It is thought that Na+ and K+ homeostasis is crucial for salt-tolerance in plants. To better understand the Na+ and K+ homeostasis in important crop rice (Oryza sativa L.), a cDNA homologous to the wheat HKT1 encoding K+-Na+ symporter was isolated from japonica rice, cv Nipponbare (Ni-OsHKT1). We also isolated two cDNAs homologous to Ni-OsHKT1 from salt-tolerant indica rice, cv Pokkali (Po-OsHKT1, Po-OsHKT2). The predicted amino acid sequence of Ni-OsHKT1 shares 100% identity with Po-OsHKT1 and 91% identity with Po-OsHKT2, and they are 66–67% identical to wheat HKT1. Low-K+ conditions (less than 3 mm) induced the expression of all three OsHKT genes in roots, but mRNA accumulation was inhibited by the presence of 30 mm Na+. We further characterized the ion-transport properties of OsHKT1 and OsHKT2 using an expression system in the heterologous cells, yeast and Xenopus oocytes. OsHKT2 was capable of completely rescuing a K+-uptake deficiency mutation in yeast, whereas OsHKT1 was not under K+-limiting conditions. When OsHKTs were expressed in Na+-sensitive yeast, OsHKT1 rendered the cells more Na+-sensitive than did OsHKT2 in high NaCl conditions. The electrophysiological experiments for OsHKT1 expressed in Xenopus oocytes revealed that external Na+, but not K+, shifted the reversal potential toward depolarization. In contrast, for OsHKT2 either Na+ or K+ in the external solution shifted the reversal potential toward depolarization under the mixed Na+ and K+ containing solutions. These results suggest that two isoforms of HKT transporters, a Na+ transporter (OsHKT1) and a Na+- and K+-coupled transporter (OsHKT2), may act harmoniously in the salt tolerant indica rice.
It has been reported that K+ starvation induces low-affinity Na+ uptake and reduces the selectivity of root membranes for K+ over Na+ (Ding and Zhu, 1997; Kochian et al., 1985; Pitman et al., 1968; Pitman, 1967). Recently Buschmann et al. (2000) found that K+ starvation caused an enhancement of instantaneous Na+ currents in wheat root cortex cells. The sos (salt overly sensitive) mutants of Arabidopsis thaliana were specifically hypersensitive to high external Na+ or Li+ and also unable to grow under very low external K+ concentrations (Zhu et al., 1998). Taken together, it has been suggested that K+ and Na+ homeostasis is important for salt tolerance in plants, and that K+ availability is crucial for inducing Na+ uptake.
A number of cDNAs encoding plant K+ channels and transporters have been isolated and characterized (Anderson et al., 1992; Fairbairn et al., 2000; Fu and Luan, 1998; Kim et al., 1998; Santa-María et al., 1997; Schachtman and Schroeder, 1994; Sentenac et al., 1992; Uozumi et al., 2000). A cDNA of HKT1 encoding K+-Na+ symporter was isolated from wheat roots (Triticum aestivum, TaHKT1;Schachtman and Schroeder, 1994), and, it was found to be a high-affinity K+-Na+ cotransporter using Saccharomyces cerevisiae and Xenopus laevis oocytes (Gassmann et al., 1996; Rubio et al., 1995). However, K+ uptake mediated by TaHKT1 was blocked and low-affinity Na+ uptake occured in the physiologically detrimental concentrations of Na+, suggesting that TaHKT1 may be one of the pathways for Na+ uptake, which leads to Na+ toxicity under saline conditions. It was reported that HvHAK1, a high-affinity K+ transporter of barley (Hordeum vulgare), mediated Na+ uptake under high millimolar concentrations of Na+, and that K+ uptake by the dual affinity K+ transporter AtKUP1 of A. thaliana was inhibited by external Na+ at both high- and low-affinity phases (Fu and Luan, 1998; Santa-María et al., 1997). These findings are consistent with earlier studies stating that K+ uptake by plant roots is inhibited under high Na+ conditions (Epstein, 1973). The A. thaliana HKT1 gene homolog, AtHKT1, with some different properties from TaHKT1 was reported (Uozumi et al., 2000). First, the TaHKT1 gene could complement null mutations of the high-affinity K+ transporter genes TRK1 and TRK2 in S. cerevisiae strain CY162 (Anderson et al., 1992; Ko and Gaber, 1991), whereas AtHKT1 could not. Second, in the analysis with Na+-extruding ATPase deficient mutant of S. cerevisiae (strain G19; Quintero et al., 1996), yeast cells expressing AtHKT1 were more sensitive to Na+ than those expressing TaHKT1. Finally, the voltage-clamp experiments with AtHKT1-expressing oocytes of X. laevis indicated that, unlike TaHKT1-expressing ones, the presence of K+ did not have a clear effect on the reversal potentials. More recently, two distinct HKT1 gene homologs, EcHKT1 and EcHKT2, were isolated from Eucalyptus tree, Eucalyptus camaldulensis, demonstrating that K+ and Na+ could be transported (Fairbairn et al., 2000).
