JXB Advance Access published online on March 5, 2008
Journal of Experimental Botany, doi:10.1093/jxb/ern033
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© 2008 The Author(s).
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RESEARCH PAPER |
Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance
CSIRO Plant Industry, Canberra, Australian Capital Territory 2601, Australia
To whom correspondence should be addressed: E-mail: rana.munns{at}csiro.au
Received 21 September 2007; Revised 7 January 2008 Accepted 9 January 2008
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Abstract |
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Salt tolerance of plants depends on HKT transporters (High-affinity K+ Transporter), which mediate Na+-specific transport or Na+-K+ co-transport. Gene sequences closely related to rice HKT genes were isolated from hexaploid bread wheat (Triticum aestivum) or barley (Hordeum vulgare) for genomic DNA southern hybridization analysis. HKT gene sequences were mapped on chromosomal arms of wheat and barley using wheat chromosome substitution lines and barley–wheat chromosome addition lines. In addition, HKT gene members in the wild diploid wheat ancestors, T. monococcum (Am genome), T. urartu (Au genome), and Ae. tauschii (Dt genome) were investigated. Variation in copy number for individual HKT gene members was observed between the barley, wheat, and rice genomes, and between the different wheat genomes. HKT2;1/2-like, HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, and HKT1;5-like genes were mapped to the wheat–barley chromosome groups 7, 7, 2, 6, 2, and 4, respectively. Chromosomal regions containing HKT genes were syntenic between wheat and rice except for the chromosome regions containing the HKT1;5-like gene. Potential roles of HKT genes in Na+ transport in rice, wheat, and barley are discussed. Determination of the chromosome locations of HKT genes provides a framework for future physiological and genetic studies investigating the relationships between HKT genes and salt tolerance in wheat and barley.
Key words: Barley, comparative mapping, HKT, rice, salt tolerance, sodium transport, wheat
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Introduction |
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Soil salinity is one of the major abiotic environmental problems affecting agricultural production. The problem of salinization is increasing due to land clearing or irrigation (Rengasamy, 2006). To meet this challenge, it is important to understand the mechanisms of salt tolerance to improve further salt tolerance of crops such as wheat and barley (Colmer et al., 2005). In wheat, sodium exclusion is one of the major mechanisms conferring salt tolerance (Gorham et al., 1990b; Munns et al., 2006). Bread wheat (Triticum aestivum, AABBDD) has a low rate of Na+ transport to the shoot and maintains a high ratio of K+/Na+ in the leaves (Gorham et al., 1990b). This trait is conferred, at least partially, by the Kna1 gene on chromosome 4DL (Dubcovsky et al., 1996). Durum wheat (Triticum turgidum ssp. durum, AABB) is more salt-sensitive than bread wheat (Rawson et al., 1988) due to its poorer ability to exclude Na+ from the shoot (Gorham et al., 1990b). However, an unusual durum wheat, Line 149, has the low Na+ concentrations and high K+/Na+ ratios in the leaf blade typical of bread wheat. Line 149 is derived from a cross between Triticum monococcum (AmAm) accession C68-101 and the durum wheat cultivar Marrocos (The, 1973). The Na+ exclusion trait in Line 149 is controlled by two major genes (Munns et al., 2003), namely Nax1 and Nax2, interacting via net Na+ xylem loading and net leaf sheath sequestration (Davenport et al., 2005). In barley (Hordeum vulgare), Na+ exclusion and K+/Na+ selectivity is lower than in bread wheat (Gorham et al., 1990a); however, in barley leaves, Na+ is compartmentalized into the vacuole and therefore its toxicity is reduced (Greenway and Munns, 1980). Furthermore, K+ in barley leaves is partitioned more into mesophyll cells than into epidermal cells, resulting in a higher K+:Na+ ratio in the cytoplasm of mesophyll cells, which is beneficial for maintaining photosynthesis (James et al., 2006b).
