Plant and Cell Physiology Advance Access published March 15, 2006

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1 Plant and Cell Physiology Advance Access published March 15, 2006 Running title: Different Na + sensitivities of athkt1 and sos3 mutants *Corresponding author: Julian I. Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, California Telephone: ; julian@biomail.ucsd.edu; Fax: Subject areas: (2) Environmental and stress responses Number of figures: Black and white (3); Color (4) Plant and Cell Physiology 2006 The Japanese Society of Plant Physiologists (JSPP); all rights reserved. 1

2 Calcium Regulation of Sodium Hypersensitivities of sos3 and athkt1 Mutants. Tomoaki Horie, Rie Horie, Wai-Yin Chan, Ho-Yin Leung, Julian I. Schroeder* Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California San Diego, 9500 Gilman Drive, La Jolla, California , USA Abbreviations: MS, Murashige-Skoog; HKT, high-affinity K + transporter; SOS, salt overly sensitive; ICP-OES, inductively coupled plasma optical emission spectroscopy 2

3 Abstract T-DNA disruption mutations in the AtHKT1 gene have been previously shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na + ) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na + sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na + hypersensitivity compared to wild type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to athkt1 plants, which showed chlorosis in leaves and stems. The Na + content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na + over-accumulation in response to salinity stress. Salinity stress analyses using basic minimal nutrient medium and MS medium revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mm external Ca 2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mm external Ca 2+. Taken together multiple analyses demonstrate that the external Ca 2+ concentration strongly impacts the Na + stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance such that sos3 more strongly causes Na + over-accumulation in roots, whereas athkt1 causes an increase in Na + levels in the xylem sap and shoots and a concomitant Na + reduction in roots. Keywords: Salinity, HKT/Trk/Ktr transporter, Na + transport, Na + sensitivity, Arabidopsis thaliana 3

4 Introduction Glycophytes including most crop plants are sensitive to saline conditions. Excessive salt build up in soils is an increasingly detrimental problem that causes growth impairment and is toxic to shoot and root growth (Glenn et al. 1999, Munns 2002, Zhu 2002, Horie and Schroeder 2004). Increasing soil salinity in irrigated lands is a major threat to agricultural production. Elevated Na + levels directly or indirectly perturb important cellular processes including the functions of vital enzymes (Murguía et al. 1995), K + absorption (Rains and Epstein 1965, Epstein 1998) and photosynthesis (Tsugane et al. 1999). The HKT1 cdna, isolated from wheat, encodes a high-affinity K + /Na + co-transporter, and at high [Na + ] a Na + influx transporter in heterologous systems (Schachtman and Schroeder 1994, Rubio et al. 1995, Gassman et al. 1996). Analyses of HKT transporters from other plants and in bacteria have also shown Na + -stimulated K + uptake (Tholema et al. 1999, Fairbairn et al. 2000, Kawano et al. 2000, Horie et al. 2001, Matsuda et al. 2004, Tholema et al. 2005). Highaffinity K + /Na + co-transport has been observed as a major K + uptake activity in aquatic plants (Walker and Sanders 1991, Maathuis et al. 1996), but in higher plants a K + /Na + uptake activity has not been detected (Maathuis et al. 1996). Rather, K + uptake is mediated by multiple overlapping K + influx pathways (Quintero and Blatt 1997, Santa-Maria et al. 1997, Fu and Luan 1998, Hirsch et al. 1998, Kim et al. 1998, Spalding et al. 1999, Santa-Maria et al. 2000, Reintanz et al. 2002, Ahn et al. 2004, Gierth et al. 2005). Potassium uptake mediated by the distinct family of KUP/HAK/KT transporters is inhibited by extracellular Na +, illustrating a strong K + vs. Na + competition for uptake in this family of transporters (Santa-María et al. 1997, Fu and Luan 1998). At high sodium concentrations K + transport via HKT1 is blocked. Furthermore, at high Na + HKT1 mediates selective Na + influx in oocytes, yeast and wheat (Rubio et al. 1995, Gassman et al. 1996, Diatloff et al. 1998, Rubio et al. 1999, Liu et al. 2000, Laurie et al. 2002). HKT1 homologs have been isolated from many plant species and functionally characterized (Fairbairn et al. 2000, Uozumi et al. 2000, Horie et al. 2001, Golldack et al. 2002, Garciadeblás et al. 2003, Su et al. 2003). 4

5 The Arabidopsis genome encodes a single HKT1 gene, AtHKT1 (Uozumi et al. 2000). Expression of the Arabidopsis transporter AtHKT1 in Xenopus laevis oocytes and Saccharomyces cerevisiae has shown a relatively Na + selective transport activity of AtHKT1 (Uozumi et al. 2000, Mäser et al. 2002b, Berthomieu et al. 2003). Loss-of-function mutations in AtHKT1 render plants Na + hypersensitive and disturb the distribution of Na + between roots and shoots, causing Na + over-accumulation in leaves and chlorosis under salinity stress (Mäser et al. 2002a). Furthermore, an EMS mutation that causes reduced AtHKT1 activity has been shown to cause growth inhibition and Na + over-accumulation in shoots under salt stress (Berthomieu et al. 2003). AtHKT1 expression was found in vascular tissues (Mäser et al. 2002a) and in particular in phloem tissues, causing reduced Na + levels in the phloem sap of the sas2-1 allele in AtHKT1 (Berthomieu et al. 2003). Furthermore, microarray-based mutant mapping identified an athkt1 (FN1148) mutation that causes hyper-accumulation of Na + in shoots (Gong et al. 2004). These findings reveal that AtHKT1 is an important Na + resistance determinant in Arabidopsis and that Na + transport via AtHKT1 is essential for protection of plant leaves against salinity stress in Arabidopsis (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004, Sunarpi et al. 2005). Recent studies demonstrated that AtHKT1 and the close rice ortholog OsHKT8, function in reducing the Na + concentration in the xylem sap (Ren et al. 2005, Sunarpi et al. 2005). Salt overly sensitive sos1 to sos3 mutant loci were identified as essential factors for salt tolerance (Wu et al. 1996, Zhu et al. 1998). SOS3 encodes an EF hand type Ca 2+ binding protein sharing significant sequence similarity with the Ca 2+ binding domain of the calcineurin B subunit of yeast (CnB) and neuronal calcium sensors from animals (Liu and Zhu 1998, Sánchez-Barrena et al. 2005). SOS3 has been shown to physically interact with the SOS2 protein kinase in yeast (Halfter et al. 2000), and the SOS2/SOS3 complex regulates the plasma membrane Na + /H + antiporter SOS1, which is essential for plant salt tolerance (Shi et al. 2000, Qiu et al. 2002, Quintero et al. 2002, Shi et al. 2002). AtHKT1 mutations were found to suppress the Na + hypersensitivity of sos3-1 mutant seedlings (Rus et al. 2001). Interestingly, both sos3 and athkt1 single mutant alleles show Na + hypersensitivity (Liu and Zhu 1998, Mäser et al. 2002a), but athkt1 sos3-1 plants showed 5

6 resistance to salinity stress independent of whether they were grown in agar medium or as mature plants in soil (Rus et al. 2001, Rus et al. 2004). In this study we characterize the complex relationship between these essential salt tolerance determinants to uncover the mechanism underlying the non-additive and counteracting Na + sensitivities of athkt1 sos3 double mutants compared to single sos3-1 and athkt1 mutants. Detailed characterization of athkt1-1 sos3-1 and athkt1-2 sos3-1 double mutants under salt stress show that SOS3 and AtHKT1 function in distinct physiological aspects of salt tolerance and that athkt1 mutations in sos3 plants do not strictly repress the Na + hypersensitivity of sos3 plants to wild type levels, but that the external Ca 2+ concentration strongly impacts Na + stress responses of athkt1 sos3 double mutants. Results athkt1 sos3 double mutant plants show athkt1-like Na + hypersensitivity on soil Previously, two independent T-DNA insertions in the AtHKT1 gene, athkt1-1 and athkt1-2, were shown to suppress the Na + hypersensitivity of sos3-1 plants (Rus et al. 2001), including recent findings in mature soil-grown plants (Rus et al. 2004). To analyze the underlying mechanisms, mature soil-grown Columbia wild type, athkt1-1, sos3, athkt1-1 sos3 and athkt1-2 sos3 plants were subjected to NaCl stress to determine whether salt sensitivity was suppressed in mature athkt1 sos3 double mutant alleles here. Three to four week old plants were watered with 100 mm NaCl twice during 1 week and subsequently watered with de-ionized water for the following week and subsequently analyzed as shown in figure 1. Interestingly, both double mutant alleles clearly showed strong Na + hypersensitivity in all experiments with soil-grown mature plants (Fig. 1D, E, I and J) compared to wild type plants (Fig. 1A, F) (> 50 plants analyzed for each genotype). These findings differ from those of Rus et al. (2004) in which soil-grown athkt1 sos3 double mutant plants showed similar NaCl sensitivity as wild type plants and repression of the sos3 Na + sensitivity. In over half of the plants, early during the salt stress phase, athkt1 sos3 mutant plants appeared less chlorotic 6

7 than athkt1. This early stress phenotype was more apparent when enriched nutrient media were used. But these plants eventually showed chlorosis after several more days, exhibiting consistent salinity stress of athkt1 sos3 double mutant plants compared to wild type controls, as illustrated in Figure 1D and E. Mutants in the AtHKT1 gene have been previously characterized as showing increased salt sensitivity under agar-grown conditions (Mäser et al. 2002a, Gong et al. 2004) and under hydroponic conditions (Berthomieu et al. 2003). Soil-grown mature salt-stressed athkt1-1 plants showed chlorotic withering as well (Fig. 1B). Under salt stress, leaves of double mutant athkt1-1 sos3 and athkt1-2 sos3 plants also showed chlorotic withering (Fig. 1D and E) and stems were also damaged, similar to athkt1-1 plants (Fig. 1G, I and J) (n=50 plants per genotype). On the other hand, salt stressed sos3 plants showed a necrotic yellowing phenotype (Fig. 1C) and normal silique development more similar to wild type than athkt1 under the imposed conditions (Fig.1 F, H). We also performed additional NaCl sensitivity analyses using soil grown plants watered with 50 mm NaCl as used in Rus et al. (2004). Experiments under these conditions also showed that athkt1 sos3 double mutant plants show chlorosis at leaf borders similar to athkt1 single mutant plants, but in contrast to wild type plants (Supplemental Figure 1). sos3 single mutant plants showed a yellowing of leaves under these conditions. athkt1 sos3 double and athkt1 mutants but not sos3 cause strong Na + over-accumulation in the xylem sap We next performed ICP analyses using soil-grown plants to compare levels of Na + accumulation in leaves. Leaves of soil-grown salt-stressed athkt1-1 plants showed a dramatically increased Na + content compared to wild type plants under both 100 mm NaCl (Fig. 2A) and 50 mm NaCl watering conditions (Supplemental Fig. 2). Furthermore, double mutant athkt1-1 sos3 and athkt1-2 sos3 plants also showed Na + over-accumulation in leaves similar to athkt1-1 plants, but unlike wild type plants (Fig. 2A and Supplemental Fig. 2). Increases in the Na + content of shoots compared to wild type were also found in soil-grown sos3 plants (Fig. 2A and Supplemental Fig. 2). 7