To understand the role of the HKT1 gene in plants further and to elucidate the mechanisms of K+ and Na+ homeostasis in crop rice (Oryza sativa), we isolated three rice HKT homologs from two different varieties of rice, one from a japonica rice, cv. Nipponbare (Ni-OsHKT1), and two from cv. Pokkali, a salt-tolerant indica rice (Po-OsHKT1 and Po-OsHKT2). Each OsHKT mRNA was found to accumulate in response to K+ starvation. However, this effect was mitigated by high concentrations of Na+. The function of each OsHKT gene was analyzed using a heterologous expression system in S. cerevisiae and X. laevis oocytes. As a result, we concluded that the OsHKT1 genes encode transporter, similar to AtHKT1 in function, while the OsHKT2 codes for a gene product most similar to the TaHKT1.
Isolation of cDNAs encoding the rice HKT proteins
Since HKT1 mRNA was found to accumulate in the roots of wheat and barley in response to K+ starvation (Wang et al., 1998), rice seedlings were grown in modified K+-free N6 medium, in which potassium salts were substituted with sodium and ammonium salts (see Experimental procedures). cDNA libraries were constructed with using mRNA derived from 4-d-old roots of Nipponbare or Pokkali. First, we screened the Nipponbare cDNA library, using TaHKT1 cDNA as a probe, and succeeded in isolating one positive clone. DNA sequence analysis revealed that the cDNA encodes a hydrophobic polypeptide of 530 amino acid residues with an expected molecular mass of 59 kDa (Figure 1a). This amino acid sequence shared 67% identity with TaHKT1 (Table 1), and thus we named the clone Nipponbare (Ni-) OsHKT1. Next, by probing with the Ni-OsHKT1 cDNA, we isolated two kinds of cDNA clones from Pokkali library, which we named Pokkali (Po-) OsHKT1 and OsHKT2 (Figure 1a). The predicted amino acid sequences of Po-OsHKT1 and Po-OsHKT2 shared 100% and 91% identity with the sequence of Ni-OsHKT1, respectively (Table 1). Hydropathy plots indicated that both OsHKT1 and OsHKT2 proteins show a high degree of similarity to TaHKT1 (Figure 1b). Any other related isoforms of OsHKT were not found in the protein database of rice.
Table 1. Percent identity of the predicted amino acid sequences between each OsHKT and TaHKT1, AtHKT1, and EcHKTs
The predicted amino acid sequences of OsHKT1 cDNA isolated from both varieties of rice are completely identical. OsHKT2 cDNA was isolated only from Pokkali rice.
Copy number of the OsHKT genes in the rice chromosome
Genomic Southern blot analysis was carried out with full length OsHKT1 cDNA as a probe under high-stringency conditions. The band patterns were found to be different between two varieties (Figure 2). In Nipponbare, a single band was detected after EcoRV digestion, but one major band and additional weak bands were detected with XbaI and HindIII digests (Figure 2a). However, upon further screening of the Nipponbare root cDNA library using Ni-OsHKT1 cDNA as probe, no other type of Ni-OsHKT cDNA was isolated. We were also unable to isolate another Ni-OsHKT homolog by genomic and RT–PCR analysis using multiple sets of primers for OsHKT1 and for OsHKT2 (data not shown). We assume that Ni-OsHKT1 is a single copy gene in Nipponbare rice, although we can not deny completely the existence of other Ni-OsHKT1 homolog in the Nipponbare genome. In pokkali, two strong hybridizing bands were detected in all digests upon probing with OsHKT1 cDNA (Figure 2b), suggesting the presence of two Po-OsHKT genes in the Pokkali genome.
