil. Although AtHKT1;1 and AtHAK5 have function in lowaffinity Na? uptake in A. thaliana, the conditions of which one plays the leading role need to be elucidated. In our experiments, during 48 h of mild salt stress (25 mm NaCl), the mrna level of AtHKT1;1 in WT roots was induced by short-term (6 h) of salt treatment, simultaneously, the transcript under 2.5 mm K? was always higher than that under 0.01 mm K? (Fig. 3), while the transcript of AtHAK5 under 2.5 mm K? was always lower than that under 0.01 mm K? (Fig. 4), suggesting AtHKT1;1 played an important role in Na? uptake under the condition of normal K? (2.5 mm). Under 0.01 mm K? plus 25 mm NaCl, net Na? uptake rate was significantly increased in both WT and athkt1;1, but it had no difference between WT and athkt1;1 (Fig. 2), implying that AtHKT1;1 did not contributed to increased net Na? uptake rate under 0.01 mm K?. However, the transcript of AtHAK5 under 0.01 mm K? was always higher than that under 2.5 mm K? during 12 h of salt stress (25 mm NaCl) (Fig. 4). In addition, during 48 h of salt stress (25 mm NaCl), the mrna level of AtHAK5 in WT roots was induced strongly by short-term (3 h) of salt treatment, then displayed a decreasing trend (Fig. 4). Fulgenzi et al. (2008) found the transcript of HvHAK1 up-regulated transitorily under salt stress, and was down-regulated gradually with prolonging of salt treatment, meanwhile the concentration of Na? not K? increased in plant during salt stress early period, demonstrating HvHAK1 related to Na? uptake directly. In conclusion, AtHAK5 played an important role in Na? uptake under the condition of low K? (0.01 mm). In athkt1;1 roots, under 0.01 mm K? plus 25 mm NaCl, the expression level of AtHAK5 increased continuously after 6 h of salt stress, while it decreased dramatically under 2.5 mm K? with 25 mm NaCl (Figs. 5, 6), indicating the decisive role of AtHAK5 in low-affinity Na? uptake. According to the present work, our results strongly support the view that AtHKT1;1 might mediate lowaffinity Na? uptake in A. thaliana at 2.5 mm K? concentrations and AtHAK5 might mediate low-affinity Na? uptake at 0.01 mm K? concentrations under mild salt stress. These two transporters could replace each other s function when one of them loses its function. Acknowledgments This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2014CB138701), the National Natural Science Foundation of China (Grant No ) and Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No ).

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