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Journal of Experimental Botany 2007 58(15-16):4387-4395; doi:10.1093/jxb/erm306
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Cloning and functional comparison of a high-affinity K+ transporter gene PhaHKT1 of salt-tolerant and salt-sensitive reed plants

Ryuichi Takahashi1, Shenkui Liu2 and Tetsuo Takano1,*

1Asian Natural Environmental Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishitokyo-shi, Tokyo 188-0002, Japan
2Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin 150040, PR China

* To whom correspondence should be addressed: E-mail: takano{at}anesc.u-tokyo.ac.jp

Received 20 September 2007; Revised 29 October 2007 Accepted 31 October 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
To understand the mechanisms of ion homeostasis in salt-tolerant and salt-sensitive plants, cDNAs for a high-affinity K+ transporter PhaHKT1 were isolated from salt-sensitive (Utsunomiya) and salt-tolerant (Nanpi, Enchi) reed plants. A cDNA of Utsunomiya (PhaHKT1-u) contained two insertions in the region corresponding to the first and second introns of the PhaHKT1 gene, which resulted in a sequence 141 amino acid residues shorter than that of Nanpi. Expression of PhaHKT1 mRNA was detected in the roots of Nanpi and Enchi plants under K+ starvation conditions and also under Na+ treatment conditions, whereas it was only slightly detected in the roots of Utsunomiya plants under each of these conditions. In the upper parts, PhaHKT1 expression was detected in the Utsunomiya plants, and two signals were obtained in the Nanpi and Enchi plants under all and K+ starvation conditions, respectively. Yeasts expressing the PhaHKT1 of Nanpi (PhaHKT1-n) or the PhaHKT1 of Enchi (PhaHKT1-e) grew better in the presence of NaCl than yeast expressing PhaHKT1-u. Furthermore, yeast expressing a chimeric cDNA containing the 5' region of the Utsunomiya gene and the 3' region of the Nanpi gene had partial salt tolerance, and yeast expressing a chimeric cDNA containing the 5' region of the Nanpi gene and the 3' region of the Utsunomiya gene had a reduced ability to take up ions. These results suggest that PhaHKT1 plays an important role in the acquisition of K+ and maintenance of ion balance under saline conditions.

Key words: HKT, PhaHKT1, potassium transporter, reed plants, salt stress, salt tolerance, sodium influx


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Potassium is an essential nutrient in plants because it is required for maintaining enzyme activity, osmoregulation, and electroneutrality. Thus, plants need to acquire K+ continuously and to maintain K+-Na+ selectivity for growth and salt tolerance. Under saline conditions, Na+ is thought to enter plant cells mainly through voltage-independent channels (Blumwald, 2000), but also through several K+ transporters (Schachtman and Liu, 1999; Maser et al., 2002c).

High affinity K+ transporter (HKT) cDNAs have been isolated from many kinds of plants (Schachtman and Schroeder, 1994; Fairbairn et al., 2000; Uozumi et al., 2000; Horie et al., 2001; Su et al., 2003; Ren et al., 2005). HKT belongs to a superfamily of yeast TRKs and bacterial KtrBs, and is considered to mediate high-affinity K+ transport (Tholema et al., 1999; Rodriguez-Navarro, 2000). HKT-type transporters have been shown to function as K+-Na+ co-transporters (Rubio et al., 1995, 1999; Gassmann et al., 1996; Liu et al., 2001), but the Arabidopsis homologue AtHKT1 (Uozumi et al., 2000) and the rice homologue OsHKT1 (Horie et al., 2001; Garciadeblas et al., 2003) and OsHKT8 (Ren et al. 2005) were found to transport only Na+. HKT proteins contain four MPM motifs, each of which consists of two transmembrane domains (M1 and M2) and a pore-forming region (P) (Durell and Guy, 1999; Durell et al., 1999). The glycine in the first P-loop is considered to contribute to the transporter's K+ selectivity (Maser et al., 2002b). Recent studies have suggested that, in Arabidopsis, AtHKT1 is mainly involved in long-distance transport of Na+ and that it does not have an important role in the uptake of K+ in root (Maser et al., 2002a; Berthomieu et al., 2003; Sunarpi et al., 2005).