8 Expression of the AtHKT1 gene in vascular tissues was shown using transgenic plants carrying AtHKT1 promoter-gus gene constructs (Mäser et al. 2002a, Berthomieu et al. 2003). The sas2-1 mutant, which has a transport-reducing point mutation in the athkt1 gene, shows lower Na + content in the phloem sap compared to wild type (Berthomieu et al. 2003). A recent study has shown that athkt1 single mutants caused an increase in the Na + level of xylem sap and that the AtHKT1 protein is targeted to the plasma membrane of xylem parenchyma cells (Sunarpi et al. 2005). Here, we analyzed whether the Na + content of the xylem sap of athkt1 sos3 plants is affected during salt stress. Xylem sap was collected from soil-grown wild type (n=8) and athkt1-1 plants (n=7) watered with 100 mm NaCl or 1/20 MS solution (non-stress control condition), and the Na + content of the xylem sap was measured by ICP-OES. To evaluate the relative purity of xylem sap samples, we analyzed sucrose levels of the xylem sap by HPLC analysis. Sucrose levels provide a measure of the level of contamination from phloem sap, as phloem sap in Arabidopsis contains high millimolar sucrose levels (Deeken et al. 2002). The average sucrose content of xylem sap samples was 8.02±1.75 µm (n=3). Average sucrose content concentrations have been reported in the phloem sap of Arabidopsis of 341±52 mm (Deeken et al. 2002). Thus our xylem sap samples contain negligibly low (< 0.01%) phloem sap contamination. We measured the Na + content in xylem sap samples of sos3, athkt1, athkt1-1 sos3, and athkt1-2 sos3 soil-grown plants in the presence and absence of salinity stress. Interestingly, xylem sap samples from athkt1 sos3 double mutant plants contained dramatically higher Na + than those from sos3 single mutant and wild type plants (Fig. 2B). The athkt1 sos3 double mutant plants showed a similar phenotype to athkt1-1 (Fig. 2B). In contrast single sos3 mutant plants showed a moderate increase in xylem Na + content similar to wild type plants (Fig. 2B), supporting an important role of AtHKT1 (Sunarpi et al. 2005) in maintaining low Na + concentrations in xylem vessels during salinity stress. The purity of our xylem sap samples was also supported by the finding that athkt1 mutants increase the Na + content in xylem sap (Fig. 2B), but decrease the Na + content in phloem sap (Berthomieu et al. 2003, Sunarpi et al. 2005). These results show that athkt1-1 sos3 and athkt1-2 sos3 double mutant plants display athkt1-1-like xylem sap Na + levels, 8

9 which correlates with the Na + hypersensitivity and leaf chlorosis in these mutants when grown on soil (Fig. 1 and Supplemental Fig. 1). athkt1 sos3 double mutant plants show sos3-like Na + hypersensitivity on minimal medium To further evaluate the Na + sensitivity of athkt1 sos3 double mutant plants in response to salt stress, we analyzed the Na + sensitivity of mature plants grown on agar medium. The Na + hypersensitive chlorotic phenotype of athkt1 shoots was identified using minimal agar medium containing high concentrations of NaCl (Mäser et al. 2002a). Minimal medium was developed to more closely mimic physiological conditions compared to nutrient rich MS medium, while allowing plant growth (see Materials and Methods). Plants grown for days on agar plates were transferred onto minimal agar medium supplemented with 75 mm NaCl and grown for 16 additional days. In minimal medium agar-grown plants, sos3 showed necrotic lesions in the middle of the stress period and eventually showed chlorotic leaf death (Fig. 3C). Similarly, in soil-grown sos3 plants, more severe salt stress (continuous 100 mm NaCl treatment for 2 weeks) caused sos3 leaves to turn black, reflecting leaf necrosis (data not shown). athkt1-1 sos3 and athkt1-2 sos3 plants grown in agar medium showed Na + hypersensitivity under the imposed conditions as well and did not show a recovery from salt sensitivity approaching the wild type phenotype (Fig. 3 A, D and E; n=3 experiments, each for athkt1-1 sos3 and athkt1-2 sos3). More surprisingly, double mutant plants showed sos3-like features under the imposed conditions, exhibiting necrotic leaf lesions in the middle of the 16-day stress-period followed by chlorosis. To quantitatively analyze this observed phenotype, leafchlorophyll content was determined by measuring absorbance at nm and nm (Porra et al. 1989) as shown in Figure 3F. The data demonstrate that athkt1 sos3 double mutant plants show a similar time course of Na + stress-induced chlorophyll content reduction to that of sos3 plants, with athkt1 plants showing an intermediate Na + sensitivity to that of wild type plants and athkt1 sos3 double mutants (Fig. 3F). We next measured Na + contents of shoots and roots of minimal medium grown plants to compare Na + accumulation among the different genotypes. Plants were grown under hydroponic 9

10 conditions on minimal medium for 2-3 weeks and then subjected to 75 mm NaCl stress for 2 days. Na + contents of shoots and roots were measured by ICP-OES. athkt1-1 plants overaccumulated Na + in shoots (Fig. 4A, n=8) and maintained lower Na + levels in roots (Fig. 4B, n=8) compared to wild type plants. In the case of sos3 plants, no significant difference was found in the Na + content of shoots compared to wild type plants under these conditions (Fig. 4A; P>0.08). However, roots of sos3 plants accumulated more Na + compared to wild type (P<1.0x10-6 ) and dramatically more Na + compared to athkt1 mutants (P<1.0x10-13 ) (Fig. 4B). athkt1-1 sos3 and athkt1-2 sos3 double mutant plants, on the other hand, showed interesting Na + accumulation profiles in which Na + over-accumulation appeared in both shoots (P<1.3x10-5 ) and roots (P<1.7x10-4 ), indicating that double mutant plants potentially have detrimental features derived from both single mutant loci: shoot Na + over-accumulation (athkt1-like) and root Na + overaccumulation (sos3-like) under the imposed conditions (Fig. 4, A and B), which correlates with the strong Na + sensitivity of athkt1 sos3 double mutant plants (Fig. 3). sos3-like or athkt1-like Na + hypersensitivities in athkt1 sos3 double mutants are primarily controlled by extracellular Ca 2+ Results from soil-grown athkt1-1 sos3 and athkt1-2 sos3 plants showed that double mutant plants exhibit an athkt1-1-like Na + sensitivity, shoot Na + accumulation and elevated Na + levels in the xylem sap (Figs. 1 and 2). Furthermore plants grown in minimal medium in agar showed that the Na + sensitivity of double mutant plants exhibited a more sos3-like sensitivity to Na + (Fig. 3); or in hydroponic medium the double mutants exhibited a Na + sensitivity reflecting a combination of both athkt1 and sos3 impairments (Fig. 4). Previous research demonstrated suppression of the sos3 phenotype in seedlings by athkt1 in nutrient rich Murashige-Skoog (MS) agar medium (Rus et al. 2001). To analyze whether Na + sensitivities depend on the nutrient status of agar-grown plants, we performed Na + sensitivity analyses using MS medium. Mature day old MS medium agar-grown plants were transferred onto MS medium supplemented with 75 mm NaCl and grown for 18 days. A portion of leaves of athkt1-1 plants showed chlorosis (Supplemental Figure 3B) and leaves of sos3 plants showed necrotic 10

11 yellowing (Supplemental Figure 3C). athkt1-1 sos3 and athkt1-2 sos3 double mutant plants showed athkt1-like Na + sensitivity exhibiting chlorosis (white bleaching) in a portion of the leaves and a partial visual repression of the sos3-1 Na + sensitivity (Supplemental Figure 3D and E). We subsequently performed additional experiments to determine whether specific components or the overall enrichment of nutrients in MS medium is responsible for showing sos3-like or athkt1-like Na + hypersensitivities of double mutants. We compared compositions and concentrations of all substances that differed between minimal medium and MS medium by testing each component individually using synthetic MS media in which the target substance was altered (n=2 experiments, 8 plants per condition). These agar growth analyses showed three ion species that affected the NaCl sensitivity of Arabidopsis: K +, NH + 4 and Ca 2+. These analyses revealed that the higher Ca 2+ concentration in normal MS medium (3 mm Ca 2+ ) compared to minimal medium (1 mm Ca 2+ ) affected the phenotypes of athkt1 sos3 double mutant plants. We prepared minimal agar medium containing 3 mm Ca 2+ and 75 mm Na + and performed further experiments. athkt1 sos3 double mutant and athkt1 plants showed chlorosis on minimal medium containing 3 mm Ca 2+ and 75 mm Na + (Fig. 5B, D and E). athkt1 sos3 double mutant plants also showed a significant reduction in the chlorophyll content in response to 75 mm NaCl stress (Fig. 5F). The difference in chlorophyll reduction between athkt1 and sos3 plants in 3 mm Ca 2+ is much smaller compared to that in 1 mm Ca 2+ because athkt1 plants become more sensitive and sos3 plants become more insensitive to Na + in terms of the chlorophyll reduction rate at 3 mm Ca 2+ (compare Figs. 3F and 5F; see also Supplemental Figure 4). We next performed ICP analyses using plants grown hydroponically on minimal medium containing 3 mm Ca 2+ and 75 mm Na +. athkt1 sos3 plants accumulated more Na + than WT in shoots similar to athkt1 plants (Fig. 6A, P>0.20 for athkt1-1 vs. athkt1-1 sos3, P>0.14 for athkt1-1 vs. athkt1-2 sos3; n=8). Interestingly, athkt1 sos3 double mutant plants accumulated less Na + in roots than wild type plants in the presence of 3 mm Ca 2+ (Fig. 6B; P<1 x 10-4 for wild type vs. athkt1-1 sos3; P<3 x 10-4 for wild type vs. athkt1-2 sos3; n=8). The reduced Na + accumulation in roots of athkt1 sos3 plants with 3 mm Ca 2+ in minimal medium (Fig. 6B) lies in contrast to experiments with 1 mm Ca 2+ in minimal medium (Fig. 4B), and may explain the suppression of 11

12 sos3 by athkt1 in seedling growth assays with 3 mm Ca 2+ in MS medium (Rus et al., 2001). To test the above hypothesis and compare seedling phenotypes with those of Rus et al. (2001), we performed additional Na + sensitivity analyses using immature agar-grown plants. Tenday old seedlings germinated on MS plates were directly transferred onto minimal medium containing 75 mm NaCl and 1 mm or 3 mm Ca(NO 3 ) 2 and grown for 7 additional days (Fig. 7). In the presence of 1 mm Ca 2+, athkt1-1 sos3 and athkt1-2 sos3 double mutant plants again showed a sos3-like phenotype (Fig. 7C; and left panels in Fig. 7D and E). In the presence of 3 mm Ca 2+, sos3-like necrosis was suppressed in athkt1 sos3 double mutant plants as reported by Rus et al. (2001) (right panels in Fig. 7D and E). Furthermore, athkt1 sos3 plants showed curled leaves, as also did athkt1-1 under the imposed conditions (Fig. 7B; and right panels in Fig. 7D and E), but wild type plants did not show curled leaves (Fig. 7A). sos3 seedlings showed a severe root growth defect (Rus et al. 2004), however, athkt1 plants did not (Mäser et al. 2002a) under these conditions (Fig. 7B and C). athkt1 sos3 double plants showed a severe root growth defect similar to sos3 plants in 75 mm Na + and 1 mm Ca 2+ condition, but showed less inhibition of root growth in 75 mm Na + and 3 mm Ca 2+ condition (Fig. 7D and E, compare left and right panels). We measured the Ca 2+ content of the soil solution extracted from soil used for Na + sensitivity analyses because soil grown athkt1 sos3 plants showed more athkt1-1-like Na + hypersensitivity (Figs. 1 and 2; and Supplemental Figs. 1 and 2). Soil solution extracted from 3 independent soil samples contained 6.89ア1.40 mm Ca 2+. Together these results demonstrate that extracellular Ca 2+ controls whether athkt1 sos3 double mutant plants display a more athkt1-like or a more sos3-like phenotype and that Ca 2+ strongly impacts Na + homeostasis in athkt1 sos3 double mutants. Discussion In the present study we demonstrate that athkt1 sos3 double mutant plants show differential Na + hypersensitivities and that the concentration of extracellular Ca 2+ controls 12

