| Potassium is an essential element for plant, and high-affinity K~+ uptake system plays a crucial role in potassium absorption and transportation. Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH and KdpA transporter subunits and fungal Trk proteins. These proteins have received extensive attention during recent years and a clear insight of their functions is emerging. Function studies of HKT transporters showed that members of this family played important roles in long-distance Na~+ transport and plant salt tolerance. Most HKT-like genes have been cloned from nonhalophytes such as Arabidopsis thaliana, Oryza sativa, Eucalyptus camaldulensis and Triticum aestivum. Only McHKT1 and McHKT2 were cloned from the facultitive CAM plant (Mesembryanthemum crystallinum). Data regarding the characterizations of HKT proteins from halophytes have been limited until now. Here we studied the the effect of K~+ uptake inhibitor (NEM and TEA) on the Na~+ content and the effect of K~+ nutrition and NaCl stress on K~+ content of Suaeda salsa plants first. Then we reported the cloning of HKT1 homolog from C3 halophyte Suaeda salsa (L.) (SsHKT1), anlysing the expression pattern and its functions using different expression systems.The results were shown as follows:1. NaCl stress did not affect K~+ content of Suaeda salsa under low K~+ concentrationK~+ nutrition plays an important role in the growth of Suaeda salsa. K~+ content in leaves and roots was increased gradually with the increase of K~+ concentration in medium. The NaCl stress markedly reduced the K~+ content in leaves and roots of plants grown in 0.7, 1, 6 mmol/L K~+ medium but hardly affected K~+ content in 0, 0.1 mmol/L K~+ medium. Moreover, 400 mmol/L NaCl stress did not reduce K~+ content markedly compared with 100 mmol/L NaCl stress whether under low K~+ concentration or under high K~+ concentration. This is very different from the nonhalophytes. This proved that there may be an effective mechanism of K~+ homeostasis in Suaeda salsa under salt stress, which was important in the salt tolerance of Suaeda salsa.2. Effect of K~+ uptake system inhibitor on the Na~+ content in Suaeda salsaNEM (3 mmol/L) is a specific inhibitor of the high affinity K~+ uptake system. It had little effect on the Na~+ content of Suaeda salsa at low K~+ concentration (0, 0.1, 0.4 mmol/L). This indicated that Na~+ entry into the plants was not mediated by the high affinity K~+ uptake system under low K~+ application.TEA (6mmol/L) is a specific inhibitor of the low affinity K~+ uptake system. It inhibited the Na~+ uptake and accumulation significantly at high K~+ concentration (6, 8, 10 mmol/L). This suggested that Na~+ entry into Suaeda salsa plants, at least, was partly mediated by low affinity K~+ uptake system under high K~+ application.3. cDNA cloning and sequence analysis of the cation transporter SsHKT1 in halophyte Suaeda salsaHere we report the cloning and characterization of a HKT1 homolog from C3 halophyte Suaeda salsa (L.) (SsHKT1), particularly under low K~+ treatment. Degenerate PCR primers were designed based on the deduced amino acid sequences of plant HKT homologs. Reverse transcriptase (RT)-PCR amplification and 5'and 3'RACE amplification were used to obtain the full-length transcript sequences of SsHKT1 (GenBank accession No. AY530754). The SsHKT1 cDNA was 2033 nucleotides long including 1650 bp ORF for a 550 amino acids peptide and a predicted molecular mass of 63.0 kD. The deduced amino acid sequence of SsHKT1 was 39%~64% identical to other plant HKT-like sequences. A putative filter glycine in first P-loop for K~+ selectivity is absent in SsHKT1. Computer–assisted hydropathy plots were constructed from the deduced amino acid sequence. The result suggested that the SsHKT1 from S. salsa was a transmembrane protein with ten transmembrane spans.4. Subcellular localization of SsHKT1 in S. salsa rootsA SsHKT1-specific antibody was prepared against the sequence near the N-terminal (135-204) of the deduced SsHKT1 protein. Protein gel blots showed specific recognition of a 63.0 kD polypeptide that resided exclusively in the plasma membrane of S. salsa roots.5. Expression analysis of SsHKT1 in halophyte S. salsa under K~+-starvation Reverse transcriptase-PCR analysis showed that SsHKT1 was mainly expressed in leaf tissues and to a lesser extent, in root tissues. Amounts of SsHKT1 transcript were developmentally controlled and significantly up-regulated by K~+ deprivation. Expression of SsHKT1 continued to increase until 48 h of K~+ deprivation. By 72 h, SsHKT1 mRNA expression declined slightly from the 48 h peak levels. Amounts of SsHKT1 transcript in younger seedlings were more than that in older seedlings.6. Expression analysis of SsHKT1 in halophyte S. salsa under salt stress SsHKT1 expression in leaves was upregulated by K~+-starvation and salt-stress compared with the controls grown only under K~+ starvation conditions. No significant changes in SsHKT1 transcription were observed between 200 and 400 mmol/L NaCl stress.7. SsHKT1mainly mediates K~+ transport in heterologous yeast expression systems.To determine the SsHKT1 function, we expressed the gene in Saccharomyces cerevisiae mutant strain CY162, which had defect in high affinity K~+ uptake system. The result showed that SsHKT1 could complement the K~+ uptake defection of yeast strain CY162. The results demonstrated that SsHKT1 mediated high-affinity K~+ uptake in the mutant strain CY162. We also expressed the gene in S. cerevisiae mutant strain G19, which displays increased Na~+ sensitivity due to disruption of the four tandemly repeated P-type Na~+-extruding ATPase genes (ENA) (Δena1–4). The G19 transformants expressing SsHKT1 didn't displayed more sensitivity to Na~+ than control cells at SC-U plate containing 200,300 and 400 mmol/L NaCl. This result suggested that SsHKT1 didn't mediated Na~+ uptake in the yeast mutant strain G19.8. Overexpression of SsHKT1 increases the salt tolerance of transgenic ArabidopsisThe results of over-expression of SsHKT1 in Arabidopsis showed that the transgenic plants were more salt-tolerant than the wild types. No difference was observed in growth between transgenic plants and wild type plants in the normal MS medium, which indicated that the insertion and overexpression of SsHKT1 had no effect on the normal growth of Arabidopsis plants. However, if the plants grown in MS medium containing 0mmol/L K~+, different concentrations of NaCl or LiCl, the colour of leaves, the development of lateral roots of transgenic plants were all much better than those of wild type plants when grown for two weeks. The shoot K~+ concentrations of transgenic Arabidopsis plants is significantly higher than those of wild type plants under different treatments (p<0.05). However, no significant changes in leaf Na~+ concentration were observed between wild type and transgenic Arabidopsis plants under similar conditions (p>0.05).Summarily, all these results demonstrated that SsHKT1 functioned as a K~+ transporter but not a Na~+ transporter in the yeast cells. Transgenic Arabidopsis plants overexpressed of SsHKT1 were more salt tolerant than wild type plants, which further indicated the important role of SsHKT1 for maintenancing K~+ homeostasis in plant salt tolerance.The main innovation points of this study were generalized as follows:1. It was the first time to clone the SsHKT1 gene from halophyte S. salsa.2. For the first time to confirm that the SsHKT1 expression in S. salsa was induced by K~+-starvation and salt stress.3. For the first time to confirm that the salt tolerance of transgenic yeast expressing SsHKT1 gene was enhanced and SsHKT1 functioned as a K~+ transporter but not a Na~+ transporter.4. The transgenic Arabidopsis plants overexpressing SsHKT1 were generated. It was the first time to indicate that overexpression of SsHKT1 in nonhalophyte could confer the plant salt-tolerance.
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