Expression of the OsHKT genes are regulated by K+ and Na+ concentration
It was reported that mRNA levels of the TaHKT1 gene, the barley HvHAK1 gene, and the Arabidopsis AtKUP3 gene were increased under the conditions of K+ starvation (Kim et al., 1998; Santa-María et al., 1997). Northern blot analysis for Ni-OsHKT1 mRNA in roots was carried out using the coding region of Ni-OsHKT1 cDNA as a probe (Figure 3a). Ni-OsHKT1 mRNA accumulation was found to be significant below 3 mm K+ and maximal under K+-free condition. However, the high level of Ni-OsHKT1 mRNA normally induced by 0.3 mm K+ decreased in the presence of 30 mm Na+.
Because the DNA sequence of Po-OsHKT1 cDNA displayed 93% identity to Po-OsHKT2 cDNA (data not shown), we performed RT–PCR analysis to discriminate between the expression patterns of two Po-OsHKT genes. Two primer sets, in which two bases at each 3′ end of the primers are different from one another (Figure 3b), were constructed and they amplified specific DNA fragments corresponding to each OsHKT cDNA (data not shown). The results of RT–PCR analysis suggested that Po-OsHKTs were regulated in a manner similar to Ni-OsHKT1. Both Po-OsHKT mRNAs were found to accumulate in response to K+ starvation and, as was the case with Ni-OsHKT1 transcripts, this effect was mitigated by the presence of 30 mm Na+, even in low external K+ conditions (Figure 3b).
Functional analysis of the OsHKT genes with yeast systems
Previous studies indicated that plant K+ channels/transporters could rescue the high-affinity K+ uptake deficient trk1 and trk2 mutations of S. cerevisiae strain CY162 (Anderson et al., 1992; Fu and Luan, 1998; Santa-María et al., 1997; Schachtman and Schroeder, 1994). The TaHKT1 cDNA was isolated by functional complementation using this strain. However, the AtHKT1 gene was not able to complement trk mutations (Schachtman and Schroeder, 1994; Uozumi et al., 2000). We tried the same complementation assay using our isolates in CY162 cells and found a significant difference between the ability of the two types of OsHKT to complement trk mutations. While expression of OsHKT1 was incapable of rescuing the trk mutations, the mutant host expressing OsHKT2 gene could grow on medium containing 0.1 mm KCl (Figure 4a). Even though the amino acid sequences of the OsHKT1 and OsHKT2 proteins shared 91% identity with each other, the K+ uptake properties of the transporters seems to be different in yeast.
Overexpression of TaHKT1 or AtHKT1 in yeast cells caused Na+ hypersensitivity due to increased Na+ uptake (Rubio et al., 1995, 1999; Uozumi et al., 2000). The Na+ uptake properties of OsHKT1 and OsHKT2 was studied by the growth inhibition test using S. cerevisiae strain G19, which displays increased Na+ sensitivity due to disruption of ENA1 to ENA4) encoding the Na+-extruding ATPases. The G19 transformants expressing OsHKT1 or OsHKT2 displayed more sensitivity to Na+ than TaHKT1-expressing cells (Figure 4b). Growth inhibition of OsHKT-expressing cells could be observed at 50 mm NaCl, and was complete at 150 mm. Interestingly, expression of OsHKT1 seemed to make G19 cells more sensitive to Na+ than OsHKT2 at 50 mm and 100 mm NaCl conditions (Figure 4b). Furthermore, overexpression of OsHKTs caused growth inhibition of S. cerevisiae W303, which contains wild type ENA genes, on medium containing 300 mm NaCl, on which TaHKT1 transformants and vector clones could grow (data not shown).
Electrophysiological measurements of OsHKT1- and OsHKT2-mediated currents in X. laevis oocytes
To investigate functional differences between OsHKT1 and OsHKT2, OsHKT-mediated currents were recorded from X. laevis oocytes using voltage-clamp technique. Figure 5 shows the amplitudes of the steady-state inward currents recorded in OsHKT-expressing oocytes measured at −120 mV, where the external solution contained 100 mm alkali cation. Large inward currents were elicited by perfusing the Na+ containing solution for both OsHKT1- and OsHKT2-expressing oocytes. These findings are consistent with the previous reports on other HKT1 transporters (Gassmann et al., 1996; Uozumi et al., 2000).