13 whether athkt1 sos3 double mutant plants display a more athkt1-like or a more sos3-like phenotype. Furthermore, the present findings also demonstrate distinct physiological roles of AtHKT1 and SOS3 in protecting plants from salinity stress. athkt1 sos3 double and athkt1 single mutants but not sos3-1, cause significantly increased Na + levels in the xylem sap (Fig. 2B) (Sunarpi et al. 2005), resulting in reduction in root Na + content and Na + over-accumulation in leaves and eventual chlorosis. In contrast, the sos3 mutation more strongly caused Na + overaccumulation in roots (Fig. 4 and 6). athkt1 sos3 double mutants show increased xylem sap Na + content under salt stress athkt1 mutant alleles have been found to show Na + hypersensitivity and Na + overaccumulation in shoots under salt stress (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004, Rus et al. 2004). Decreased Na + levels in the phloem sap were found in the reduced function point mutation athkt1 allele, sas2-1, compared to wild type, but no effect was found in the Na + content of the xylem sap of the mutant (Berthomieu et al. 2003). Moreover, phloem tissue expression of AtHKT1 was found in promoter GUS analyses and in situ hybridization (Berthomieu et al. 2003). A Na + recirculation model for AtHKT1 has been proposed based on these observations, in which AtHKT1 functions in loading Na + into the phloem cells in shoots and unloading Na + from the phloem in roots (Berthomieu et al. 2003). Xylem sap collected from athkt1 sos3 double and athkt1 single mutant alleles contained dramatically higher Na + compared to sos3-1 and wild type controls (Fig. 2B). These findings support an expanded model for AtHKT1 functions in reducing Na + in the xylem sap under salt stress, thus preventing plants from over-accumulating Na + in shoots (Sunarpi et al. 2005). The rice gene OsHKT8 is a close orthologue of AtHKT1 and has recently been demonstrated to also control Na + levels in the xylem sap (Ren et al. 2005). Direct immunoelectron microscopy of AtHKT1 demonstrated localization of the AtHKT1 protein in the plasma membrane of xylem parenchyma cells (Sunarpi et al. 2005). Note that these data do not exclude the complementary function of AtHKT1 in enhancing Na + levels in the phloem (Berthomieu et al. 2003, Sunarpi et al. 2005). AtHKT1 may cooperate in both xylem and phloem Na + content control thus mediating Na + 13

14 recirculation to roots. Recirculation of Na + from the xylem sap via the phloem to roots protects leaves from salinity stress and may allow plants to subsequently excrete Na + from roots. Na + recirculation may also enhance the overall vacuolar Na + sequestration in different types of root cells via tonoplast H + /Na + exchangers (Blumwald and Poole 1985, Apse et al. 1999, Gaxiola et al. 1999). Important questions, including how AtHKT1 functions in both the xylem and the phloem and whether the activity and expression of AtHKT1 is differentially regulated in these tissues will be an important subject of investigation. Salinity stress also caused an increase in the xylem sap of sos1-1 plants (Shi et al. 2002), but had a more limited effect in sos3-1 plants compared to athkt1 mutants, with sos3-1 xylem levels of Na + being closer to those in wild type plants (Fig. 2B; P>0.06 for sos3-1 compared to WT). The dramatically increased Na + accumulation in roots of sos3 plants (Fig. 4B and 6B) thus appears to result in a mild increase in the Na + content in the xylem sap. athkt1 sos3 plants show an athkt1-like phenotype in 3 mm external Ca 2+, which can account for suppression of Na + hypersensitivity of sos3-1 seedlings athkt1-1 and athkt1-2 alleles function as genetic suppressor alleles of sos3-1 seedlings and AtHKT1 was proposed to function as a Na + uptake pathway into roots (Rus et al. 2001). However, athkt1 mutant alleles were subsequently shown to cause Na + hypersensitivity in leaves (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004). No expression of AtHKT1 in the root epidermis or root cortex has been observed thus far (Mäser et al. 2002a, Berthomieu et al. 2003). Moreover, detailed tracer influx experiments using 22 Na + showed no measurable disruption of Na + influx into Arabidopsis roots due to athkt1 mutations, despite analyzing several conditions (Berthomieu et al. 2003, Essah et al. 2003). Thus the mechanism by which athkt1 mutations suppress the Na + sensitivity of sos3-1 has remained unclear. The present study shows that athkt1 sos3 plants show an athkt1-like phenotype with 3 mm Ca 2+ in the nutrient medium in 2 to 4 weeks old plants (Figs. 5, 6 and 7). Under these conditions athkt1 alleles completely suppressed the characteristic Na + over-accumulation in roots of the sos3 mutant (Fig. 6B). Our analyses suggest that this protective effect of athkt1 on root Na + levels may explain why athkt1 14

15 mutations suppress the Na + hypersensitivity of sos3-1 seedlings (Rus et al. 2001). A more recent study showed however that athkt1 mutations also reverted sos3-1- dependent Na + -induced damage of leaves of mature soil-grown plants (Rus et al. 2004). It was shown that mature soil-grown athkt1 sos3 plants are more salt tolerant than athkt1 single mutant plants, exhibiting similar growth and leaf health as wild type control plants under salinity stress conditions (Rus et al. 2004). However, our results obtained from mature soil-grown plants were inconsistent with these findings (Figs. 1 and 2; Supplemental Figs. 1 and 2). In the present study, mature soil-grown athkt1 sos3 plants showed Na + hypersensitivity similar to athkt1 mutants (Figs.1 and 2; Supplemental Figs. 1 and 2). The reason for this discrepancy is not clear and may depend on growth conditions. Nevertheless, the physiological effects of athkt1 in athkt1 sos3 double mutant plants on Na + accumulation in leaves (Fig. 2A and Supplemental Fig. 2), xylem sap (Fig. 2B), and shoots (Figs. 4 and 6), and on Na + -induced chlorophyll reduction (Figs. 3F and 5F), make it unlikely that athkt1 mutations can protect mature sos3-1 mutant plants from salinity stress and the repression of sos3-1 Na + hypersensitivity in mature plants by athkt1 loss-offunction mutations appears unlikely under most physiological conditions. Ca 2+ -dependence of SOS3 Ca 2+ sensor and Na + hypersensitivities of athkt1 sos3 double mutants Na + influx channels and currents into root cells have been characterized in electrophysiological studies (Amtmann et al. 1997, Roberts and Tester 1997, Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000, Maathuis and Sanders 2001). However, transporter mutants that cause major reduction in root Na + influx have not yet been found and the underlying genes remain to be identified. Several electrophysiological studies have shown partial block of Na + influx by Ca 2+ (Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000), suggesting that multiple transporters contribute to Na + influx in roots. These findings correlate with classical studies showing that calcium enhances the relative absorption of K + at high Na + concentrations (Epstein 1961, LaHaye and Epstein 1969, Epstein 1998). SOS3 encodes a Ca 2+ sensor that regulates the activity of the plasma membrane SOS1 Na + /H + exchanger, thus mediating cellular Na + extrusion (Liu and Zhu 1998, Qiu et al. 2002, Shi et al. 2002). Thus 15

16 calcium appears to impact several mechanisms in mediating salt tolerance. Findings presented here demonstrate that AtHKT1 mutations do not strictly repress the Na + hypersensitivity of sos3 plants to wild type levels, but that athkt1 sos3 plants exhibit more athkt1-like or more sos3-like Na + hypersensitivities, depending on the external Ca 2+ concentration. sos3-like Na + hypersensitivity became predominant in athkt1 sos3 mutants under salt stress in the presence of 1 mm Ca 2+ (Figs. 3 and 4). On the other hand, AtHKT1 was the strongest salt tolerance determinant in athkt1 sos3 mutants in the presence of 3 mm Ca 2+ as athkt1-like Na + hypersensitivity becomes more prevalent (Figs. 5 and 6). The partial Ca 2+ block of Na + influx channels contributes to reduction in salinity stress (Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000). Furthermore, the Ca 2+ - dependent differential Na + hypersensitivities of athkt1 sos3 double mutants can likely be explained by a model in which an increase in cytoplasmic Ca 2+ can partially suppress the sos3-1 phenotype. This model is supported by results shown in Supplemental Figures S-3C, S-3F and S- 4E. Note that young sos3 seedlings did not show this effect possibly because the effect is not clearly detected in young seedlings (Fig. 7C right panel). The SOS3 gene encodes a Ca 2+ binding protein that is essential for plant salt tolerance (Liu and Zhu 1998). Recently, the threedimensional structure of the SOS3 protein was reported (Sánchez-Barrena et al. 2005). The SOS3 protein includes four EF-hand Ca 2+ -binding motifs and forms a dimer when Ca 2+ concentrations are increased (Sánchez-Barrena et al. 2005). The sos3-1 mutation causes a three amino acid deletion in the third EF-hand (Liu and Zhu 1998, Sánchez-Barrena et al. 2005). This third EFhand is hypothesized to stabilize the dimer structure of the SOS3 protein upon Ca 2+ binding (Sánchez-Barrena et al. 2005). The SOS3-1 protein shows a reduced affinity for Ca 2+ (Ishitani et al. 2000). Thus increasing extracellular Ca 2+ may partially activate the SOS3-1 protein, thus reducing the sos3-1 phenotype resulting in the observed athkt1 like phenotypes of athkt1 sos3 plants at 3 mm external Ca 2+ (Figs. 5, 6 and 7) (Rus et al. 2001). Furthermore, at high Ca 2+ SOS3-independent mechanisms may be activated, as the Arabidopsis genome encodes 9 homologues of the SOS3 gene (Zhu 2002, Kolukisaoglu et al. 2004). In summary, the present findings show that increasing the external Ca 2+ concentration 16

17 strongly impacts the response of athkt1 sos3 double mutant plants to Na + stress. Ca 2+ -dependent sos3-like and athkt1-like Na + hypersensitivities of athkt1 sos3 plants suggest that both the SOS pathway and the AtHKT1 pathway are mechanistically independent strong salt tolerance determinants in Arabidopsis. Under physiological conditions both pathways mediate distinguishable Na + tolerance mechanisms and loss-of-function mutations in AtHKT1 are unlikely to cause physiological Na + resistance of mature Arabidopsis plants. Furthermore, comparative xylem sap analyses in sos3 and athkt1 single and double mutants show that athkt1 sos3 double mutants show enhanced Na + levels in xylem sap, whereas sos3 alone did not greatly affect xylem sap Na + levels. Studies into the question whether specific signaling pathways control the activity of AtHKT1 and whether cross talk with the SOS pathway exists will be interesting subjects of future inquiry. Materials and Methods Cultivation of Arabidopsis Seeds were surface sterilized and sown on MS agar medium supplemented with 1% sucrose. All mutant alleles were confirmed to have the reported homozygous mutations by sequencing genomic DNA at the SOS3 and AtHKT1 loci. Ten to twelve day-old seedlings were transferred onto soil (Professional Blend; McConkey Co., Sumner, WA) for experiments using soil-grown plants, or onto a 10 µm pore-nylon mesh (Spectrum laboratories, Inc., Rancho Dominguez, CA) placed on fresh MS medium or minimal medium (Mäser et al. 2002a) for the agar-growth analysis, or directly onto minimal medium for the short-term agar-growth analysis, or onto a plug (Jaece Industries, Inc., North Tonawanda, NY) soaked in minimal medium for ion content measurements for plants grown on hydroponic medium. Minimal medium contained 0.5 mm KH 2 PO 4, 1.25 mm KNO 3, 0.5 mm MgSO 4, 20 µm FeNa-EDTA, and the additional micronutrients 7 µm H 3 BO 3, 1.4 µm MnSO 4, 1 µm ZnSO 4, 4.5 µm KI, 0.1 µm CuSO 4, 0.2 µm Na 2 MoO 4, 10 nm CoCl 2 supplemented with the indicated amounts of NaCl and Ca(NO 3 ) 2. Plants were subsequently grown for 2-3 weeks on soil, or 5 days on MS medium or minimal medium in the case of agar growth analyses, or for 2-3 weeks with a solution change every 3 days in the case of hydroponic cultures, before salt stress was imposed. Surgical tape (3M, Santa Fe Springs, CA) was used to tape plates instead of a paraffin film in all agar growth analyses. 17

