Function Analysis Of A Sodium Transporter,OsHKT2;4,in Rice

Over800million hectares of land worldwide is affected by salinity, comprising nearly7%of the world’s total land area. Irrigation systems are particularly prone to salinization, with nearly one third of irrigated land being severely affected. Despite persistent questioning of the sustainability of irrigation as a method to increase food production, it is highly unlikely that such practices will be stopped in the near future. Most crops are glycophytes, thus are not capable of growing in high concentrations of salt in the soil. Given the above trends, improving crop salt tolerance is fast becoming one of the key aspects of plant breeding in the future. However, all attempts so far towards improving crop salt tolerance through conventional breeding programs have met with very limited success, primarily because of the physiological and genetic complexity of this trait.Recent progress in molecular genetics and plant electrophysiology suggests that the ability of plants to maintain a high cytosolic K/Na ratio appears to be critical to plant salt tolerance, which can be achieved by either restricting Na accumulation in plant tissues or by preventing K loss from the cell. Most of Plant breeders and cell physiologists have focused on the former mechanism. With the development of molecular biology, more and more physiological mechanism of K/Na channels and carriers are identified, which are attributable to improving crops salt tolerance.Members of HAK family and HKT family are well characterized carriers for K and Na uptake and transport in plants. HAK genes functioned as K high affinity transporters whereas HKT family as Na low affinity transporters. However, only a few genes in these families have been well characterized so far. In rice there are26members in the HAK family with only OsHAK1investigated thoroughly. Rice HKT family have9members (OsHKT1;1-1;5and OsHKT2;1-2;4) but only one (AtHKT1;1) in Arabidopsis. Despite that AtHKT1;1and OsHKT2;1have been intensively studied, some of their functions were still open to debate.In the present study, the interactive effects of K and Na on rice growth and expression of K/Na transporter genes were first investigated for revealing the key dominant K/Na involved in salt tolerance in rice. Based on performance of its two knockout mutants, OsHKT2;4, a member of HKT family in charge of Na uptake and transport in rice, was further examined in details to address its expression patterns and physiological functions. The main results are summarized as follows:Germinated rice (Oryza Sativa CV. Nipponbare L.) was pre-treated with normal nutrient solution for one month followed by treatments of three different levels of Na (0mM,25mM and100mM) without or with supply of K for9d. We found that high Na (100mM) resulted in significant decrease of root and shoot growth, root activity and total K uptake. However, it increased root to shoot biomass ratio of the seedlings. Short period removal of K supply did not affect root morphology and root to shoot biomass ratio, but decreased the activity of roots grown only if high Na solution was supplied simultaneously. The K deficiency resulted in enhanced uptake of Na and increased transport of K from roots to shoots. Moreover, expression of OsHAKl, a putative K transporter gene, was up-regulated by low Na (25mM) and down-regulated by high Na (100mM) in the roots. In the leaves, its expression was suppressed by the Na treatments when K supply was maintained. Expression of OsHKT2;1, which acts mainly as a Na transporter gene, was down-regulated by high Na, but was enhanced by K deficiency both in the roots and leaves. Expression of other five putative K/Na transporter genes, OsHKT1;1,OsHKT1;2, OsHKT2;3, OsNHXl, OsSOS1, did not show affected expression by the treatments. The results suggest that OsHAKl and OsHKT2;1are involved in the interactive effects of K and Na on their uptake and distribution in rice.Most of genes in HKT family are not expressed in rice root. Interestingly, OsHKT2;3was so highly homologous (over95%) to OsHKT2;4, both in Open Read Frame and promoter region that we did not find gene specific primers to tell them apart. Primers of OsHKT2;3were only available30bp more sequence in3’-UTR of mRNA than that of OsHKT2;4. As a result, OsHKT2;4, also not express in root, was selected in HKT family to characterize its physiological function in rice plant.Two lines of TOS17inserted mutants of OsHKT2;4gene were obtained from National Institute of Agrobiological Sciences, Japan. PCR analyses confirmed that they are truly homozygous knockout mutants by Tos17insertion at different extron of the gene. The mutants were treated with external low Na level (2mM) or high Na level (100mM) with its wild type (WT) as a control. In the experiment of2mM NaCl treatment for15d, the mutants exhibited remarkably inhibitive growth in roots. However, their Na accumulation was enhanced in roots and aerial parts compared with WT. In addition, the mutation increased transcripts of OsHKT2;3, which was highly homologous to OsHKT2;4. When treated with100mM Na and K-starved for3d after cultivated with normal nutrients for30d, both oshkt2;4lines showed more salt tolerance and lower Na accumulation in whole rice plants than that of WT.Analyses GUS expression driven by OsHKT2;3and OsHKT2;4promoter in their transgenic rice plants showed that both genes were expressed in vascular systems and guard cells of shoots, not in roots. WT and oshkt2;4s seedlings were cultured in normal nutrient solution for78d, their net photosynthetic rate, stomata conductance and transpiration rate, which indicate the extent of stomatal open indirectly, were determined within a day (9:00-15:00). The values of the three parameters were all smaller in WT than that of the mutants at12:00and13:00. When treated with100mM NaCl for3d after cultured in normal nutrient solution for75d, similar results were obtained except the determination time was from9:00to12:00. The results represent that OsHKT2;4may regulate Na translocation and stomatal movement of leaf guard cells, thus affect rice growth. The high similarity of both sequences and expression place and pattern suggest that OsHKT2;3and OsHKT2;4might play the similar but not redundant functions in Na transport and stomatal movement in rice.In summary, OsHKT2;4regulates Na transport in shoot and stomatal movement of leaves in rice, knockout of the gene, therefore, could enhance tolerance to Na stress and improved growth of rice.