Salt tolerance of plants could depend on HKT transporters (High-affinity K+ Transporter), which mediate Na+-specific transport or Na+-K+ transport and play a key role in regulation of Na+ homeostasis (Rodríguez-Navarro and Rubio, 2006; Munns and Tester, 2008). In Arabidopsis thaliana there is only one HKT gene (Uozumi et al., 2000). In rice (Oryza sativa) there are eight HKT genes (Horie et al., 2001; Garciadeblás et al., 2003). Based on amino acid sequence similarity, HKT genes have been grouped into two main subfamilies (Platten et al., 2006). The division into the two subfamilies is associated with differences in a key amino acid in the first pore loop of the protein (Mäser et al., 2002b; Garciadeblás et al., 2003); all gene members of subfamily 1 have a serine residue which is replaced by glycine in most members of subfamily 2. The division is also associated with differences in Na+ and K+ selectivity (Horie et al., 2001; Mäser et al., 2002a; Garciadeblás et al., 2003).
Gene members of subfamily 1 are all Na+-specific transporters. Some of them are expressed in cells in the stele rather than the root cortex, and regulate root-to-shoot transport of Na+ by removing Na+ from the xylem sap as it flows to the shoot. The AtHKT1;1 (AtHKT1) transporter plays an important role in regulation of Na+ homeostasis (Rus et al., 2001; Mäser et al., 2002a), but its mechanism of action is not fully resolved (Munns and Tester, 2008). AtHKT1;1 is expressed in xylem parenchyma cells in roots and leaves (Sunarpi et al., 2005), and athkt1;1 mutants have a higher concentration of Na+ in xylem sap than their wild type (Berthomieu et al., 2003; Sunarpi et al., 2005). A quantitative flux analysis study using 22Na+ showed that AtHKT1;1 controls the rate of Na+ transport from root to shoot by the retrieval of Na+ from the xylem in the roots (Davenport et al., 2007). AtHKT1;1 expression has also been detected in phloem tissue (Berthomieu et al., 2003; Sunarpi et al., 2005), but there is no evidence that AtHKT1;1 contributes significantly to recirculation in the phloem and thereby the control of shoot Na+ concentration in Arabidopsis (Davenport et al., 2007).
The rice gene OsHKT1;5 (OsHKT8), first identified as the quantitative trait locus SKC1 (Lin et al., 2004), codes for a transporter that unloads Na+ from the root xylem (Ren et al., 2005). In wheat, HKT1;5-like (HKT8-like) genes are likely candidates for two major genes controlling Na+ exclusion from leaves: Nax2 in durum wheat, and Kna1 in bread wheat (Byrt et al., 2007). Nax2 is associated with Na+ exclusion from leaves via the retrieval of Na+ from the xylem in roots (James et al., 2006a), and Kna1 with Na+ exclusion from leaves via the control of net xylem loading in roots (Gorham et al., 1990b). In wheat also, an HKT1;4-like (HKT7-like) gene is a candidate for the major gene Nax1 (Huang et al., 2006). Nax1 was first identified as a quantitative trait locus for Na+ exclusion in durum wheat by Lindsay et al. (2004), and is associated with the retrieval of Na+ from the xylem in roots and leaf bases (James et al., 2006a).
Gene members of subfamily 2 are Na+-K+ co-transporters or Na+ and K+ uni-porters, except OsHKT2;2 (OsHKT2). Some of them are specifically expressed in the root cortex, and may serve to scavenge Na+ under conditions of K+ deficiency and so provide ionic homeostasis. Under saline conditions the expression of those genes may be down-regulated. This was recently shown to be the case for OsHKT2;1 (OsHKT1) (Horie et al., 2007). OsHKT2;1 mediated the transport of Na+ into roots of K+-starved plants and enhanced their growth, but was down-regulated when plants were exposed to 30 mM NaCl (Horie et al., 2007). In wheat and barley roots, TaHKT2;1 (TaHKT1) and HvHKT2;1 (HvHKT1) also mediated Na+ uptake into roots of K+-starved plants (Laurie et al., 2002; Haro et al., 2005).