Reed plants (Phragmites australis Trinius) are hydrophilous perennial grasses growing in fresh and brackish water areas of swamps, riversides, estuaries, and coasts. Furthermore, the plants can adapt to adverse environments such as drought and salinity regions. Reed plants growing in saline environments maintain low Na+ levels by restricting the intrusion of Na+ into the shoot or by preferentially excluding Na+ from the shoot base to the root (Matoh et al., 1988; Matsushita and Matoh, 1991, 1992). It has been found that reed plants from a riverside location (Utsunomiya) were salt sensitive and unable to grow even in the presence of 50 mM NaCl, whereas reed plants from a high salinity region (Nanpi) tolerated 150 mM NaCl (Takahashi et al., 2007a). The salt-tolerant reed plants contained low sodium and high potassium, whereas the salt-sensitive ones contained high sodium and low potassium in the presence of NaCl (Takahashi et al., 2007a, b). These data suggested that salt-tolerant reed plants have an ability to select potassium over sodium, and that this ability helps to maintain ion homeostasis in spite of the salinity conditions.

In this study, cDNAs were isolated for an HKT-type potassium transporter (PhaHKT1) from salt-sensitive and salt-tolerant reed plants to understand the function of HKT1 in relation to the salt tolerance of reed plants.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant material and growth conditions
Reed plants (Phragmites australis Trinius) were collected from the arid and high salinity regions at Nanpi (38°1’ N; 116°44’ E) and Enchi (37°50’ N; 107°15’ E) in China, and from a riverside at Utsunomiya and Fuchu in Japan. Nanpi is located about 100 km inland from the Bohai Sea, and Enchi is located in the middle of the Yellow River. The soil of Nanpi and Enchi contains about 0.4% and 1.0% of Na+, respectively. Utsunomiya is a city on the Kinugawa River in the Tochigi prefecture, and the riverbank soil contains <0.01% Na+. Fuchu is a city on the Nogawa River in the Tokyo prefecture, and the riverbank soil is also not affected by salinity.

Reed plants were grown as described previously (Takahashi et al., 2007c). For the salt stress treatment, NaCl was added to this solution to a final concentration of 50 mM. For the K+-starved treatment, KCl was removed from the standard solution. Each stress treatment was continued for 7 d.

Isolation and cloning of PhaHKT1 genes
Total RNA was extracted from reed plants grown in K+-starved medium for 7 d using ISOGEN (Nippon gene, Japan) according to the manufacturer's instructions. The RNA was reverse transcribed by using an oligo dT primer and MMLV-RT. The reverse transcription products were amplified by PCR, using the degenerate primers (Degenerate-Forward, Degenerate-Reverse) deduced from the conserved regions of HKT (Table 1). Amplified cDNA fragments were subcloned into a pBluescript plasmid vector, and were sequenced. To obtain the full-length cDNAs of the genes, 5'-RACE and 3'-RACE were performed with a Marathon cDNA amplification kit (Clontech, USA) according to the manufacturer's instructions. Full-length cDNAs of the PhaHKT1 genes containing the open reading frames (ORFs) were obtained by PCR using the Full-F2 and Full-R4 primers (Table 1).


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  		Table 1. Oligonucleotide primers used for PCR reactions in this study

 
The PCR products were subcloned into the pT7-Blue vector (Novagen, Germany) and sequenced. They were inserted also into the pAUR123 vector (TaKaRa, Japan) for expression in yeast cells.

To determine the nucleotide sequence of the genomic DNA corresponding to the HKT cDNAs, PCR was performed using the Full-F2 and Full-R4 primers (Table 1) and genomic DNAs as templates. The PCR products were subcloned into the pT7-Blue vector and sequenced.

Southern blot analysis
Genomic DNA was prepared from leaves of each reed plant using a DNeasy Plant Maxi Kit (QIAGEN, Germany). 5 µg of DNA was digested with BamHI, HindIII, and PstI. The digests were separated on 0.7% agarose gel, and blotted onto a nylon membrane. The membrane was hybridized overnight in hybridization buffer containing DIG-labelled full-length PhaHKT1-n cDNA with a high concentration of SDS. The membrane was washed twice for 10 min with wash buffer 1 (2x SSC, 0.1% SDS), washed twice for 15 min at 45 °C or 65 °C with wash buffer 2 (0.1x SSC, 0.1% SDS), treated with anti-digoxigenin-AP for 30 min, treated with CDP-star (Amersham, UK) and imaged in an image analyser (LAS-1000plus; FUJIFILM, Japan).