18 Imposition of salt stress Pots of all soil-grown plants were placed on plastic trays and irrigated by adding irrigation solutions (de-ionized water or 1/20 MS basal salt solution). Mature soil-grown plants at the bolting stage were irrigated two times with 100 mm NaCl added to one-twentieth MS basal salts-solution (Shi et al. 2002) over a period of 6 days for Na + content determination, for 2 days for xylem sap collection experiments, or for 7 days for NaCl sensitivity analyses. Plants were subsequently irrigated with de-ionized water for another 7 days after the stress treatment in the case of NaCl sensitivity analyses and the NaCl sensitivities were subsequently analyzed. Plants were watered with one-twentieth MS basal salts solution in parallel as a negative control in all experiments using soil-grown plants. For the NaCl sensitivity analyses of agar-grown plants, meshes on which mature plants were laying were transferred onto the indicated media containing 75 mm NaCl. Plants were subjected to NaCl stress for days. Hydroponically grown plants were subjected to minimal medium containing 75 mm NaCl (ph was adjusted to 5.5 with N-methyl-D-Glucamine) for 2 days. Xylem sap collection and sucrose content determination Xylem sap samples were collected as described previously (Gaymard et al. 1998, Shi et al. 2002). All rosette leaves were removed and the inflorescence stem was cut with a very sharp razor blade from soil-grown 4 to 5 week-old plants. The tray and plants were then covered with a transparent plastic dome to maintain high humidity. Xylem sap drops that accumulated at the cut surface of the inflorescence stem were then collected using a micropipette. For sucrose content determinations xylem sap samples were first purified before HPLC analysis by passing through a Dionex H cartridge (Dionex corp., CA). The run-through and 3 x 0.2 ml water washes were collected in the same container and dried in a Speedvac and then redissolved in an appropriate amount of de-ionized water. Separation and detection of the sugars were achieved by injecting an aliquot of the sample into a Dionex DX 500 HPLC system (Dionex corp., CA) using the CarboPac MA1 column (Dionex corp., CA) in an isocratic mobile phase (480 mm NaOH at 0.4ml/min. for 50 min.) and electrochemical detector at the UCSD Glycobiology Center (UCSD School of Medicine). Chlorophyll content determination Chlorophyll content was determined by the method of Porra et al. (1989). Leaves of plants were harvested at the indicated time points and 4 leaf-disks were punched out of leaves. Punched disks were soaked in 3 ml of Dimethyl formamide (Fisher, Fair lawn, NJ) and kept at 4 C for hrs. Absorbances at nm and nm were then measured by photo 18

19 absorption spectrometry (Spectronic GENESYS5; Milton Roy USA, Ivyland, PA). Contents of chlorophyll a, b and a+b were calculated with formulas described by Porra et al. (1989). Ion content determination Ion content of the described samples was measured by inductively coupled plasma-optic emission spectroscopy (ICP-OES) at the Scripps Institution of Oceangraphy UCSD analytical facility. Shoot and root samples were isolated as described previously (Mäser et al. 2002a). Xylem sap samples were diluted in 5% HNO 3 and directly measured. Acknowledgements We thank Nigel Crawford (UCSD) and Jared Young (UCSD) for comments on the manuscript. We thank Mike Hasegawa (Purdue University) for providing seeds of athkt1-1 and athkt1-1 sos3-1 and athkt1-2 sos3-1 double mutants, Annette Deyhle (Scripps Institute Oceanography, University of California, San Diego) for help with ICP-OES measurements, and Anup Datta and Delia Matriano (UCSD School of Medicine, Glycobiology Center, University of California, San Diego) for help with HPLC analyses of sucrose content in xylem samples. This work was supported by the U.S. Department of Energy (grant no. DOE-DE-FG02-03ER15449) to J.I.S. References Ahn, S.J., Shin, R. and Schachtman, D.P. (2004) Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K + uptake. Plant Physiol. 134: Amtmann, A., Laurie, S., Leigh, R. and Sanders, D. (1997) Multiple inward channels provide flexibility in K + /Na + discrimination at the plasma membrane of barley suspension culture cells. J Exp Bot. 48: Apse, M.P., Aharon, G.S., Snedden, W.A. and Blumwald, E. (1999) Salt tolerance conferred by overexpression of a vacuolar Na + /H + antiport in Arabidopsis. Science. 285: Berthomieu, P., Conéjéro, G., Nublat, A., Brackenbury, W.J., Lambert, C., Savio, C., Uozumi, N., Oiki, S., Yamada, K., Cellier, F., Gosti, F., Simonneau, T., Essah, P.A., Tester, M., Véry, A.A., Sentenac, H. and Casse, F. (2003) Functional analysis of AtHKT1 in Arabidopsis shows that Na + recirculation by the phloem is crucial for salt tolerance. Embo J. 22: Blumwald, E. and Poole, R. (1985) Na + /H + -antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiol. 78:

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23 Bot. 48: Uozumi, N., Kim, E.J., Rubio, F., Yamaguchi, T., Muto, S., Tsuboi, A., Bakker, E.P., Nakamura, T. and Schroeder, J.I. (2000) The Arabidopsis HKT1 gene homolog mediates inward Na + currents in Xenopus laevis oocytes and Na + uptake in Saccharomyces cerevisiae. Plant Physiol. 122: Walker, N.A. and Sanders, D. (1991) Sodium-coupled solute transport I charophyte algae: a general mechanism for transport energization in plant cells? Planta. 185: Wu, S.J., Ding, L. and Zhu, J.K. (1996) SOS1, a Genetic Locus Essential for Salt Tolerance and Potassium Acquisition. Plant Cell. 8: Zhu, J.K. (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol. 53: Zhu, J.K., Liu, J. and Xiong, L. (1998) Genetic analysis of salt tolerance in Arabidopsis. Evidence for a critical role of potassium nutrition. Plant Cell. 10: Legends to figures Figure 1. Increased salt sensitivity of athkt1-1 sos3 and athkt1-2 sos3 plants grown on soil. Plants were irrigated twice with one-twentieth MS basal salt-solution with 100 mm NaCl (right panels in A to E, and panels F to J) or without NaCl (left panels in A to E). Visual phenotypes are shown of leaves and shoots of (A, F) wild type/columbia; (B, G) athkt1-1; (C, H) sos3; (D, I) athkt1-1 sos3; and (E, J) athkt1-2 sos3. Plants were watered twice during a 7-day period, and then all plants were irrigated with de-ionized water for the following 7 days prior to taking photographs. Figure 2. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants over-accumulate Na + in the xylem sap similar to athkt1-1, but not sos3. Na + contents of leaves and xylem sap, derived from soilgrown plants. (A) Na + content measurements in leaves of plants watered with one-twentieth MS basal salt solution with 100 mm NaCl (black bars) or without NaCl (white bars) for 6 days. (B) Na + content measurements in the xylem sap of soil-grown plants watered for 2 days with the same solutions used in (A). Na + content was measured by ICP-OES. Error bars represent standard deviations (n=5 to 8). Figure 3. Agar-grown athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show sos3-like Na + hypersensitivity under salt stress in the presence of 1 mm Ca to 17 day-old mature plants were transferred onto minimal medium containing 75 mm NaCl and 1 mm Ca 2+. (A-E) Plants were grown for 16 additional days and photographs were taken at the end of this period. (F) Contents of chlorophyll a + b extracted from agar-grown plants, which were subjected to 75 mm NaCl stress for 0, 3, 6, 9, 12 or 15 days on minimal medium. Error bars represent standard deviations (n=3 to 4). 23

24 Figure 4. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants over-accumulate Na + in both roots and shoots in the presence of 1 mm Ca 2+ showing cumulative detrimental phenotypes of the two single athkt1 and sos3 mutations. (A) Na + contents in shoots. (B) Na + contents in roots. Plants were hydroponically grown using minimal medium for 2-3 weeks. Mature, bolting plants were then subjected to 75 mm NaCl stress for 2 days. The Na + content was measured by ICP-OES. Error bars represent standard deviations (n=7 to 8). Figure 5. Agar-grown athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show Na + hypersensitivity under salt stress in the presence of 3 mm Ca to 17 day-old mature plants were transferred onto minimal medium containing 75 mm NaCl and 3 mm Ca 2+. (A-E) Plants were grown for 16 additional days and photographs were then taken. (F) Contents of chlorophyll a + b extracted from agar-grown plants, which were subjected to 75 mm NaCl stress for 0, 3, 6, 9, 12 or 15 days on minimal medium containing 3 mm Ca 2+. Error bars represent standard deviations (n=3 to 4). Figure 6. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show ICP profiles similar to single athkt1 mutants in 3 mm Ca 2+ and 75 mm NaCl. (A) Na + contents in shoots. (B) Na + contents in roots. Plants were hydroponically grown using minimal medium containing 1 mm Ca 2+ for 2-3 weeks. Mature, bolting plants were then subjected to 75 mm NaCl stress for 2 days in the presence of 3 mm Ca 2+. Error bars represent standard deviations (n=7 to 8). Figure 7. Young athkt1-1 sos3 double mutant seedlings exhibit sos3-like or athkt1-like phenotypes on minimal agar medium, depending on the external Ca 2+ concentration. The salt sensitivities of 17-day old seedlings are shown of (A) Wild type, (B) athkt1-1, (C) sos3, (D) athkt1-1 sos3 and (E) athkt1-2 sos3. Seedlings were grown on MS agar medium for 10 days and then transferred to minimal agar medium for 7 days containing 75 mm NaCl and 1 mm Ca 2+ (left in each panel) or 3 mm Ca 2+ (right in each panel). Photographs of 2 representative plants from each condition are shown. Twenty seedlings per line were analyzed in 3 independent experiments and all plants showed the depicted phenotypes. 24

25 Running title: Different Na + sensitivities of athkt1 and sos3 mutants *Corresponding author: Julian I. Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California at San Diego, 9500 Gilman Drive, La Jolla, California Telephone: ; julian@biomail.ucsd.edu; Fax: Subject areas: (2) Environmental and stress responses Number of figures: Black and white (3); Color (4) 1

26 Calcium Regulation of Sodium Hypersensitivities of sos3 and athkt1 Mutants. Tomoaki Horie, Rie Horie, Wai-Yin Chan, Ho-Yin Leung, Julian I. Schroeder Division of Biological Sciences, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California San Diego, 9500 Gilman Drive, La Jolla, California , USA Abbreviations: MS, Murashige-Skoog; HKT, high-affinity K + transporter; SOS, salt overly sensitive; ICP-OES, inductively coupled plasma optical emission spectroscopy 2