In determining the copy number of individual HKT genes and their chromosome locations in wheat and barley, the rice genome sequence provides a useful reference for comparative mapping (Yu et al., 2002). Using the available HKT gene sequences in rice (Horie et al., 2001; Garciadeblás et al., 2003), closely related EST sequences in wheat and barley were identified, and mapped by Southern hybridization. The complex hexaploid wheat genome (Fig. 1) originates from the hybridization of three wild diploid species: T. urartu (A genome), an unknown species closely related to Aegilops speltoides (B genome), and Aegilops tauschii (D genome). Wheat ancestors and wild relatives are a likely source of new genes for salt tolerance in wheat (Colmer et al., 2006). HKT genes were therefore also examined in T. monococcum, T. urartu, and Ae. tauschii.
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This study presents information on the copy number of individual HKT gene members in the barley genome, the different genomes of bread wheat, and related wild diploid wheats, and their chromosome locations of individual HKT members in the barley and bread wheat genomes. The potential role of HKT genes in Na+ transport and salt tolerance in rice, wheat, and barley is discussed.
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Materials and methods |
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Genetic materials
Genetic materials used for HKT gene mapping included the hexaploid bread wheat cv. Chinese Spring, the tetraploid durum wheat cvs Langdon and Tamaroi, the diploid wheats Triticum urartu AUS1789 and AUS1790, Triticum monococcum C68-101 and DV92, Aegilops tauschii AUS18913, and the barley cv. Betzes. For wheat chromosome mapping, nulli-tetrasomic and ditelosomic aneuploid stocks developed in Chinese Spring were used (Sears et al., 1954). In the nulli-tetrasomic lines, a deleted pair of chromosomes is compensated for by two copies of a pair of homoeologous chromosomes. Ditelosomic lines carry a centromeric deletion of one chromosome arm (Sears et al., 1954). For barley chromosome mapping, wheat–barley addition lines were used. These were developed by adding one barley chromosome from Betzes barley (chromosome addition line) or chromosome arm (ditelosomic addition line) into Chinese Spring (Islam et al., 1981).
Database searches
In rice, there are eight HKT-like genes (OsHKT2;1–4, OsHKT1;1, OsHKT1;3–5; Horie et al., 2001; Garciadeblás et al., 2003). OsHKT2;1 has been annotated in the Nipponbare genome sequence as Os06g48810 on chromosome 6 and shares 93% identity at the nucleotide level with OsHKT2;2 (AB061313), a gene isolated from salt-tolerant cultivar Pokkali but which could not be identified in the japonica or indica rice genome sequences. OsHKT2;3 (AJ491820; Os01g34850) was positioned on chromosome 1 and was 95% identical to OsHKT2;4 (AJ491855; Os06g48800) on chromosome 6. OsHKT2;4 is therefore tightly linked to OsHKT2;1 but has only 73–76% sequence identity in three regions of OsHKT2;1 at positions 669–855, 1016–1170, and 1366–1502 (Fig. 2). OsHKT1;1 on chromosome 4 (Os4g51820) was separated by 
3 kb from a pseudogene OsHKT1;2. OsHKT1;1 and OsHKT1;2 gene sequences are 80% identical at the nucleotide level. OsHKT1;3 was located on chromosome 2 (Os02g07830), OsHKT1;4 on chromosome 4 (Os4g51830), and OsHKT1;5 on chromosome 1 (Os01g20160). Garciadeblás et al. (2003) described the sequence similarity between OsHKT genes using phylogenetic trees. NCBI (www.ncbi.nlm.nih.gov) and the Gramene (www.gramene.org) database were used to search closely related wheat or barley sequences for probe design (Table 1).
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Cloning and sequencing of wheat or barley ESTs related to HKT genes in rice
Primers were designed on the basis of wheat or barley EST sequences that were closely related to rice HKT genes (Table 1). The amplified products from wheat or barley were cloned using pGEM-T Easy vector system (Promega) and confirmed by sequencing. The probe developed from the wheat orthologue TaHKT2;1 was not expected to hybridize to HKT2;3 or HKT2;4-like genes in wheat, although OsHKT2;1 has some similarity (73–76% identity) at the positions 669–855, 1016–1170, and 1366–1502 with OsHKT2;3/4 in rice (Fig. 2).