RT-PCR analysis
Total RNAs prepared from roots or the upper parts of plants were reverse transcribed as described above. Amplification reactions were carried out with Chimeric-F1 and Full-R4 primers (Table 1) and KOD DNA polymerase (TOYOBO, Japan) in a volume of 50 µl. After denaturation (1 min, 94 °C), samples were subjected to 35 cycles of 30 s at 94 °C, 30 s at 55 °C, and 1 min at 68 °C. The amplified fragments were cloned into pT7-Blue vector and sequenced. The actin gene was also amplified as an internal standard using Actin-F1 and Actin-R1 primer (Table 1). PCR products were separated on 0.8% TAE agarose gels.

Construction of chimeric cDNA
Two chimeric genes, one containing the 5' region of the Utsunomiya gene connected to the 3' region of the Nanpi gene, and the other containing the 5' region of the Nanpi gene connected to the 3' region of the Utsunomiya gene, were constructed. Partial cDNA fragments were obtained by PCR using the full-length cDNA of Utsunomiya or Nanpi as templates. For this purpose, two primers (Chimeric-F1 and -R1) were designed (Table 1). The 5' fragment was amplified by using the Full-F2 and Chimeric-R1 primers, and the 3' fragment was amplified by using the Chimeric-F1 and Full-R4 primers. Finally, the full-length cDNAs for chimeric transporters were obtained by using these two PCR fragments as templates and by performing a final PCR with primers for the full length.

Ion uptake experiment
Full-length cDNAs for PhaHKT1 were inserted into the protein expression vector pAUR123, and introduced into the yeast Saccharomyces cerevisiae strain 9.3 (ATCC, USA, No. 201409), in which the original potassium transporters (TRK1, 2) and P-type ATPase involved in Na+ extrusion (ENA1–4) were deleted.

Transformed yeast cells were grown in SD medium (Dropout medium without histidine), and amplified until the OD600 value reached about 0.7 (1.0x108 cells). For K+ and Na+ uptake experiments, cells were suspended in an uptake buffer [2.0% glucose, 10 mM MES, and pH 6.0 adjusted with Ca(OH)2], and incubated for 2 h. Cells were rinsed twice in distilled water, and were acid-extracted overnight in 0.1 M HCl. Cell samples were collected by centrifuge at 5000 g for 5 min, and the K+ and Na+ concentrations in the supernatant were determined. The K+ and Na+ contents were determined with an atomic absorption spectrophotometer (AA-670G; Shimadzu, Japan).

Control experiments were performed with the yeast 9.3 strain transformed with plasmid pAUR123 without an insert. Three replicates of each treatment were performed.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cloning of PhaHKT1 cDNAs
By performing RT-PCR using degenerate primers, cDNA homologues of a HKT high-affinity K+ transporter were isolated from salt-sensitive reed plants (Utsunomiya) and from salt-tolerant reed plants (Nanpi and Enchi). The lengths of the amplified cDNA fragments were about 500 bp, and the translated amino acid sequences showed high homology to TaHKT1 (Schachtman and Schroeder, 1994). The full-length cDNAs of each reed plant were obtained by 5' and 3' RACE. The Utsunomiya, Nanpi, and Enchi genes were designated as PhaHKT1-u (Accession No. AB234303), PhaHKT1-n (AB234304), and PhaHKT1-e (AB234305), respectively.

PhaHKT1-n and PhaHKT1-e contained open reading frames (ORFs) of 1593 bp and 1671 bp, respectively, encoding 531 and 557 amino acid residues, respectively (Fig. 1). The nucleotide sequence of PhaHKT1-u was highly homologous to the sequence of PhaHKT1-n and PhaHKT1-e in the 5' region, but contained two insertions in the 3' region. The regions of the PhaHKT1 genes were amplified and sequenced using genomic DNA as templates from each reed plant, and it was found that each insertion corresponds to an intron of the PhaHKT1 gene (Fig. 2). The first insertion corresponds to 60 bp (from bp 1258–1317) of intron 1 in the genome containing the PhaHKT1 gene (PhaHKT1-genome), and the second insertion completely corresponds to intron 2 (Fig. 2). A stop codon was generated within intron 1 of PhaHKT1-u cDNA, resulting in an ORF of 390 amino acids lacking the normal C-terminus of the protein. Since each PhaHKT1 has a glycine at a certain position of the P-loop of the first MPM motif, they are considered K+-Na+-type rather than Na+-Na+-type transporters (Maser et al., 2002b).