27 Abstract T-DNA disruption mutations in the AtHKT1 gene have been previously shown to suppress the salt sensitivity of the sos3 mutant. However, both sos3 and athkt1 single mutants show sodium (Na + ) hypersensitivity. In the present study we further analyzed the underlying mechanisms for these non-additive and counteracting Na + sensitivities by characterizing athkt1-1 sos3 and athkt1-2 sos3 double mutant plants. Unexpectedly, mature double mutant plants grown in soil clearly showed an increased Na + hypersensitivity compared to wild type plants when plants were subjected to salinity stress. The salt sensitive phenotype of athkt1 sos3 double mutant plants was similar to athkt1 plants, which showed chlorosis in leaves and stems. The Na + content in xylem sap samples of soil-grown athkt1 sos3 double and athkt1 single mutant plants showed dramatic Na + over-accumulation in response to salinity stress, which was not observed to this degree in sos3-1 plants. We then performed salinity stress analyses using basic minimal nutrient medium and MS medium. These analyses revealed that athkt1 sos3 double mutant plants show a more athkt1 single mutant-like phenotype in the presence of 3 mm external Ca 2+, but show a more sos3 single mutant-like phenotype in the presence of 1 mm external Ca 2+. Taken together results from chlorophyll content, Na + accumulation and Na + sensitivity analyses in high and low Ca 2+ demonstrate that the external Ca 2+ concentration strongly impacts the Na + stress response of athkt1 sos3 double mutants. Furthermore, the presented findings show that SOS3 and AtHKT1 are physiologically distinct major determinants of salinity resistance in Arabidopsis. Keywords: Salinity, HKT/Trk/Ktr transporter, Na + transport, Na + sensitivity, Arabidopsis thaliana 3

28 Introduction Glycophytes including most crop plants are sensitive to saline conditions. Excessive salt build up in soils is an increasingly detrimental problem that causes growth impairment and is toxic to shoot and root growth (Glenn et al. 1999, Munns 2002, Zhu 2002, Horie and Schroeder 2004). Increasing soil salinity in irrigated lands is a major threat to agricultural production. Elevated Na + levels directly or indirectly perturb important cellular processes including the functions of vital enzymes (Murguía et al. 1995), K + absorption (Rains and Epstein 1965, Epstein 1998) and photosynthesis (Tsugane et al. 1999). The HKT1 cdna, isolated from wheat, encodes a high-affinity K + /Na + co-transporter, and at high [Na + ] a Na + influx transporter in heterologous systems (Schachtman and Schroeder 1994, Rubio et al. 1995, Gassman et al. 1996). Analyses of HKT transporters from other plants and in bacteria have also shown Na + -stimulated K + uptake (Tholema et al. 1999, Fairbairn et al. 2000, Kawano et al. 2000, Horie et al. 2001, Matsuda et al. 2004, Tholema et al. 2005). High-affinity K + /Na + co-transport has been observed as a major K + uptake activity in aquatic plants (Walker and Sanders 1991, Maathuis et al. 1996), but in higher plants a K + /Na + uptake activity has not been detected (Maathuis et al. 1996). Rather, K + uptake is mediated by multiple overlapping K + influx pathways (Quintero and Blatt 1997, Santa-Maria et al. 1997, Fu and Luan 1998, Hirsch et al. 1998, Kim et al. 1998, Spalding et al. 1999, Santa-Maria et al. 2000, Reintanz et al. 2002, Ahn et al. 2004, Gierth et al. 2005). Potassium uptake mediated by the distinct family of KUP/HAK/KT transporters is inhibited by extracellular Na +, illustrating a strong K + vs. Na + competition for uptake in this family of transporters (Santa-María et al. 1997, Fu and Luan 1998). At high sodium concentrations K + transport via HKT1 is blocked. Furthermore, at high Na + HKT1 mediates selective Na + influx in oocytes, yeast and wheat (Rubio et al. 1995, Gassman et al. 1996, Diatloff et al. 1998, Rubio et al. 1999, Liu et al. 2000, Laurie et al. 2002). HKT1 homologs have been isolated from many plant species and functionally characterized (Fairbairn et al. 2000, Uozumi et al. 2000, Horie et al. 2001, Golldack et al. 2002, Garciadeblás et al. 2003, Su et al. 2003). 4

29 The Arabidopsis genome encodes a single HKT1 gene, AtHKT1 (Uozumi et al. 2000). Expression of the Arabidopsis transporter AtHKT1 in Xenopus laevis oocytes and Saccharomyces cerevisiae has shown a relatively Na + selective transport activity of AtHKT1 (Uozumi et al. 2000, Mäser et al. 2002b, Berthomieu et al. 2003). Loss-of-function mutations in AtHKT1 render plants Na + hypersensitive and disturb the distribution of Na + between roots and shoots, causing Na + over-accumulation in leaves and chlorosis under salinity stress (Mäser et al. 2002a). Furthermore, an EMS mutation that causes reduced AtHKT1 activity has been shown to cause growth inhibition and Na + over-accumulation in shoots under salt stress (Berthomieu et al. 2003). AtHKT1 expression was found in vascular tissues (Mäser et al. 2002a) and in particular in phloem tissues, causing reduced Na + levels in the phloem sap of the sas2-1 allele in AtHKT1 (Berthomieu et al. 2003). Furthermore, microarray-based mutant mapping identified an athkt1 (FN1148) mutation that causes hyper-accumulation of Na + in shoots (Gong et al. 2004). These findings reveal that AtHKT1 is an important Na + resistance determinant in Arabidopsis and that Na + transport via AtHKT1 is essential for protection of plant leaves against salinity stress in Arabidopsis (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004, Sunarpi et al. 2005). Recent studies demonstrated that AtHKT1 and the close rice ortholog OsHKT8, function in reducing the Na + concentration in the xylem sap (Ren et al. 2005, Sunarpi et al. 2005). Salt overly sensitive sos1 to sos3 mutant loci were identified as essential factors for salt tolerance (Wu et al. 1996, Zhu et al. 1998). SOS3 encodes an EF hand type Ca 2+ binding protein sharing significant sequence similarity with the Ca 2+ binding domain of the calcineurin B subunit of yeast (CnB) and neuronal calcium sensors from animals (Liu and Zhu 1998, Sánchez-Barrena et al. 2005). SOS3 has been shown to physically interact with the SOS2 protein kinase in yeast (Halfter et al. 2000), and the SOS2/SOS3 complex regulates the plasma membrane Na + /H + anti-porter SOS1, which is essential for plant salt tolerance (Shi et al. 2000, Qiu et al. 2002, Quintero et al. 2002, Shi et al. 2002). AtHKT1 mutations were found to suppress the Na + hypersensitivity of sos3-1 mutant seedlings (Rus et al. 2001). Interestingly, both sos3 and athkt1 single mutant alleles show Na + hypersensitivity (Liu and Zhu 1998, Mäser et al. 2002a), but athkt1 sos3-1 plants showed 5

30 resistance to salinity stress independent of whether they were grown in agar medium or as mature plants in soil (Rus et al. 2001, Rus et al. 2004). In this study we characterize the complex relationship between these essential salt tolerance determinants to uncover the mechanism underlying the non-additive and counteracting Na + sensitivities of athkt1 sos3 double mutants compared to single sos3-1 and athkt1 mutants. Detailed characterization of athkt1-1 sos3-1 and athkt1-2 sos3-1 double mutants under salt stress show that SOS3 and AtHKT1 function in distinct physiological aspects of salt tolerance and that athkt1 mutations in sos3 plants do not strictly repress the Na + hypersensitivity of sos3 plants to wild type levels, but that the external Ca 2+ concentration strongly impacts Na + stress responses of athkt1 sos3 double mutants. Results athkt1 sos3 double mutant plants show athkt1-like Na + hypersensitivity on soil Previously, two independent T-DNA insertions in the AtHKT1 gene, athkt1-1 and athkt1-2, were shown to suppress the Na + hypersensitivity of sos3-1 plants (Rus et al. 2001), including recent findings in mature soil-grown plants (Rus et al. 2004). To analyze the underlying mechanisms, mature soil-grown Columbia wild type, athkt1-1, sos3, athkt1-1 sos3 and athkt1-2 sos3 plants were subjected to NaCl stress to determine whether salt sensitivity was suppressed in mature athkt1 sos3 double mutant alleles here. Three to four week old plants were watered with 100 mm NaCl twice during 1 week and subsequently watered with de-ionized water for the following week and subsequently analyzed as shown in figure 1. Interestingly, both double mutant alleles clearly showed strong Na + hypersensitivity in all experiments with soil-grown mature plants (Fig. 1D, E, I and J) compared to wild type plants (Fig. 1A, F) (> 50 plants analyzed for each genotype). These findings differ from those of Rus et al. (2004) in which soil-grown athkt1 sos3 double mutant plants showed similar NaCl sensitivity as wild type plants and repression of the sos3 Na + sensitivity. In over half of the plants, early during the salt stress phase, athkt1 sos3 mutant plants appeared less chlorotic than 6

31 athkt1. This early stress phenotype was more apparent when enriched nutrient media were used. But these plants eventually showed chlorosis after several more days, exhibiting consistent salinity stress of athkt1 sos3 double mutant plants compared to wild type controls, as illustrated in Figure 1D and E. Mutants in the AtHKT1 gene have been previously characterized as showing increased salt sensitivity under agar-grown conditions (Mäser et al. 2002a, Gong et al. 2004) and under hydroponic conditions (Berthomieu et al. 2003). Soil-grown mature salt-stressed athkt1-1 plants showed chlorotic withering as well (Fig. 1B). Under salt stress, leaves of double mutant athkt1-1 sos3 and athkt1-2 sos3 plants also showed chlorotic withering (Fig. 1D and E) and stems were also damaged, similar to athkt1-1 plants (Fig. 1G, I and J) (n=50 plants per genotype). On the other hand, salt stressed sos3 plants showed a necrotic yellowing phenotype (Fig. 1C) and normal silique development more similar to wild type than athkt1 under the imposed conditions (Fig.1 F, H). We also performed additional NaCl sensitivity analyses using soil grown plants watered with 50 mm NaCl as used in Rus et al. (2004). Experiments under these conditions also showed that athkt1 sos3 double mutant plants show chlorosis at leaf borders similar to athkt1 single mutant plants, but in contrast to wild type plants (Supplemental Figure 1). sos3 single mutant plants showed a yellowing of leaves under these conditions. athkt1 sos3 double and athkt1 mutants but not sos3 cause strong Na + over-accumulation in the xylem sap We next performed ICP analyses using soil-grown plants to compare levels of Na + accumulation in leaves. Leaves of soil-grown salt-stressed athkt1-1 plants showed a dramatically increased Na + content compared to wild type plants under both 100 mm NaCl (Fig. 2A) and 50 mm NaCl watering conditions (Supplemental Fig. 2). Furthermore, double mutant athkt1-1 sos3 and athkt1-2 sos3 plants also showed Na + over-accumulation in leaves similar to athkt1-1 plants, but unlike wild type plants (Fig. 2A and Supplemental Fig. 2). Increases in the Na + content of shoots compared to wild type were also found in soil-grown sos3 plants (Fig. 2A and Supplemental Fig. 2). Expression of the AtHKT1 gene in vascular tissues was shown using transgenic plants 7

32 carrying AtHKT1 promoter-gus gene constructs (Mäser et al. 2002a, Berthomieu et al. 2003). The sas2-1 mutant, which has a transport-reducing point mutation in the athkt1 gene, shows lower Na + content in the phloem sap compared to wild type (Berthomieu et al. 2003). A recent study has shown that athkt1 single mutants caused an increase in the Na + level of xylem sap and that the AtHKT1 protein is targeted to the plasma membrane of xylem parenchyma cells (Sunarpi et al. 2005). Here, we analyzed whether the Na + content of the xylem sap of athkt1 sos3 plants is affected during salt stress. Xylem sap was collected from soil-grown wild type (n=8) and athkt1-1 plants (n=7) watered with 100 mm NaCl or 1/20 MS solution (non-stress control condition), and the Na + content of the xylem sap was measured by ICP-OES. To evaluate the relative purity of xylem sap samples, we analyzed sucrose levels of the xylem sap by HPLC analysis. Sucrose levels provide a measure of the level of contamination from phloem sap, as phloem sap in Arabidopsis contains high millimolar sucrose levels (Deeken et al. 2002). The average sucrose content of xylem sap samples was 8.02±1.75 µm (n=3). Average sucrose content concentrations have been reported in the phloem sap of Arabidopsis of 341±52 mm (Deeken et al. 2002). Thus our xylem sap samples contain negligibly low (< 0.01%) phloem sap contamination. We measured the Na + content in xylem sap samples of sos3, athkt1, athkt1-1 sos3, and athkt1-2 sos3 soil-grown plants in the presence and absence of salinity stress. Interestingly, xylem sap samples from athkt1 sos3 double mutant plants contained dramatically higher Na + than those from sos3 single mutant and wild type plants (Fig. 2B). The athkt1 sos3 double mutant plants showed a similar phenotype to athkt1-1 (Fig. 2B). In contrast single sos3 mutant plants showed a moderate increase in xylem Na + content similar to wild type plants (Fig. 2B), supporting an important role of AtHKT1 (Sunarpi et al. 2005) in maintaining low Na + concentrations in xylem vessels during salinity stress. The purity of our xylem sap samples was also supported by the finding that athkt1 mutants increase the Na + content in xylem sap (Fig. 2B), but decrease the Na + content in phloem sap (Berthomieu et al. 2003, Sunarpi et al. 2005). These results show that athkt1-1 sos3 and athkt1-2 sos3 double mutant plants display athkt1-1-like xylem sap Na + levels, which correlates with the Na + hypersensitivity and leaf chlorosis in these mutants when grown on 8