DNA extraction and Southern hybridization
Plants were grown in soil for 4 weeks. The leaves were harvested for DNA extraction as described by Lagudah et al. (1991). DNAs were digested with different restriction enzymes (EcoRI, EcoRV, HindIII, NcoI) and electrophoretically fractionated in 1% agarose gel and transferred to Hybond N+ nylon membranes (Amersham) by capillary transfer. Prehybridization and hybridization were performed in a rotary hybridization chamber at 65 °C in a solution containing 1% sodium dodecyl sulphate (SDS), 50 mM 1 TRIS–HCl (pH 8.0), 10 mM EDTA, 3.3x SSC buffer, 10% dextran sulphate, 0.1% BSA, 0.1% PVP, 0.1% Ficoll-400, and 0.03% salmon DNA. The immobilized DNAs were hybridized overnight in buffer at 65 °C with probes 32P-labelled by the random primer method using Megaprime DNA Labelling Kit (Amersham). The membranes were washed at 65 °C twice, 20 min each time, in 2x SSC/0.1% SDS, once for 20 min in 1x SSC/0.1% SDS and once for 15 min in 0.5x SSC/0.1% SDS. Autoradiograms were exposed for 1–3 d at –80 °C with intensifying screens.
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Results |
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Chromosome mapping
DNA probes were used to localize each HKT-like gene to a specific chromosome or chromosome arm in wheat and barley, utilizing the nulli-tetrasomic and ditelosomic aneuploid stocks developed in Chinese Spring hexaploid wheat. The mapping results for each HKT-like gene are given in detail below and are summarized in Table 2 and Fig. 3. A representative set of blots is shown for the HKT2;1/2-like gene and described in detail in the legend of Fig. 4A–C. HKT gene map locations in hexaploid wheat and barley are shown in Fig. 5A, B, respectively.
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HKT2;1/2-like genes in wheat and barley
The HKT2;1/2 probe which was developed from TaHKT2;1 (TaHKT1, Genbank accession: U16709) hybridized to five bands in genomic DNA of hexaploid wheat (see Fig. 4) suggesting that up to five members of the HKT2;1/2-like family could be present in the bread wheat genome, which is consistent with results of Laurie et al. (2002). Up to two bands were mapped on the long arm of chromosomes 7A and 7B, while only one band mapped to chromosome 7D (Figs 3, 4
). In barley, only one band was detected (Table 2; Fig. 5), which was mapped on chromosome 7H (Fig. 3). Two bands were detected in the A genome of the diploid T. urartu, but only one band was present in the A genome of the diploid T. monococcum (Table 2; Fig. 4). In the D genome of the diploid Ae. tauschii, two bands were detected (Table 2; Fig. 4). The location of HKT1/2-like genes within the syntenic wheat region to rice chromosome 6 suggests that these genes are orthologous to OsHKT2;1 (Sorrells et al., 2003).
HKT2;3/4-like genes in wheat and barley
In rice, OsHKT2;3 (AJ491820) and OsHKT2;4 (AJ491855) are 95% identical at the nucleotide sequence level and are located on chromosomes 1 and 6, respectively. Using a probe developed from a closely related barley EST (DR733562), three bands were detected in hexaploid wheat (Table 2; Fig. 5) with one band mapping to chromosomes 7A, 7B, and 7D (Fig. 3). In barley, two bands were detected and mapped to chromosome 7H (Table 2; Fig. 5). There was one band present in T. monococcum, T. urartu, and Ae. tauschii, indicating that only one member of HKT2;3/4-like genes were present in those diploid wheats (Table 2). The location of HKT2;3/4-like genes on the long arm of wheat chromosome 7 suggests that these genes are orthologues of OsHKT2;4 (Os06g48800) located within the syntenic region on rice chromosome 6 (Sorrells et al., 2003) but not of OsHKT2;3 (Os01g34850) on chromosome 1 (Table 3). An HKT2;3-like orthologue could be absent from the wheat genome.