Figure 1
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  		Fig. 1. Amino acid sequence alignment of HKT from reed plants (Utsunomiya, Nanpi, Enchi), wheat (TaHKT1), and Arabidopsis (AtHKT1). Black and grey backgrounds indicate identical residues and similar residues, respectively. The bar indicates the region changed as chimeric cDNA.

 

Figure 2
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  		Fig. 2. Structures of HKT1 gene (PhaHKT1-genome), HKT1 cDNA of Utsunomiya (PhaHKT1-u), and HKT1 cDNA of Nanpi (PhaHKT1-n). PhaHKT1-u contains two insertions corresponding to the introns of the HKT gene of reed plants, whereas PhaHKT1-n does not have these insertions. The first insertion in PhaHKT1-u was incomplete (60 bp of 85 bp) and shorter than intron 1 in PhaHKT1-genome, whereas the second insertion completely corresponded to intron 2. A stop codon was generated in PhaHKT1-u (asterisks) upstream of the stop codon of PhaHKT1-n (open triangles) because of the failure to remove intron 1. The locations of the primers (Primers 3–8 in Table 1) are indicated. White and black bars indicate exons and introns, respectively.

 
Genomic Southern blot analysis was carried out with full-length PhaHKT1-n cDNA as a probe. Although the PhaHKT1-genome has no HindIII site, each PhaHKT1-genome has one PstI site and the PhaHKT1-genome of Nanpi plants has one BamHI site. In the Nanpi lane, one major band was detected after BamHI digestion, and two or three bands were detected with HindIII digests, suggesting that the Nanpi plants have 2–3 copies of the HKT gene (Fig. 3). On the other hand, two bands were detected with PstI digests in the Utsunomiya lane, suggesting that the Utsunomiya plants have 1–2 copies of the HKT gene (Fig. 3).


Figure 3
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  		Fig. 3. Southern blot analysis of genomic DNA from reed plants. DNA (5 µg) was digested with BamHI (B), HindIII (H), and PstI (P). The membrane was washed at 45 °C (A) or 65 °C (B). Full-length of DIG-labelled PhaHKT1-n cDNA was used as probe.

 
Expression of HKT
PhaHKT1 transcripts were detected at moderate levels in the roots of Enchi plants under control conditions and in the roots of Nanpi and Enchi plants under K+-starvation and Na+-treatment conditions, and a larger transcript was slightly detected in the roots of Utsunomiya plants under both conditions (Fig. 4A).


Figure 4
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  		Fig. 4. RT-PCR analysis of the PhaHKT1 of Utsunomiya (U), Nanpi (N), and Enchi (E) plants. RNA was extracted from the roots (A) or upper parts (B) of plants after 1 week of KCl starvation or 50 mM NaCl treatment, and the reverse transcription products were amplified. Under control conditions, KCl concentration in the medium was 1 mM. (C) Amplified cDNA fragments of the PhaHKT1 of Utsunomiya (U), Nanpi (N), and Fuchu (F). Primer pair Chimeric-F1 and Full-R4 was used (Table 1).

 
In the upper parts, two signals were obtained in the Nanpi plants under all conditions and in the Enchi plants under K+-starvation conditions (Fig. 4B). The larger transcripts were due to the presence of two introns (compare with Fig. 1 with Fig. 2), and the shorter transcripts, which do not contain introns were detected only in salt-tolerant reed plants. Only larger transcripts were detected in the Utsunomiya plants, and in the other salt-sensitive reed plants, Fuchu (Fig. 4C). PhaHKT1 transcripts were strongly detected in the upper parts of Utsunomiya plants under control and K+-starvation conditions and weakly detected under Na+-treatment conditions (Fig. 4B). In the Utsunomiya plants, there was an additional band under Na+ treatment-conditions. To investigate the existence of other splice variants in the Utsunomiya plants, 50 clones between primer 5 and primer 6 (Fig. 2) were sequenced using cDNA from the Utsunomiya plants as template. None of the clones spliced out intron 1 completely. Thirty-seven clones included 60 bp of intron 1, as in PhaHKT1-u, and the other 13 clones contained less than 60 bp of intron 1 (data not shown).