Next, the effect of external K+ on the OsHKT-mediated currents was studied. Currents were measured in the presence of 1 mm Na+ with increasing K+ concentrations from 0.3 to 9 mm. The current-voltage relationships obtained by a ramp command (see Experimental procedures) are shown in Figure 6. In OsHKT1-expressing oocytes, the reversal potentials shifted only slightly as external K+ concentration was increased (Figure 6a), indicating that Na+ currents mediated by OsHKT1 was a little sensitive to external K+. In contrast, for OsHKT2-expressing oocytes, increasing K+ concentrations led to the prominent positive shifts in the reversal potentials. Thus, OsHKT2 mediates inward K+ flux in the presence of external Na+. On the other hand, it is noted that in Figure 5(b) negligible currents were elicited in high K+ solution in the absence of external Na+. Thus, K influx turned on only if external solution contained low concentration of Na. Currents were also measured in the presence of 1 mm K+ with increasing Na+ concentrations (Figure 6c,d). For both OsHKT1- and OsHKT2-expressing oocytes the positive shifts in the reversal potentials indicate that OsHKTs mediate Na+ influx in the presence of external K+. Taken together, it is concluded that (1) both OsHKT1 and OsHKT2 mediate Na+ flux in the physiological concentration range (Figure 6c,d); (2) OsHKT1 does not mediate K+ flux (Figure 6a); (3) OsHKT2 mediates K+ flux in the presence of external Na+ (Figure 6b). Therefore, OsHKT1 is a Na+ transporter and OsHKT2 is a Na+ and K+ coupled transporter.
We isolated two different types of cDNAs encoding rice HKT proteins from cv. Nipponbare (Ni-OsHKT1) and cv. Pokkali (Po-OsHKT1 and Po-OsHKT2) of O. sativa. All of OsHKT cDNAs encoded 530 amino acids and predicted amino acid sequences of Ni-OsHKT1 and Po-OsHKT1 were completely identical. OsHKT1 and OsHKT2 share 91% sequence identity and show similar hydrophobic profiles (Table 1; Figure 1b). However the K+ transport properties of OsHKT2 differed from OsHKT1 when they expressed in both yeast and X. laevis oocytes. As shown in Figure 4(a), the complementation assays in S. cerevisiae CY162 cells indicated that OsHKT2 was capable of rescuing the trk mutations in low-K+ conditions, whereas OsHKT1 could not. These ion specificities of the transport activity of OsHKTs were further investigated by electrophysiological techniques using X. laevis oocytes. It was demonstrated that OsHKT1 mediates Na+ influx, but not K+ influx (Figure 5, Figure 6c), and OsHKT1-mediated currents do not depend on external K+ (Figure 6a). On the other hand, OsHKT2 exhibited intriguing behavior in its ion specificity. In physiological conditions the shift of the reversal potentials indicate that OsHKT2 can mediate both Na+ and K+ influx (Figure 6b,d). However, OsHKT2 does not mediate K+ influx in the high K+ solution without Na+ (Figure 5). The opposite was not true: External Na+ elicited large inward current in the absence of external K+. Thus, we concluded that, at least in these heterologous systems, OsHKT1 is a Na+ transporter without mediating K+ flux, which corresponds to the transporting properties of AtHKT1. On the other hand, OsHKT2 is a Na+ and K+ coupled transporter, the character of which is similar to those of TaHKT1. These properties should be related to the molecular mechanism of the transport, such as binding affinity and stoichiometry of the transporting ions.
Although the Na+ uptake ability of OsHKT2 displayed a relatively similar profile to that of OsHKT1, we observed that OsHKT2-expressing S. cerevisiae G19 cells were less sensitive than OsHKT1-expressing cells in medium containing 50 mm and 100 mm NaCl (Figure 4b). This could be because the internal Na+/K+ concentration ratio (Rubio et al., 1995, 1999) of OsHKT2-expressing cells is lower than that of OsHKT1-expressing cells because of the differences of the K+ uptake ability between OsHKT1 and OsHKT2 in their imposed conditions.