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HKT1;1/2-like genes in wheat and barley
The barley probe which was derived from EST BJ472463 detected two members of the HKT1;1/2-like gene family present in hexaploid wheat (Table 2; Fig. 5). Those two members were mapped on the long arm of chromosome 2B and 2D, respectively (Fig. 3), but no HKT1;1/2-like gene was detected in the A genome of hexaploid wheat (Table 2). In the A genome of T. monococcum and T. urartu, an HKT1;1/2-like gene was also absent (Table 2). In the D genome of Ae. tauschii, one copy of HKT1;1/2-like gene was detected (Table 2). In barley, one band was present and mapped on the long arm of chromosome 2H (Table 2; Figs 3, 5). The map location of HKT1;1/2-like genes in wheat is syntenic to rice chromosome 4 (Sorrells et al., 2003) containing OsHKT1;1 (Os04g51820) and OsHKT1;2 (Table 3).
HKT1;3-like genes in wheat and barley
Two members of the HKT1;3-like genes were detected in hexaploid wheat (Table 2; Fig. 5) and mapped to the short arm of chromosomes 6B and 6D, respectively (Fig. 3). No HKT1;3-like gene was detected in the A genome of bread wheat (Table 2); however, in the A genomes of T. monococcum and T. urartu, at least one copy of the HKT1;3-like gene was present in each genome (Table 2). This result differs from that of the HKT1;1/2-like genes, which are absent from all homoeologous A genomes of bread wheat, T. monococcum and T. urartu (Table 2). In Ae. tauschii, one HKT1;3-like gene was found (Table 2). In barley, one copy of HKT1;3-like gene was present and mapped on the short arm of chromosome 6H (Table 2; Figs 3, 5). The location of HKT1;3-like genes on wheat chromosome 6 is syntenic to rice chromosome 2 (Sorrells et al., 2003) that contains OsHKT1;3 (Os02g07830) (Table 3).
HKT1;4-like genes in wheat and barley
The HKT1;4 probe showed that up to eight bands were present in hexaploid wheat (Table 2; Fig. 5). Three hybridization bands were mapped to the long arm of chromosomes 2B and 2D, respectively, and two bands were mapped on the long arm of chromosome 2A (Figs 4, 5) consistent with the previous report (Huang et al., 2006). Two bands were present in the A genomes of T. monococcum and T. urartu (Table 2), the same number as in the A genome of hexaploid wheat. One HKT1;4-like gene in T. monococcum (EF062819) is a strong candidate for Nax1, a gene conferring sodium exclusion in durum wheat (Huang et al., 2006; James et al., 2006a). In Ae. tauschii, three bands were detected, the same number as for the D genome of hexaploid wheat (Table 2). In barley, two bands were detected and mapped to the long arm of chromosome 2H (Table 2; Figs 3, 5). The location of HKT1;4-like genes on the long arm of chromosome 2 is syntenic to rice chromosome 4 (Sorrells et al., 2003) containing OsHKT1;4 (Os04g51830) (Table 3).
HKT1;5-like genes in wheat and barley
Up to four bands of HKT1;5-like genes were detected in hexaploid wheat using HKT1;5 probe (Table 2; Fig. 5). Three hybridization bands were mapped on the long arm of chromosome 4B and one band was mapped on the long arm of chromosome 4D (Fig. 3). Notably, the HKT1;5-like gene is absent in the A genome of hexaploid wheat (Table 2). A single copy HKT1;5-like gene was present in T. monococcum while no member was found in the accession of T. urartu used in the study. Byrt et al. (2007) previously showed that the single copy HKT1;5-like gene found in T. monococcum was located in the distal region of the long arm of chromosome 5A, reflecting the ancient reciprocal translocation which occurred between the distal segment of the long arm of chromosomes 4A and 5A in an ancestral wheat genome. The HKT1;5-like gene in T. monococcum (DQ646339) was considered a strong candidate for Nax2, a gene conferring sodium exclusion in durum wheat (James et al., 2006a; Byrt et al., 2007). The HKT1;5-like gene in the D genome of hexaploid wheat is a candidate for Kna1 (DQ646342), a gene conferring sodium exclusion in hexaploid wheat (Gorham et al., 1990b; Dubcovsky et al., 1996; Byrt et al., 2007). One band was also detected in Ae. tauschii (Table 2). In barley, one band was detected and mapped to the long arm of chromosome 4H (Table 2; Figs 3, 5).