Ion uptake in transformed yeast
To characterize PhaHKT1 functionally, each PhaHKT1 cDNA was introduced and expressed in yeast cells and the ion uptake characteristics of the cells were analysed. Since a stop codon was generated in the PhaHKT1-u cDNA upstream of the stop codon of PhaHKT1-n, it is expected that the C-terminus of PhaHKT1-u is shorter than the normal C-terminus of the protein. To clarify the role of the C-terminus region which is deleted in PhaHKT1-u, chimeric cDNAs were constructed in which the 5' region of PhaHKT1-u is connected to the 3' region of PhaHKT1-n (U-N) and in which the 5' region of PhaHKT1-n is connected to the 3' region of PhaHKT1-u (N-U). The cDNAs were then expressed in yeast cells.

The yeast strains expressing PhaHKT1-n or PhaHKT1-e grew better than the strain expressing PhaHKT1-u under micromolar K+ concentrations (Fig. 5). The yeast strain expressing U-N complemented the K+ uptake deficiency phenotype, whereas the yeast strain expressing N-U didn't. Furthermore, the yeast strain expressing PhaHKT1-n, PhaHKT1-e, and U-N grew even in the presence of 100 mM NaCl, whereas the yeast strain expressing PhaHKT1-u and N-U were suppressed.


Figure 5
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  		Fig. 5. Growth of yeast cells expressing PhaHKT1 of reed plants or chimeric cDNA. The strain 9.3, in which the original potassium transporter was deleted, was transformed with the empty plasmid pAUR123 or with the plasmid containing PhaHKT1 or chimeric cDNA. YPD medium or SD medium supplemented with 10 µM KCl, 50 µM KCl, 50 µM KCl and 50 mM NaCl or 1 mM KCl and 100 mM NaCl were inoculated with serially diluted yeast cell suspensions.

 
The yeast strains expressing PhaHKT1-n or PhaHKT1-e showed a high K+ accumulation rate, whereas the yeast strain expressing PhaHKT1-u showed a lower K+ accumulation rate, even in the presence of 1 mM K+ (Fig. 6). The rate at which PhaHKT1-e-transformed yeast accumulated K+ was the same as that of PhaHKT1-n-transformed yeast at K+ concentrations above 0.5 mM, and was almost the same as that of PhaHKT1-u-transformed yeast at K+ concentrations in the micromolar range. The rate at which the yeast strain expressing N-U chimeric cDNA accumulated K+ was the same as that of the strain expressing PhaHKT1-u under all conditions, whereas the rate at which the yeast strain expressing U-N accumulated K+ was between the rates of the PhaHKT1-n and PhaHKT1-u-expressing yeasts.


Figure 6
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  		Fig. 6. Concentration dependence of K+ uptake in yeast expressing PhaHKT1 of reed plants or chimeric cDNA. Strain 9.3 yeast cells were transformed with empty plasmid (open diamonds) or with a plasmid containing PhaHKT1-u (closed circles), PhaHKT1-n (closed triangles), PhaHKT1-e (closed squares), U-N chimeric cDNA (asterisk), and N-U chimeric cDNA (open circles), respectively. Results are expressed as means ±SE (n=3).

 
The rate at which the yeast strains expressing PhaHKT1-n or PhaHKT1-e accumulated K+ decreased in the presence of NaCl (Fig. 7A), but at all the Na+ concentrations examined, it was still higher than the K+ accumulation rate of the strain expressing PhaHKT1-u. In the presence of NaCl, the yeast strain expressing N-U accumulated K+ at a low rate similar to that of the control and the strain expressing U-N accumulated K+ at a rate between the rates of the strains expressing PhaHKT1-n and PhaHKT1-u. On the other hand, the rates at which the yeast strains accumulated Na+ were not significantly different (Fig. 7B).


Figure 7
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  		Fig. 7. Concentration dependence of K+ uptake (A) and Na+ influx (B) in yeast expressing PhaHKT1 or chimeric cDNA in the presence of 1 mM KCl and 5, 10, 25, 50 and 100 mM of NaCl. Symbols are the same as those in Fig. 6. Results are expressed as means ±SE (n=3).