33 soil (Fig. 1 and Supplemental Fig. 1). athkt1 sos3 double mutant plants show sos3-like Na + hypersensitivity on minimal medium To further evaluate the Na + sensitivity of athkt1 sos3 double mutant plants in response to salt stress, we analyzed the Na + sensitivity of mature plants grown on agar medium. The Na + hypersensitive chlorotic phenotype of athkt1 shoots was identified using minimal agar medium containing high concentrations of NaCl (Mäser et al. 2002a). Minimal medium was developed to more closely mimic physiological conditions compared to nutrient rich MS medium, while allowing plant growth (see Materials and Methods). Plants grown for days on agar plates were transferred onto minimal agar medium supplemented with 75 mm NaCl and grown for 16 additional days. In minimal medium agar-grown plants, sos3 showed necrotic lesions in the middle of the stress period and eventually showed chlorotic leaf death (Fig. 3C). Similarly, in soil-grown sos3 plants, more severe salt stress (continuous 100 mm NaCl treatment for 2 weeks) caused sos3 leaves to turn black, reflecting leaf necrosis (data not shown). athkt1-1 sos3 and athkt1-2 sos3 plants grown in agar medium showed Na + hypersensitivity under the imposed conditions as well and did not show a recovery from salt sensitivity approaching the wild type phenotype (Fig. 3 A, D and E; n=3 experiments, each for athkt1-1 sos3 and athkt1-2 sos3). More surprisingly, double mutant plants showed sos3-like features under the imposed conditions, exhibiting necrotic leaf lesions in the middle of the 16-day stress-period followed by chlorosis. To quantitatively analyze this observed phenotype, leaf-chlorophyll content was determined by measuring absorbance at nm and nm (Porra et al. 1989) as shown in Figure 3F. The data demonstrate that athkt1 sos3 double mutant plants show a similar time course of Na + stress-induced chlorophyll content reduction to that of sos3 plants, with athkt1 plants showing an intermediate Na + sensitivity to that of wild type plants and athkt1 sos3 double mutants (Fig. 3F). We next measured Na + contents of shoots and roots of minimal medium grown plants to compare Na + accumulation among the different genotypes. Plants were grown under hydroponic conditions on minimal medium for 2-3 weeks and then subjected to 75 mm NaCl stress for 2 days. 9

34 Na + contents of shoots and roots were measured by ICP-OES. athkt1-1 plants over-accumulated Na + in shoots (Fig. 4A, n=8) and maintained lower Na + levels in roots (Fig. 4B, n=8) compared to wild type plants. In the case of sos3 plants, no significant difference was found in the Na + content of shoots compared to wild type plants under these conditions (Fig. 4A; P>0.08). However, roots of sos3 plants accumulated more Na + compared to wild type (P<1.0x10-6 ) and dramatically more Na + compared to athkt1 mutants (P<1.0x10-13 ) (Fig. 4B). athkt1-1 sos3 and athkt1-2 sos3 double mutant plants, on the other hand, showed interesting Na + accumulation profiles in which Na + over-accumulation appeared in both shoots (P<1.3x10-5 ) and roots (P<1.7x10-4 ), indicating that double mutant plants potentially have detrimental features derived from both single mutant loci: shoot Na + over-accumulation (athkt1-like) and root Na + over-accumulation (sos3-like) under the imposed conditions (Fig. 4, A and B), which correlates with the strong Na + sensitivity of athkt1 sos3 double mutant plants (Fig. 3). sos3-like or athkt1-like Na + hypersensitivities in athkt1 sos3 double mutants are primarily controlled by extracellular Ca 2+ Results from soil-grown athkt1-1 sos3 and athkt1-2 sos3 plants showed that double mutant plants exhibit an athkt1-1-like Na + sensitivity, shoot Na + accumulation and elevated Na + levels in the xylem sap (Figs. 1 and 2). Furthermore plants grown in minimal medium in agar showed that the Na + sensitivity of double mutant plants exhibited a more sos3-like sensitivity to Na + (Fig. 3); or in hydroponic medium the double mutants exhibited a Na + sensitivity reflecting a combination of both athkt1 and sos3 impairments (Fig. 4). Previous research demonstrated suppression of the sos3 phenotype in seedlings by athkt1 in nutrient rich Murashige-Skoog (MS) agar medium (Rus et al. 2001). To analyze whether Na + sensitivities depend on the nutrient status of agar-grown plants, we performed Na + sensitivity analyses using MS medium. Mature day old MS medium agar-grown plants were transferred onto MS medium supplemented with 75 mm NaCl and grown for 18 days. A portion of leaves of athkt1-1 plants showed chlorosis (Supplemental Figure 3B) and leaves of sos3 plants showed necrotic yellowing (Supplemental Figure 3C). athkt1-1 sos3 and athkt1-2 sos3 double mutant plants showed 10

35 athkt1-like Na + sensitivity exhibiting chlorosis (white bleaching) in a portion of the leaves and a partial visual repression of the sos3-1 Na + sensitivity (Supplemental Figure 3D and E). We subsequently performed additional experiments to determine whether specific components or the overall enrichment of nutrients in MS medium is responsible for showing sos3-like or athkt1-like Na + hypersensitivities of double mutants. We compared compositions and concentrations of all substances that differed between minimal medium and MS medium by testing each component individually using synthetic MS media in which the target substance was altered (n=2 experiments, 8 plants per condition). These agar growth analyses showed three ion species that affected the NaCl sensitivity of Arabidopsis: K +, NH + 4 and Ca 2+. These analyses revealed that the higher Ca 2+ concentration in normal MS medium (3 mm Ca 2+ ) compared to minimal medium (1 mm Ca 2+ ) affected the phenotypes of athkt1 sos3 double mutant plants. We prepared minimal agar medium containing 3 mm Ca 2+ and 75 mm Na + and performed further experiments. athkt1 sos3 double mutant and athkt1 plants showed chlorosis on minimal medium containing 3 mm Ca 2+ and 75 mm Na + (Fig. 5B, D and E). athkt1 sos3 double mutant plants also showed a significant reduction in the chlorophyll content in response to 75 mm NaCl stress (Fig. 5F). The difference in chlorophyll reduction between athkt1 and sos3 plants in 3 mm Ca 2+ is much smaller compared to that in 1 mm Ca 2+ because athkt1 plants become more sensitive and sos3 plants become more insensitive to Na + in terms of the chlorophyll reduction rate at 3 mm Ca 2+ (compare Figs. 3F and 5F; see also Supplemental Figure 4). We next performed ICP analyses using plants grown hydroponically on minimal medium containing 3 mm Ca 2+ and 75 mm Na +. athkt1 sos3 plants accumulated more Na + than WT in shoots similar to athkt1 plants (Fig. 6A, P>0.20 for athkt1-1 vs. athkt1-1 sos3, P>0.14 for athkt1-1 vs. athkt1-2 sos3; n=8). Interestingly, athkt1 sos3 double mutant plants accumulated less Na + in roots than wild type plants in the presence of 3 mm Ca 2+ (Fig. 6B; P<1 x 10-4 for wild type vs. athkt1-1 sos3; P<3 x 10-4 for wild type vs. athkt1-2 sos3; n=8). The reduced Na + accumulation in roots of athkt1 sos3 plants with 3 mm Ca 2+ in minimal medium (Fig. 6B) lies in contrast to experiments with 1 mm Ca 2+ in minimal medium (Fig. 4B), and may explain the suppression of sos3 by athkt1 in seedling growth assays with 3 mm Ca 2+ in MS medium (Rus et al., 2001). 11

36 To test the above hypothesis and compare seedling phenotypes with those of Rus et al. (2001), we performed additional Na + sensitivity analyses using immature agar-grown plants. Ten-day old seedlings germinated on MS plates were directly transferred onto minimal medium containing 75 mm NaCl and 1 mm or 3 mm Ca(NO 3 ) 2 and grown for 7 additional days (Fig. 7). In the presence of 1 mm Ca 2+, athkt1-1 sos3 and athkt1-2 sos3 double mutant plants again showed a sos3-like phenotype (Fig. 7C; and left panels in Fig. 7D and E). In the presence of 3 mm Ca 2+, sos3-like necrosis was suppressed in athkt1 sos3 double mutant plants as reported by Rus et al. (2001) (right panels in Fig. 7D and E). Furthermore, athkt1 sos3 plants showed curled leaves, as also did athkt1-1 under the imposed conditions (Fig. 7B; and right panels in Fig. 7D and E), but wild type plants did not show curled leaves (Fig. 7A). sos3 seedlings showed a severe root growth defect (Rus et al. 2004), however, athkt1 plants did not (Mäser et al. 2002a) under these conditions (Fig. 7B and C). athkt1 sos3 double plants showed a severe root growth defect similar to sos3 plants in 75 mm Na + and 1 mm Ca 2+ condition, but showed less inhibition of root growth in 75 mm Na + and 3 mm Ca 2+ condition (Fig. 7D and E, compare left and right panels). We measured the Ca 2+ content of the soil solution extracted from soil used for Na + sensitivity analyses because soil grown athkt1 sos3 plants showed more athkt1-1-like Na + hypersensitivity (Figs. 1 and 2; and Supplemental Figs. 1 and 2). Soil solution extracted from 3 independent soil samples contained 6.89±1.40 mm Ca 2+. Together these results demonstrate that extracellular Ca 2+ controls whether athkt1 sos3 double mutant plants display a more athkt1-like or a more sos3-like phenotype and that Ca 2+ strongly impacts Na + homeostasis in athkt1 sos3 double mutants. Discussion In the present study we demonstrate that athkt1 sos3 double mutant plants show differential Na + hypersensitivities and that the concentration of extracellular Ca 2+ controls whether athkt1 sos3 double mutant plants display a more athkt1-like or a more sos3-like phenotype. 12