No syntenic relationship between HKT1;5-like genes in wheat and rice was found (Table 3) since HKT1;5 (Os01g20160) is located on rice chromosome 1, which is syntenic to wheat chromosome 3 (Sorrells et al., 2003).
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Discussion |
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In the three different genomes of bread wheat (Fig. 1), variations in copy numbers of HKT genes were observed (Table 2). Differences in copy number were also found between the A genome of the wild diploid relatives (T. monococcum and T. urartu) and the A genome of bread wheat. There were variations in copy number of HKT2;1/2-like, HKT2;3/4-like, HKT1;3-like, and HKT1;5-like genes (Table 2). It is not known whether these variations in numbers are widely present in the A genome of bread wheat and ancestor diploid relatives, or confined to the individual genotypes that were used in this study. It is possible that the variation in copy number may affect salt tolerance of wheat, and that allelic variations in gene sequence may also affect salt tolerance.
The syntenic chromosome regions between wheat and rice have been described comprehensively by Sorrells et al. (2003). In the present study, HKT2;1/2-like, HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, and HKT1;4-like genes were mapped on wheat and barley chromosome groups 7L, 7L, 2L, 6S, and 2L, respectively, which are synentic to chromosome regions containing putative orthologues of HKT genes in rice (Table 3). No synteny between wheat and rice is known for the chromosomal regions containing HKT1;5-like genes (Table 3).
The Na+-K+ co-transporters—subfamily 2
These are high-affinity transporters of K+ and/or Na+, and are important in K+-deficient conditions where they may take up Na+ and thereby promote growth. Some of them are specifically expressed in plasma membrane of cells in the epidermis and cortex of roots and their expression could be down-regulated in conditions of salinity.
TaHKT2;1 (TaHKT1) was the first HKT gene isolated from higher plants (Schachtman and Schroeder, 1994). Bread wheat could have five copies of HKT2;1-like genes on the basis of DNA hybridization from the present study (Fig. 3) and the previous report (Laurie et al., 2002). TaHKT2;1 is probably one of the two copies located on the B genome as (i) the wheat EST (BE428877) isolated from roots of tetraploid durum wheat was 100% identical to TaHKT2;1 (U16709), and (ii) primers designed on the basis of TaHKT2;1 amplified a product only from the long arm of chromosome 7B (Mullan et al., 2007). TaHKT2;1 was found to be expressed in cortical cells of bread wheat roots by in situ hybridization (Schachtman and Schroeder, 1994), but this finding may be confounded by potential cross hybridization with other HKT2;1-like members in bread wheat.
In barley, there is only a single copy of the HKT2;1-like gene (HvHKT1, AM000056) (Haro et al., 2005), which is consistent with the present study (Table 2). HvHKT2;1 and TaHKT2;1 have 92% identity at nucleotide sequence level and both encoded as a Na+-K+ co-transporter in a yeast transformation system (Rubio et al., 1995; Haro et al., 2005). In a root uptake system, however, HvHKT2;1 and TaHKT2;1 functioned as a putative Na+ uniport (Haro et al., 2005; Fig. 6). The reason for the different behaviour in yeast is considered to be an alternative initiation of translation, which produced a different protein with different kinetic properties from that in roots (Haro et al., 2005). This hypothesis was supported by another study using a TaHKT2;1 anti-sense transgenic line (Laurie et al., 2002). The down-regulation of TaHKT2;1 in wheat was associated with an increase in shoot fresh weight in 200 mM NaCl under conditions of K+ deficiency (Laurie et al., 2002). Following the down-regulation of TaHKT2;1, the transgenic wheat had smaller Na+-induced depolarization in root cortical cells than the control, and lower 22Na+ influx, indicating that TaHKT2;1 mediates Na+ influx (Laurie et al., 2002; Fig. 6).