 
The K+/Na+ ratio was calculated using the data of Fig. 7A and B. At NaCl concentrations less than 100 mM, the K+/Na+ ratios of the yeast strains expressing PhaHKT1-n and PhaHKT1-e were much higher than the ratio of the strain expressing PhaHKT1-u (Fig. 8). Yeast expressing PhaHKT1-n and PhaHKT1-e maintained a high K+/Na+ ratio under saline conditions because of their high rates of K+ accumulation, although the rate of Na+ accumulation was not low.


Figure 8
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  		Fig. 8. K+ to Na+ ratio of yeast cells transformed with empty plasmid (open bars) or plasmids containing PhaHKT1 genes from reed plants from Utsunomiya, Nanpi, Enchi, U-N chimeric cDNA, and N-U chimeric cDNA in the presence of 1 mM KCl and 5, 10, 25, 50, and 100 mM NaCl. Results are expressed as means ±SE (n=3). Asterisks indicate a significant difference from the control at P <0.05 by Student's t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, the cDNAs were cloned for PhaHKT1 which encoded the high-affinity K+ transporters from salt-tolerant and salt-sensitive reed plants. PhaHKT1-n and PhaHKT1-e showed 72% and 69% identity to TaHKT1 (Schachtman and Schroeder, 1994) at the amino acid level, respectively, but PhaHKT1-u contained two insertions in the region corresponding to the first and second introns of the PhaHKT1 gene, which, because one of the insertions introduced a stop codon, resulted in a truncated protein that was 141 amino acids shorter than the protein of Nanpi (Fig. 1).

To confirm the occurrence of alternate splicing, RT-PCR was performed with a primer pair designed to amplify the region containing the exon–intron junction sites. Most (37/50) of the sequences of the PCR products matched the sequence of the PhaHKT1-u cDNA, and no difference was found in the exon–intron junction sites of the PhaHKT1-genome (data not shown). Interestingly, the other 13 clones contained a shorter insertion than that of PhaHKT1-u, and none of the clones spliced out intron 1 completely. Our finding that unspliced PhaHKT1 was also expressed in the upper parts of Nanpi and Enchi plants, in addition to the spliced transcripts (Fig. 4B), suggests that the Nanpi and Enchi plants can splice the two introns of the PhaHKT1 gene during mRNA maturation, whereas the Utsunomiya plants do not. Since unspliced transcripts were only detected in the other salt-sensitive plants Fuchu (Fig. 4C), there is a possibility that the inability to splice the introns of PhaHKT1 is common to salt-sensitive reed plants. Alternate splicing of the HKT gene has been reported in OsHKT1 of rice (Golldack et al., 2002). Larger transcripts, which contain one intron and encode a shorter and dysfunctional HKT protein because of the presence of a stop codon in the intron, were detected only in the salt-sensitive rice line IR29 in the presence of millimolar concentrations of K+, Rb+ or Cs+ (Golldack et al., 2002). In the salt-tolerant rice line Pokkali, no transcripts with an intron were detected. Golldack et al. (2002) suggested that incomplete splicing was a possible mechanism for retaining transcripts in the nucleus or reducing translatable transcripts under stress conditions. The results in rice plants are compatible with our results in that the salt-tolerant plants have more functional HKT protein than salt-sensitive plants. There have been no reports about alternate splicing in the other ion channels or ion transporters. Further studies are needed to understand the relation between the alternate splicing of HKT genes and salt-tolerance, and to understand the function of PhaHKT1 in the salt-tolerance mechanism of reed plants.

Some amino acid residues in TaHKT1 or AtHKT1 have been identified as being involved in determining K+ and Na+ binding affinity (Rubio et al., 1995, 1999; Diatloff et al., 1998; Kato et al., 2001), and these same amino acid residues are present in each of the PhaHKT1s. Thus, the dysfunction of PhaHKT1-u is not due to substituted amino acids but to the lack of the C-terminal portion. The C-terminal portion has around 141 amino acids and contains the M2 domains of the third and fourth MPM motifs. Growth of the yeast strain expressing PhaHKT1-u and N-U was suppressed in the presence of micromolar concentrations of K+ (Fig. 5), and the K+ contents of the PhaHKT1-u- and N-U-transformed yeast strains were very low compared with those of the PhaHKT1-n- and PhaHKT1-e-transformed yeast strains, whereas in the yeast strain expressing U-N, the ability to take up K+ was partially restored (Fig. 6). These results suggest that PhaHKT1-u is a non-functional form of the transporter. The protein product of an incompletely spliced OsHKT1 transcript also had no HKT activity (Golldack et al., 2002), although the structure of the protein was not shown.