37 Furthermore, the present findings also demonstrate distinct physiological roles of AtHKT1 and SOS3 in protecting plants from salinity stress. athkt1 sos3 double and athkt1 single mutants but not sos3-1, cause significantly increased Na + levels in the xylem sap (Fig. 2B) (Sunarpi et al. 2005), resulting in reduction in root Na + content and Na + over-accumulation in leaves and eventual chlorosis. In contrast, the sos3 mutation more strongly caused Na + over-accumulation in roots (Fig. 4 and 6). athkt1 sos3 double mutants show increased xylem sap Na + content under salt stress athkt1 mutant alleles have been found to show Na + hypersensitivity and Na + over-accumulation in shoots under salt stress (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004, Rus et al. 2004). Decreased Na + levels in the phloem sap were found in the reduced function point mutation athkt1 allele, sas2-1, compared to wild type, but no effect was found in the Na + content of the xylem sap of the mutant (Berthomieu et al. 2003). Moreover, phloem tissue expression of AtHKT1 was found in promoter GUS analyses and in situ hybridization (Berthomieu et al. 2003). A Na + recirculation model for AtHKT1 has been proposed based on these observations, in which AtHKT1 functions in loading Na + into the phloem cells in shoots and unloading Na + from the phloem in roots (Berthomieu et al. 2003). Xylem sap collected from athkt1 sos3 double and athkt1 single mutant alleles contained dramatically higher Na + compared to sos3-1 and wild type controls (Fig. 2B). These findings support an expanded model for AtHKT1 functions in reducing Na + in the xylem sap under salt stress, thus preventing plants from over-accumulating Na + in shoots (Sunarpi et al. 2005). The rice gene OsHKT8 is a close orthologue of AtHKT1 and has recently been demonstrated to also control Na + levels in the xylem sap (Ren et al. 2005). Direct immunoelectron microscopy of AtHKT1 demonstrated localization of the AtHKT1 protein in the plasma membrane of xylem parenchyma cells (Sunarpi et al. 2005). Note that these data do not exclude the complementary function of AtHKT1 in enhancing Na + levels in the phloem (Berthomieu et al. 2003, Sunarpi et al. 2005). AtHKT1 may cooperate in both xylem and phloem Na + content control thus mediating Na + recirculation to roots. Recirculation of Na + from the xylem sap via the phloem to roots protects 13

38 leaves from salinity stress and may allow plants to subsequently excrete Na + from roots. Na + recirculation may also enhance the overall vacuolar Na + sequestration in different types of root cells via tonoplast H + /Na + exchangers (Blumwald and Poole 1985, Apse et al. 1999, Gaxiola et al. 1999). Important questions, including how AtHKT1 functions in both the xylem and the phloem and whether the activity and expression of AtHKT1 is differentially regulated in these tissues will be an important subject of investigation. Salinity stress also caused an increase in the xylem sap of sos1-1 plants (Shi et al. 2002), but had a more limited effect in sos3-1 plants compared to athkt1 mutants, with sos3-1 xylem levels of Na + being closer to those in wild type plants (Fig. 2B; P>0.06 for sos3-1 compared to WT). The dramatically increased Na + accumulation in roots of sos3 plants (Fig. 4B and 6B) thus appears to result in a mild increase in the Na + content in the xylem sap. athkt1 sos3 plants show an athkt1-like phenotype in 3 mm external Ca 2+, which can account for suppression of Na + hypersensitivity of sos3-1 seedlings athkt1-1 and athkt1-2 alleles function as genetic suppressor alleles of sos3-1 seedlings and AtHKT1 was proposed to function as a Na + uptake pathway into roots (Rus et al. 2001). However, athkt1 mutant alleles were subsequently shown to cause Na + hypersensitivity in leaves (Mäser et al. 2002a, Berthomieu et al. 2003, Gong et al. 2004). No expression of AtHKT1 in the root epidermis or root cortex has been observed thus far (Mäser et al. 2002a, Berthomieu et al. 2003). Moreover, detailed tracer influx experiments using 22 Na + showed no measurable disruption of Na + influx into Arabidopsis roots due to athkt1 mutations, despite analyzing several conditions (Berthomieu et al. 2003, Essah et al. 2003). Thus the mechanism by which athkt1 mutations suppress the Na + sensitivity of sos3-1 has remained unclear. The present study shows that athkt1 sos3 plants show an athkt1-like phenotype with 3 mm Ca 2+ in the nutrient medium in 2 to 4 weeks old plants (Figs. 5, 6 and 7). Under these conditions athkt1 alleles completely suppressed the characteristic Na + over-accumulation in roots of the sos3 mutant (Fig. 6B). Our analyses suggest that this protective effect of athkt1 on root Na + levels may explain why athkt1 mutations suppress the Na + hypersensitivity of sos3-1 seedlings (Rus et al. 2001). 14

39 A more recent study showed however that athkt1 mutations also reverted sos3-1- dependent Na + -induced damage of leaves of mature soil-grown plants (Rus et al. 2004). It was shown that mature soil-grown athkt1 sos3 plants are more salt tolerant than athkt1 single mutant plants, exhibiting similar growth and leaf health as wild type control plants under salinity stress conditions (Rus et al. 2004). However, our results obtained from mature soil-grown plants were inconsistent with these findings (Figs. 1 and 2; Supplemental Figs. 1 and 2). In the present study, mature soil-grown athkt1 sos3 plants showed Na + hypersensitivity similar to athkt1 mutants (Figs.1 and 2; Supplemental Figs. 1 and 2). The reason for this discrepancy is not clear and may depend on growth conditions. Nevertheless, the physiological effects of athkt1 in athkt1 sos3 double mutant plants on Na + accumulation in leaves (Fig. 2A and Supplemental Fig. 2), xylem sap (Fig. 2B), and shoots (Figs. 4 and 6), and on Na + -induced chlorophyll reduction (Figs. 3F and 5F), make it unlikely that athkt1 mutations can protect mature sos3-1 mutant plants from salinity stress and the repression of sos3-1 Na + hypersensitivity in mature plants by athkt1 loss-of-function mutations appears unlikely under most physiological conditions. Ca 2+ -dependence of SOS3 Ca 2+ sensor and Na + hypersensitivities of athkt1 sos3 double mutants Na + influx channels and currents into root cells have been characterized in electrophysiological studies (Amtmann et al. 1997, Roberts and Tester 1997, Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000, Maathuis and Sanders 2001). However, transporter mutants that cause major reduction in root Na + influx have not yet been found and the underlying genes remain to be identified. Several electrophysiological studies have shown partial block of Na + influx by Ca 2+ (Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000), suggesting that multiple transporters contribute to Na + influx in roots. These findings correlate with classical studies showing that calcium enhances the relative absorption of K + at high Na + concentrations (Epstein 1961, LaHaye and Epstein 1969, Epstein 1998). SOS3 encodes a Ca 2+ sensor that regulates the activity of the plasma membrane SOS1 Na + /H + exchanger, thus mediating cellular Na + extrusion (Liu and Zhu 1998, Qiu et al. 2002, Shi et al. 2002). Thus calcium appears to impact several mechanisms in mediating salt tolerance. 15

40 Findings presented here demonstrate that AtHKT1 mutations do not strictly repress the Na + hypersensitivity of sos3 plants to wild type levels, but that athkt1 sos3 plants exhibit more athkt1-like or more sos3-like Na + hypersensitivities, depending on the external Ca 2+ concentration. sos3-like Na + hypersensitivity became predominant in athkt1 sos3 mutants under salt stress in the presence of 1 mm Ca 2+ (Figs. 3 and 4). On the other hand, AtHKT1 was the strongest salt tolerance determinant in athkt1 sos3 mutants in the presence of 3 mm Ca 2+ as athkt1-like Na + hypersensitivity becomes more prevalent (Figs. 5 and 6). The partial Ca 2+ block of Na + influx channels contributes to reduction in salinity stress (Tyerman et al. 1997, Buschmann et al. 2000, Davenport and Tester 2000). Furthermore, the Ca 2+ -dependent differential Na + hypersensitivities of athkt1 sos3 double mutants can likely be explained by a model in which an increase in cytoplasmic Ca 2+ can partially suppress the sos3-1 phenotype. This model is supported by results shown in Supplemental Figures S-3C, S-3F and S-4E. Note that young sos3 seedlings did not show this effect possibly because the effect is not clearly detected in young seedlings (Fig. 7C right panel). The SOS3 gene encodes a Ca 2+ binding protein that is essential for plant salt tolerance (Liu and Zhu 1998). Recently, the three-dimensional structure of the SOS3 protein was reported (Sánchez-Barrena et al. 2005). The SOS3 protein includes four EF-hand Ca 2+ -binding motifs and forms a dimer when Ca 2+ concentrations are increased (Sánchez-Barrena et al. 2005). The sos3-1 mutation causes a three amino acid deletion in the third EF-hand (Liu and Zhu 1998, Sánchez-Barrena et al. 2005). This third EF-hand is hypothesized to stabilize the dimer structure of the SOS3 protein upon Ca 2+ binding (Sánchez-Barrena et al. 2005). The SOS3-1 protein shows a reduced affinity for Ca 2+ (Ishitani et al. 2000). Thus increasing extracellular Ca 2+ may partially activate the SOS3-1 protein, thus reducing the sos3-1 phenotype resulting in the observed athkt1 like phenotypes of athkt1 sos3 plants at 3 mm external Ca 2+ (Figs. 5, 6 and 7) (Rus et al. 2001). Furthermore, at high Ca 2+ SOS3-independent mechanisms may be activated, as the Arabidopsis genome encodes 9 homologues of the SOS3 gene (Zhu 2002, Kolukisaoglu et al. 2004). In summary, the present findings show that increasing the external Ca 2+ concentration strongly impacts the response of athkt1 sos3 double mutant plants to Na + stress. Ca 2+ -dependent 16

41 sos3-like and athkt1-like Na + hypersensitivities of athkt1 sos3 plants suggest that both the SOS pathway and the AtHKT1 pathway are mechanistically independent strong salt tolerance determinants in Arabidopsis. Under physiological conditions both pathways mediate distinguishable Na + tolerance mechanisms and loss-of-function mutations in AtHKT1 are unlikely to cause physiological Na + resistance of mature Arabidopsis plants. Furthermore, comparative xylem sap analyses in sos3 and athkt1 single and double mutants show that athkt1 sos3 double mutants show enhanced Na + levels in xylem sap, whereas sos3 alone did not greatly affect xylem sap Na + levels. Studies into the question whether specific signaling pathways control the activity of AtHKT1 and whether cross talk with the SOS pathway exists will be interesting subjects of future inquiry. Materials and Methods Cultivation of Arabidopsis Seeds were surface sterilized and sown on MS agar medium supplemented with 1% sucrose. All mutant alleles were confirmed to have the reported homozygous mutations by sequencing genomic DNA at the SOS3 and AtHKT1 loci. Ten to twelve day-old seedlings were transferred onto soil (Professional Blend; McConkey Co., Sumner, WA) for experiments using soil-grown plants, or onto a 10 µm pore-nylon mesh (Spectrum laboratories, Inc., Rancho Dominguez, CA) placed on fresh MS medium or minimal medium (Mäser et al. 2002a) for the agar-growth analysis, or directly onto minimal medium for the short-term agar-growth analysis, or onto a plug (Jaece Industries, Inc., North Tonawanda, NY) soaked in minimal medium for ion content measurements for plants grown on hydroponic medium. Minimal medium contained 0.5 mm KH 2 PO 4, 1.25 mm KNO 3, 0.5 mm MgSO 4, 20 µm FeNa-EDTA, and the additional micronutrients 7 µm H 3 BO 3, 1.4 µm MnSO 4, 1 µm ZnSO 4, 4.5 µm KI, 0.1 µm CuSO 4, 0.2 µm Na 2 MoO 4, 10 nm CoCl 2 supplemented with the indicated amounts of NaCl and Ca(NO 3 ) 2. Plants were subsequently grown for 2-3 weeks on soil, or 5 days on MS medium or minimal medium in the case of agar growth analyses, or for 2-3 weeks with a solution change every 3 days in the case of hydroponic cultures, before salt stress was imposed. Surgical tape (3M, Santa Fe Springs, CA) was used to tape plates instead of a paraffin film in all agar growth analyses. 17