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OsHKT2;1 functions as a relatively Na+-specific transporter that mediates Na+ influx in K+-starved roots and so promotes their growth (Horie et al., 2001, 2007; Fig. 6). It is expressed in the cortex of rice roots, and is down-regulated under saline conditions, and so does not cause Na+ toxicity (Horie et al., 2007). It is also expressed in leaves (Kader et al., 2006). OsHKT2;2 could function as a Na+-K+ co-transporter (Mäser et al., 2002a). Using a yeast transformation system, OsHKT2;2 showed Na+-K+ co-transport activity (Horie et al., 2001). Treatment with 150 mM NaCl greatly enhanced the expression of OsHKT2;2 in the leaves of salt-tolerant Pokkali, particularly in the mesophyll cells (Kader et al., 2006), although these results have to be treated with caution as the 150 mM NaCl was administered as an osmotic shock. The expression of OsHKT2;1 was down-regulated in the phloem and xylem tissues, as well as the mesophyll cells in the leaves (Kader et al., 2006). It would be interesting to investigate any tissue differential expression of HKT2;1/2-like genes in wheat, as observed for OsHKT2;1 and OsHKT2;2 in rice (Kader et al., 2006) and wheat (Mullan et al., 2007).
In rice, OsHKT2;3/4 is expressed mainly in the shoot with little expression in the roots (Garciadeblás et al., 2003). No EST matching OsHKT2;3/4 has been isolated so far from roots of wheat and barley. In wheat, OsHKT2;3/4-like ESTs (DR733562 and CA663666) were isolated from leaf and crown, suggesting that the OsHKT2;3/4-like genes were expressed in those tissues. In barley, OsHKT2;3/4-like ESTs (DN180983 and DN187639) were isolated from the leaf epidermis (Zierold et al., 2005). Future research is required to determine whether the expression of OsHKT2;3/4-like genes is specific to the epidermis. This investigation is of particular interest because differential partitioning of K+ between epidermal and mesophyll cells was observed in salt-treated barley and wheat, leading to a desirably high K/Na+ ratio in the cytoplasm of mesophyll cells (James et al., 2006b). Furthermore, Garciadeblás et al. (2003) found that OsHKT2;3 and OsHKT2;4 did not mediate any type of transport in transformed yeast, and suggested that their toxicity effects might be consistent with their expression in internal membrane and not in plasma membrane. It would be particularly interesting to investigate any HKT2;3/4-like genes targeted to the tonoplast which may be related to tissue tolerance in barley.
The Na+-specific transporters—subfamily 1
These are low-affinity transporters specific to Na+. Some of them are located in the plasma membrane of cells in the stele of roots, particularly the xylem parenchyma cells where they retrieve Na+ from the xylem sap, and so prevent it reaching the shoots.
OsHKT1;1 can be expressed in rice shoots and roots (Garciadeblás et al., 2003). In a transformed yeast system, OsHKT1;1 mediated low-affinity Na+ uptake (Garciadeblás et al., 2003). Although OsHKT1;2 is a pseudogene, a transcript was isolated from rice roots (Garciadeblás et al., 2003). In the barley genome and wheat B and D genomes, there is only one copy of the HKT1;1/2-like gene (Table 2; Fig. 3). It is not clear which gene member is absent from the wheat and barley genome. In wheat, HKT1;1/2-like ESTs (CJ594572, CJ700470, CJ594562, and CJ700475) were isolated from the shoots, showing that the HKT1;1/2-like gene can be expressed in wheat shoots. In barley, HKT1;1/2-like ESTs (BJ472463, BM816866, CD058368, BF262602, and DN17794) were isolated from leaves or leaf epidermis. Those barley ESTs could come from different regions of the same gene because they have 100% or 99% (sequence variation) identity in the overlapped region. This may provide additional information that there is only one HKT1;1/2-like gene in barley. Future research is required to test any tissue-specific expression of the HKT1;1/2-like gene in barley.
OsHKT1;3 is mainly expressed in shoots of rice, with little expression in roots (Garciadeblás et al., 2003). The present study found no wheat EST matching OsHKT1;3, although a single copy of an HKT1;3-like gene was present in the B and D genomes of bread wheat (Table 2). In barley, 18 HKT1;3-like ESTs (e.g. BJ476674) were isolated from one cDNA library from the leaves of adult plants, indicating that an HKT1;3-like gene may be highly expressed in barley leaves. From experiments with transformed yeast, Garciadeblás et al. (2003) suggested that OsHKT1;3 might not be located on the plasma membrane but on an internal membrane. In barley, a species known for its tolerance of high internal Na+ concentrations, presumably because of compartmentalization in the vacuole, the presence of an HKT1;3-like transporter on the tonoplast warrants investigation.