An amino acids sequence comparison between a pair of OsHKT2/TaHKT1 and OsHKT1/AtHKT1 was done to focus on the candidate sites responsible for K+ selectivity. We picked up nine amino acid residues, which are identical in the former pair but different in the latter pair (asterisks of Figure 1a), and one of them, position at amino acid number 88, was in existence in the conserved P-loop domain. It has been suggested that TaHKT1 is one of a member of K+ symporters, which contain four loops that are homologous to the selectivity filter-forming P loops of K+ channels (Durell and Guy, 1998; Durell et al., 1998). Each of four P loops of these transporters contains highly conserved Gly residues that are postulated to play an important role in K+ selectivity. While the 88th amino acid, Gly, in OsHKT2 is conserved, it is Ser in OsHKT1 and AtHKT1 (Figure 1c). It is suggested that the exchange of Gly for Ser in this position may cause the difference in K+ transport properties between TaHKT1 type transporters and AtHKT1 type transporters including EcHKT1 and EcHKT2 (Mäser et al., 2000; Tholema et al., 1999). It is interesting that both types of HKT transporters, OsHKT1 and OsHKT2, are found in a salt-tolerant indica rice.
In the roots of Nipponbare and Pokkali, the expression patterns of the OsHKT1 and OsHKT2 genes were nearly the same under various external K+and Na+ conditions (Figure 3). Interestingly, mRNAs of both OsHKT genes were found to accumulate under low external K+ concentrations, especially under K+-free conditions. Several earlier studies suggested that low-affinity Na+ uptake is activated by K+ starvation and reduces the selectivity of root membranes for K+ over Na+ (Ding and Zhu, 1997; Kochian et al., 1985; Pitman et al., 1968; Pitman, 1967; Buschmann et al., 2000). There is a hypothesis that plants can, to a certain extent, use Na+ to compensate for K+ deficiency (Flowers and Läuchli, 1983; Mengel and Kirkby, 1982; Rodríguez-Navarro, 2000; Buschmann et al., 2000). Therefore, it is possible that if OsHKT1 has a function as a Na+ transporter in rice roots, it may help to compensate for K+ deficiency by transporting Na+ into the cell. In saline conditions, however, it is also possible that OsHKT1 causes Na+ toxicity by allowing excessive Na+ uptake into the cell, where the gene is expressing. The growth inhibition test of yeast showed that OsHKT1 overexpression increased Na+ sensitivity not only in G19 cells (Figure 4b), but also in W303, a parent strain for G19 (data not shown). It is an interesting observation that the accumulation of OsHKT1 mRNA in the presence of 0.3 mm K+ was decreased in 30 mm Na+ compared with 0–3 mm Na+ conditions (Figure 3a,b). It is purposive action to avoid transporting excessive Na+ into the cell that the transcription of OsHKT1 gene is repressed under the high Na+ conditions.
The sos mutants of A. thaliana were specifically hypersensitive to high external Na+ or Li+ and also unable to grow under very low external K+ concentrations (Zhu et al., 1998). The N365S mutant of the TaHKT1 that was reduced inhibited high affinity K+ uptake and reduced low affinity Na+ uptake, increased Na+ tolerance in yeast (Rubio et al., 1999). These results suggest that the high-affinity K+ uptake and K+ and Na+ homeostasis are important for salt tolerance of cells. It is possible that OsHKT2 plays the role of TaHKT1 type high-affinity K+ transporter in Pokkali rice. The important question of whether the K+ uptake ability of OsHKT2 confers the salt tolerance of Pokkali should be addressed.
The pathway of Na+ uptake through the roots of rice was well characterized physiologically and genetically so far (Yadav et al., 1996; Yeo et al., 1987; Yeo et al., 1999). Salt damage in rice occurs as the result of excessive transport of harmful ions such as Na+ to the leaves. By using a fluorescent tracer Yeo et al. (1987) demonstrated the fact that excessive Na+ transport was caused by apoplastic water leakage, which is accompanied by the transpirational volume flow, without crossing biological membrane. This uncontrolled apoplastic pathway, so-called bypass-flow, could be a major factor for Na+ accumulation in aerial part of rice in saline conditions (Yeo et al., 1987). However, the question of what kind of transporter functions in a main pathway for the uptake of Na+ into cytoplasm from apoplastic spaces still remains. It is also important to address the question of how the symplastic Na+ transport contributes to the toxic Na+ accumulation in aerial part in saline conditions. It is interesting how HKT-type transporters contribute to such a Na+ transport. Intracellular localization of OsHKT proteins in plant tissues and phenotypes of transgenic plants for overexpression or silencing of the OsHKT genes will be useful in determining their exact physiological roles as transporter. The functional and expression analyses of the OsHKT genes among various rice varieties are also important to know the relationship between salt-tolerance and OsHKT functions.