One possibility is that the dysfunction of PhaHKT1-u is due to an inability of the truncated protein to reach its proper location in the membrane. PhaHKT1-u lacks the C-terminal portion. PhaHKT1-n and PhaHKT1-u are predicted to localize on the plasma membrane by the PSORT program. However, the lack of a C-terminus will change the structure of the protein, and affect the stability of the protein, which might be part of the reason why PhaHKT1-u is non-functional.

The expression of HKT1 was localized to the vascular tissues in all organs (Golldack et al., 2002; Maser et al., 2002a; Berthomieu et al., 2003; Su et al., 2003; Sunarpi et al., 2005; Kader et al., 2006). These studies suggest that HKT transporters contribute to root-shoot Na+ distribution or Na+ recirculation. Furthermore, AtHKT1 was shown to control Na+ homeostasis in Arabidopsis (Rus et al., 2001, 2004). The cytosolic K+/Na+ ratio is an important determinant of Na+ tolerance in plants, and salt-tolerant reed plants had a much higher K+/Na+ ratio than salt-sensitive reed plants (Takahashi et al., 2007a). Under K+-starvation and Na+-treatment conditions, PhaHKT1 mRNA was expressed more strongly in the roots of salt-tolerant reed plants (Fig. 4A), and mature PhaHKT1 mRNA was also detected only in salt-tolerant reed plants. It was found that Nanpi plants contain low Na+ in the upper parts and accumulate Na+ in the roots, whereas Utsunomiya plants contain high Na+ in the upper parts (Takahashi et al., 2007a). Furthermore, Na+ contents in the leaves and stems having increased, decreased to the same level as that in the control plants at 10 d in the Nanpi plants, whereas they continuously increased through 10 d in the Utsunomiya plants (Takahashi et al., 2007a). These results raise the possibility that PhaHKT1 also plays a role in the recirculation of Na+ from shoots to roots or in the distribution of Na+ to shoots and roots in salt-tolerant reed plants.

Reed plants are distributed worldwide and are categorized as glycophytes, although some reed plants, i.e. those that grow in saline soils, have high salt tolerance. This means that the reed plants have differentiated into many phenotypes including salt-tolerant phenotypes, that have adapted to different habitats during their distribution from the place of origin. The loss of the ability to splice introns of HKT genes should have occurred in the reed plants during the distribution. Although the reed plants appear to have between one and three copies of the HKT gene (Fig. 3), it is unclear how many of these are functional. Even if the Utsunomiya plants have a functional HKT gene, it is considered that salt-sensitive reed plants have been distributed with non-functional form of PhaHKT1, and a non-functional PhaHKT1-u might still seriously degrade the plant's ion transport ability and salt-tolerance. To clarify the role of PhaHKT1 in salt tolerance, functional studies of PhaHKT1 in planta, for example, physiological analysis of transgenic plants over-expressing PhaHKT1, are needed. An Agrobacterium-mediated transformation technique has been established in reed plants (Mori et al., 2000). Introduction of a functional PhaHKT1 gene into salt-sensitive reed plants, in which a functional PhaHKT1 protein is not expressed, and study of the transgenic reed plant should clarify the mechanism by which PhaHKT1 affects Na+ distribution or recirculation in reed plants. Further comparative analysis between salt-tolerant reed plants and another plant species will also help to understand the function of PhaHKT1 and the salt-tolerant mechanisms of glycophytes.


   Acknowledgements
 
We thank Dr Nobumasa Ichizen and Dr Takayoshi Nishio (Weed Science Center in Utsunomiya University) for kindly providing reed plants of Enchi, Nanpi, and Utsunomiya. This work was supported by a Grant-in-aid for Scientific Research (18658001) and by a grant from Heiwa Nakajima Foundation to TT.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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