OsHKT1;4 is mainly expressed in shoots (Garciadeblás et al., 2003). A barley OsHKT1;4-like gene (BQ739876) was also expressed in the leaves of drought-stressed plants (Ozturk et al., 2002). The matched wheat EST (BE604162) was isolated from a drought-stressed wheat leaf cDNA library, indicating it was expressed in leaf tissues. Using a comparative mapping approach, an OsHKT1;4-like gene, TmHKT1;4-A2 (TmHKT7-A2) was cloned from Triticum monococcum as the candidate for Nax1 conferring sodium exclusion and salt tolerance to durum wheat (Huang et al., 2006; James et al., 2006a). TmHKT1;4-A2 co-segregated with Nax1 and its expression pattern in roots and leaf sheath was consistent with its proposed physiological role in removing Na+ from the xylem of the roots and leaf sheaths (Huang et al., 2006; James et al., 2006a; Fig. 6). The distinctive phenotype of TmHKT1;4-A2 in wheat is a high Na+ sheath:blade ratio (Davenport et al., 2005; James et al., 2006a).
OsHKT1;5 (SKC1) regulates K+/Na+ selectivity in rice, and maintains high shoot K+ and low Na+ under salt stress by controlling the unloading of Na+ from the root xylem (Ren et al., 2005; Fig. 6). OsHKT1;5 was preferentially expressed in the parenchyma cells surrounding xylem vessels (Ren et al., 2005). Voltage-clamp analysis of Xenopus laevis oocytes showed that OsHKT1;5 functions as a Na+-selective transporter (Ren et al., 2005). HKT1;5-like genes are considered as candidates for Nax2 in durum wheat and Kna1 in bread wheat (Byrt et al., 2007). Nax2 and Kna1 have the same phenotype as SKC1, namely low leaf Na+ concentration and high K+:Na+ ratio, enhanced discrimination of K+ over Na+ in transport from roots to shoots, and no effect on root Na+ concentration (Gorham et al., 1990b; Davenport et al., 2005; James et al., 2006a). With Nax2 and Kna1 there is no effect on the sheath:blade Na+ ratio, and no evidence of removal of Na+ from the xylem in the leaf sheath (data for SKC1 is lacking). The lack of a high sheath:blade Na+ ratio distinguishes Nax2 and Kna1 from Nax1 (James et al., 2006a). In durum wheat, TmHKT1;5-A on chromosome 5A segregated perfectly with Na+ exclusion and K+/Na+ selectivity, and is the candidate gene for Nax2 (Byrt et al., 2007). In bread wheat, TaHKT1;5-D on chromosome 4D is the candidate gene for Kna1 (Byrt et al., 2007).
In summary, the copy numbers of individual HKT gene members vary between barley, wheat, and rice genomes and among different wheat genomes. HKT2;1/2-like, HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, and HKT1;5-like genes were mapped on wheat and barley chromosome groups 7L, 7L, 2L, 6S, 2L, and 4L, respectively. Chromosomal regions containing HKT-like genes between wheat and rice were syntenic except for the chromosome regions containing the HKT1;5-like gene. Some HKT1;4 and HKT1;5-like genes are candidates for Nax1, Nax2, and Kna1 in wheat, conferring salt tolerance by controlling sodium exclusion from xylem unloading (Huang et al., 2006; James et al., 2006a; Byrt et al., 2007). Further research will elucidate the full range of important functions of HKT genes in wheat and barley, and their role in salt tolerance.
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Acknowledgements |
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The help of Caitlin Byrt in providing the HKT1;5 probes and Dr AKMR Islam for providing the wheat–barley addition lines is gratefully acknowledged. This work was supported by a CSIRO Postdoctoral Fellowship to SH, and the Grains Research and Development Corporation.
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Footnotes |
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* Present address: ARC Centre of Excellence in Plant Energy Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
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