Plant growth and medium
N6 and modified N6 medium, in which K+ salts were substituted to various degrees with NH4+ or Na+ salts, were brought to pH 5.8 with Tris–HCl (pH 9.5) and used for the plant growth in this study. Rice (Oryza sativa L. cv. Nipponbare and cv. Pokkali) seeds were threshed and sterilized for 30 min in one-fifth diluted sodium hypochlorite solution and washed five times with distilled water. The seeds were then planted on plastic mesh in sterilized modified medium and grown for 4 days at 28°C on a 16-h light/8-h dark schedule.
Isolation of OsHKT cDNAs
About 2000 rice seeds of each variety were used for cDNA library construction. Total RNA was extracted from the roots of 4-d-old seedlings of Nipponbare or Pokkali grown on K+-free medium, and poly (A)+ RNA was purified by using oligo (dT)-cellulose (Amersham Pharmacia Biotech UK Ltd, Bucks., UK). The cDNA libraries were constructed in Uni-ZAP XR vector (Stratagene, La Jolla, CA, USA) and ZIP-LOX vector (GibcoBRL, MD, Gaithersburg, MD, USA) for Nipponbare and Pokkali, respectively. Plaques were replicated on nylon membranes (Hybond-N+, Amersham Pharmacia Biotech UK Ltd) and hybridization was performed at 65°C for high-stringency or 55°C for low-stringency in the buffer described by Church and Gilbert (1984). The Nipponbare cDNA library was screened by low-stringency hybridization with 32P-labeled full length of TaHKT1 cDNA, kindly donated by Dr Schroeder (Schachtman and Schroeder, 1994). One positive clone was purified, and the phage DNA was excised in vivo to yield the pBluescript SK(–) plasmid according to Strategene's protocol. The Pokkali cDNA library was subsequently screened with 32P-labeled full length of Ni-OsHKT1 cDNA (see Results) under high-stringency conditions. Positive clones were recovered by in vivo excision according to the manufacturer's manual.
Genomic DNA gel blot analysis
Approximately 5 µg of genomic DNA derived from the shoot of each rice variety were digested with XbaI, HindIII or EcoRV, and separated on a 1.0% (w/v) agarose gel. Hybridization was performed in the buffer described by Church and Gilbert (1984) at 65°C. Full length of 32P-labeled Ni-OsHKT1 cDNA was used as probe.
Detection of OsHKT transcripts
Poly (A)+ RNA were isolated from root samples of seedlings grown in modified N6 medium with varying K+ and Na+ concentrations by using of oligo (dT)-cellulose (Pharmacia).
For Northern hybridization of Nipponbare RNA, 1 µg of each RNA sample was electrophoresed on a 1.0% (w/v) agarose-formaldehyde gel, and blotted onto a nylon membrane (Hybond-N+, Amersham). Hybridization was performed as previously described (Church and Gilbert, 1984) at 65°C. A 0.8-kb EcoRI fragment (positions 243–1061) of 32P-labeled Ni-OsHKT1 cDNA was used as probe. After autoradiography, the membrane was stripped and reprobed with a 32P-labeled rice actin gene.
For RT–PCR analysis with Pokkali RNA, first-strand cDNA was synthesized from 100 ng of poly(A)+ RNA from each sample with a first-strand cDNA synthesis kit (Perkin Elmer, Branchburg, NJ, USA), and then was used as the template for the PCR reaction. To discriminate between the two different OsHKT genes in Pokkali, specific primer sets, differing by two bases in their 3′ ends from one another, were constructed. Amplification was performed with the following cycle parameters: once at 94°C for 1 min; 30 times at 94°C for 30 sec, 60°C for 30 sec, 72°C for 90 sec; and once at 72°C for 7 min. Degenerated actin primers were used as an internal control, and the set was as follows: 5′-GG (A/C) AC (C/T) G (A/G) (A/T) ATGGTCAAG-3′ and 5′-GAAGCA (C/T) TTC (A/C) TGTG (C/G) AC-3′ (Reece et al., 1990).