42 Imposition of salt stress Pots of all soil-grown plants were placed on plastic trays and irrigated by adding irrigation solutions (de-ionized water or 1/20 MS basal salt solution). Mature soil-grown plants at the bolting stage were irrigated two times with 100 mm NaCl added to one-twentieth MS basal salts-solution (Shi et al. 2002) over a period of 6 days for Na + content determination, for 2 days for xylem sap collection experiments, or for 7 days for NaCl sensitivity analyses. Plants were subsequently irrigated with de-ionized water for another 7 days after the stress treatment in the case of NaCl sensitivity analyses and the NaCl sensitivities were subsequently analyzed. Plants were watered with one-twentieth MS basal salts solution in parallel as a negative control in all experiments using soil-grown plants. For the NaCl sensitivity analyses of agar-grown plants, meshes on which mature plants were laying were transferred onto the indicated media containing 75 mm NaCl. Plants were subjected to NaCl stress for days. Hydroponically grown plants were subjected to minimal medium containing 75 mm NaCl (ph was adjusted to 5.5 with N-methyl-D-Glucamine) for 2 days. Xylem sap collection and sucrose content determination Xylem sap samples were collected as described previously (Gaymard et al. 1998, Shi et al. 2002). All rosette leaves were removed and the inflorescence stem was cut with a very sharp razor blade from soil-grown 4 to 5 week-old plants. The tray and plants were then covered with a transparent plastic dome to maintain high humidity. Xylem sap drops that accumulated at the cut surface of the inflorescence stem were then collected using a micropipette. For sucrose content determinations xylem sap samples were first purified before HPLC analysis by passing through a Dionex H cartridge (Dionex corp., CA). The run-through and 3 x 0.2 ml water washes were collected in the same container and dried in a Speedvac and then re-dissolved in an appropriate amount of de-ionized water. Separation and detection of the sugars were achieved by injecting an aliquot of the sample into a Dionex DX 500 HPLC system (Dionex corp., CA) using the CarboPac MA1 column (Dionex corp., CA) in an isocratic mobile phase (480 mm NaOH at 0.4ml/min. for 50 min.) and electrochemical detector at the UCSD Glycobiology Center (UCSD School of Medicine). Chlorophyll content determination Chlorophyll content was determined by the method of Porra et al. (1989). Leaves of plants were harvested at the indicated time points and 4 leaf-disks were punched out of leaves. Punched disks were soaked in 3 ml of Dimethyl formamide (Fisher, Fair lawn, NJ) and kept at 4 C for hrs. Absorbances at nm and nm were then measured by photo absorption spectrometry (Spectronic GENESYS5; Milton Roy USA, Ivyland, PA). Contents of chlorophyll a, 18

43 b and a+b were calculated with formulas described by Porra et al. (1989). Ion content determination Ion content of the described samples was measured by inductively coupled plasma-optic emission spectroscopy (ICP-OES) at the Scripps Institution of Oceangraphy UCSD analytical facility. Shoot and root samples were isolated as described previously (Mäser et al. 2002a). Xylem sap samples were diluted in 5% HNO 3 and directly measured. Acknowledgements We thank Nigel Crawford (UCSD) and Jared Young (UCSD) for comments on the manuscript. We thank Mike Hasegawa (Purdue University) for providing seeds of athkt1-1 and athkt1-1 sos3-1 and athkt1-2 sos3-1 double mutants, Annette Deyhle (Scripps Institute Oceanography, University of California, San Diego) for help with ICP-OES measurements, and Anup Datta and Delia Matriano (UCSD School of Medicine, Glycobiology Center, University of California, San Diego) for help with HPLC analyses of sucrose content in xylem samples. This work was supported by the U.S. Department of Energy (grant no. DOE-DE-FG02-03ER15449) to J.I.S. References Ahn, S.J., Shin, R. and Schachtman, D.P. (2004) Expression of KT/KUP genes in Arabidopsis and the role of root hairs in K + uptake. Plant Physiol. 134: Amtmann, A., Laurie, S., Leigh, R. and Sanders, D. (1997) Multiple inward channels provide flexibility in K + /Na + discrimination at the plasma membrane of barley suspension culture cells. J Exp Bot. 48: Apse, M.P., Aharon, G.S., Snedden, W.A. and Blumwald, E. (1999) Salt tolerance conferred by overexpression of a vacuolar Na + /H + antiport in Arabidopsis. Science. 285: Berthomieu, P., Conéjéro, G., Nublat, A., Brackenbury, W.J., Lambert, C., Savio, C., Uozumi, N., Oiki, S., Yamada, K., Cellier, F., Gosti, F., Simonneau, T., Essah, P.A., Tester, M., Véry, A.A., Sentenac, H. and Casse, F. (2003) Functional analysis of AtHKT1 in Arabidopsis shows that Na + recirculation by the phloem is crucial for salt tolerance. Embo J. 22: Blumwald, E. and Poole, R. (1985) Na + /H + -antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Physiol. 78: Buschmann, P.H., Vaidyanathan, R., Gassmann, W. and Schroeder, J.I. (2000) Enhancement of Na + 19

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47 Physiol. 122: Walker, N.A. and Sanders, D. (1991) Sodium-coupled solute transport I charophyte algae: a general mechanism for transport energization in plant cells? Planta. 185: Wu, S.J., Ding, L. and Zhu, J.K. (1996) SOS1, a Genetic Locus Essential for Salt Tolerance and Potassium Acquisition. Plant Cell. 8: Zhu, J.K. (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol. 53: Zhu, J.K., Liu, J. and Xiong, L. (1998) Genetic analysis of salt tolerance in Arabidopsis. Evidence for a critical role of potassium nutrition. Plant Cell. 10: Legends to figures Figure 1. Increased salt sensitivity of athkt1-1 sos3 and athkt1-2 sos3 plants grown on soil. Plants were irrigated twice with one-twentieth MS basal salt-solution with 100 mm NaCl (right panels in A to E, and panels F to J) or without NaCl (left panels in A to E). Visual phenotypes are shown of leaves and shoots of (A, F) wild type/columbia; (B, G) athkt1-1; (C, H) sos3; (D, I) athkt1-1 sos3; and (E, J) athkt1-2 sos3. Plants were watered twice during a 7-day period, and then all plants were irrigated with de-ionized water for the following 7 days prior to taking photographs. Figure 2. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants over-accumulate Na + in the xylem sap similar to athkt1-1, but not sos3. Na + contents of leaves and xylem sap, derived from soil-grown plants. (A) Na + content measurements in leaves of plants watered with one-twentieth MS basal salt solution with 100 mm NaCl (black bars) or without NaCl (white bars) for 6 days. (B) Na + content measurements in the xylem sap of soil-grown plants watered for 2 days with the same solutions used in (A). Na + content was measured by ICP-OES. Error bars represent standard deviations (n=5 to 8). Figure 3. Agar-grown athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show sos3-like Na + hypersensitivity under salt stress in the presence of 1 mm Ca to 17 day-old mature plants were transferred onto minimal medium containing 75 mm NaCl and 1 mm Ca 2+. (A-E) Plants were grown for 16 additional days and photographs were taken at the end of this period. (F) Contents of chlorophyll a + b extracted from agar-grown plants, which were subjected to 75 mm NaCl stress for 0, 3, 6, 9, 12 or 15 days on minimal medium. Error bars represent standard deviations (n=3 to 4). Figure 4. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants over-accumulate Na + in both roots and shoots in the presence of 1 mm Ca 2+ showing cumulative detrimental phenotypes of the two single athkt1 and sos3 mutations. (A) Na + contents in shoots. (B) Na + contents in roots. Plants were hydroponically grown using minimal medium for 2-3 weeks. Mature, bolting plants were then 23

48 subjected to 75 mm NaCl stress for 2 days. The Na + content was measured by ICP-OES. Error bars represent standard deviations (n=7 to 8). Figure 5. Agar-grown athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show Na + hypersensitivity under salt stress in the presence of 3 mm Ca to 17 day-old mature plants were transferred onto minimal medium containing 75 mm NaCl and 3 mm Ca 2+. (A-E) Plants were grown for 16 additional days and photographs were then taken. (F) Contents of chlorophyll a + b extracted from agar-grown plants, which were subjected to 75 mm NaCl stress for 0, 3, 6, 9, 12 or 15 days on minimal medium containing 3 mm Ca 2+. Error bars represent standard deviations (n=3 to 4). Figure 6. athkt1-1 sos3 and athkt1-2 sos3 double mutant plants show ICP profiles similar to single athkt1 mutants in 3 mm Ca 2+ and 75 mm NaCl. (A) Na + contents in shoots. (B) Na + contents in roots. Plants were hydroponically grown using minimal medium containing 1 mm Ca 2+ for 2-3 weeks. Mature, bolting plants were then subjected to 75 mm NaCl stress for 2 days in the presence of 3 mm Ca 2+. Error bars represent standard deviations (n=7 to 8). Figure 7. Young athkt1-1 sos3 double mutant seedlings exhibit sos3-like or athkt1-like phenotypes on minimal agar medium, depending on the external Ca 2+ concentration. The salt sensitivities of 17-day old seedlings are shown of (A) Wild type, (B) athkt1-1, (C) sos3, (D) athkt1-1 sos3 and (E) athkt1-2 sos3. Seedlings were grown on MS agar medium for 10 days and then transferred to minimal agar medium for 7 days containing 75 mm NaCl and 1 mm Ca 2+ (left in each panel) or 3 mm Ca 2+ (right in each panel). Photographs of 2 representative plants from each condition are shown. Twenty seedlings per line were analyzed in 3 independent experiments and all plants showed the depicted phenotypes. 24

49 A A WT/Col B athkt1-1 C Control +100 mm Na + Control +100 mm Na + sos3 D athkt1-1 sos3 E Control +100 mm Na + Control +100 mm Na + athkt1-2 sos3 Control +100 mm Na + F G H I J WT/Col athkt1-1 sos3 athkt1-1 sos3 athkt1-2 sos3 25 Figure 1

50 Na + [µmol/g DW] A WTcol WTcol+Na athkt1-1 athkt1-1 +Na sos3 sos3 +Na athkt1-1 sos3 athkt1-1 sos3+na athkt1-2 sos3 athkt1-2 sos3+na Na + [µmol/ml sap] B WTcol WTcol+Na athkt1-1 athkt1-1 +Na sos3 sos3 +Na athkt1-1 sos3 athkt1-1 sos3+na athkt1-2 sos3 athkt1-2 sos3+na 26 Figure 2

51 WT/Col athkt1-1 sos3 A B C athkt1-1 sos3 athkt1-2 sos3 D E F Chlorophyll (a+b) [µm/cm 2 ] Days WTcol athkt1-1 athkt1-1 sos3 athkt1-2 sos3 sos3 27 Figure 3

52 A Na + [µmol/g DW] Shoots 0 WTcol athkt1-1 sos3 athkt1-1 sos3 athkt1-2 sos3 B Na + [µmol/g DW] Roots WTcol athkt1-1 sos3 athkt1-1 sos3 athkt1-2 sos3 28 Figure 4

53 WT/Col athkt1-1 sos3 A B C athkt1-1 sos3 athkt1-2 sos3 D E F Chlorophyll (a+b) [µm/cm 2 ] WTcol athkt1-1 athkt1-1 sos3 athkt1-2 sos3 sos3 Days 29 Figure 5

54 A Na + [µmol/g DW] Shoots 0 WTcol athkt1-1 sos3 athkt1-1 sos3 athkt1-2 sos3 B Na + [µmol/g DW] Roots WTcol athkt1-1 sos3 athkt1-1 sos3 athkt1-2 sos3 30 Figure 6

55 A 75 mm NaCl [Ca 2+ ] 1 mm 3 mm B 75 mm NaCl 1 mm 3 mm C 75 mm NaCl 1 mm 3 mm WT athkt1-1 sos3-1 D [Ca 2+ ] 75 mm NaCl 1 mm 3 mm E 75 mm NaCl 1 mm 3 mm athkt1-1 sos3 athkt1-2 sos3 31 Figure 7