Complementation and growth inhibition assays using S. cerevisiae
Expression plasmids containing OsHKT1, OsHKT2, and TaHKT1 (Schachtman and Schroeder, 1994), under the control of the GAL1 promoter in the pYES2 vector were used for yeast complementation and growth inhibition assays. S. cerevisiae strain CY162 (MATa, trk1, trk2::pCK64, his3, ura3; Anderson et al., 1992) and G19 (MATa, his3, ura3, trp1, ade2, and ena1::HIS3::ena4; Quintero et al., 1996), and the parent strain of G19, W303 (MATa, his3, leu2, ura3, trp1, and ade2), were transformed. Ura3+ transformants were selected on Ura-selective medium containing 0.67% (w/v) yeast nitrogen base, 2% (w/v) sucrose, 100 mm KCl, and 2% agar (w/v). For complementation tests, an arginine phosphate medium containing 2% (w/v) galactose, 0.6% (w/v) sucrose, 2% (w/v) agar, 10 µm NaCl, and the indicated concentrations of KCl were used (Rodríguez-Navarro and Ramos, 1984). For growth inhibition test, a medium containing 0.67% (w/v) yeast nitrogen base, 2% (w/v) galactose, 0.6% (w/v) sucrose, 2% (w/v) agar, and the indicated concentrations of NaCl were used.
OsHKT genes expression and electrophysiology in X. laevis oocytes
The coding region of each OsHKT cDNA, with Bgl II restriction sites attached at each end by PCR, were subcloned into the Bgl II site of pXßG-ev1 (Prestone et al., 1992), a kind gift from Dr Katsuhara (University of Okayama), under the control of the T3 promoter. OsHKT mRNA was transcribed from linearized plasmid using mMESSAGE mMACHINE in vitro transcription kit (Ambion, Austin, TX, USA) and capped complementary RNA was injected into X. laevis oocytes. The oocytes were kept for 1–2 days at 18°C in standard Barth's solution containing 88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 0.33 mm Ca(NO3)2, 0.41 mm CaCl2, 0.82 mm MgSO4, and 10 mm 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES)-NaOH, pH 7.4, before electrophysiological measurements.
Two-electrode voltage clamp experiments were performed using a Dagan CA1 amplifier (Dagan Corp., Minneapolis, MN, USA). The detailed descriptions of the electrophysiological experiments are shown in Sabirov et al. (1997). Oocytes were perfused with a solution containing 6 mm MgCl2, 1.8 mm CaCl2, 10 mm 2-(N-morpholino)-ethanesulfonic acid (MES)-1,3-bis (Tris[hydroxy methyl]methylamino) propane (BTP), pH 5.5, 180 mm d-mannitol, and the indicated concentrations of NaCl and KCl. The ionic strength of the solutions for different Na+ and K+ concentrations was kept constant by adding N-methyl-d-glucamine as an ‘inert’ cation. In the solutions 100 mm of alkali cations, d-mannitol was omitted. pCLAMP (Axon Instr., CA, USA) was used for electrophysiological measurements. A ramp command was generated, in which the membrane potential was swept from −100 mV to +100 mV with the rate of 1 mV ms−1 (200 ms from −100 mV to 100 mV). Microelectrode resistances were between 0.2 and 0.8 MΩ when filled with 3 m KCl. All experiments were performed at room temperature (25°C).
We would like to thank Dr Julian I. Schroeder, University of California, San Diego, USA, for generously providing us with the wheat HKT1 cDNA and for helpful discussions, and Dr Richard F. Gaber, University of North-western, IL, USA for generously providing us with the yeast CY162 strain. We are grateful to Dr Alonso Rodríguez-Navarro, Universidad Politecnica de Madrid, Spain, for generously providing us with the yeast G19 strain and for helpful discussions. We thank Dr Maki Katsuhara, Okayama University, Japan, for generously providing us with the pXßG-ev1, and Dr Noboru Endo, TAISEI Corporation, Japan, for generously providing us with the seeds of Pokkali. We also thank Dr Nobuyuki Uozumi, Nagoya University, Japan, and Dr Masami Sekine and Dr Ko Kato from our laboratory for helpful discussions. This work was supported by the ‘Research for the Future’ Program of the Japan Society for the Promotion of Science and by a Grants-in-Aid for Scientific Research (12019249) from the Ministry of Education, Science and Culture, Japan.