Functional Analyses Of Potassium Transporter OsHAK21 And Channel OsKx In Response To Salt Stress In Rice

Cellular potassium(K~+)homeostasis is vital for the plant growth and development.K~+and Na~+ are similar in physiochemical properties.With the aggravation of soil salinization problems,promoting salt tolerance in plants has became an urgent demand for raising crop yield and promoting agricultural production.So far,sodium transporter function has been a major focus of investigations into the adaptive mechanism by which plants cope with Na~+injury,such as restricted Na~+ uptake,active Na~+ exclusion back to the soil solution,and compartmentation of excessive Na~+ in the vacuole,which are considered to be central to salt tolerance.However,when Na~+ toxicity in cells could not be relieved effectively,it would induce the disorders of potassium homeostasis in cells.In recent years,evidence has been accumulated suggesting that plants' ability to maintain a high cytosolic K~+/Na~+ ratio appears to be critical to plant salt tolerance.K~+ absorption from soil and translocation in plants under saline conditions are mediated by potassium channels and transporters.The KT/HAK/KUP trnsporter and the Shaker K~+channel are among the major K~+ acquisition systems in plants.In this study,we determined the physiological roles of OsHAK21 and OsKx transport proteins in response to salt stress.Detailed investigations,such as quantitative analysis,phenotype comparison,expression pattern and subcellular localization analyses,and the complementation tests in yeast cells and Arabidopsis plants were then carried out.The main results were listed as follows:1.We found a significant inducement of OsHAK21 expression by high salinity treatment.The Tos17 insertion mutant oshak21 exhibited more sensitive to salt stress compared with wild type,as determined by the growth inhibition and chlorosis of leaves.The transcript of OsKx induced by high Na~+ concentrations showed no changes,but the T-DNA disruption mutant oskx exhibited hypersensitivity to salt stress.2.We generated complementation and RNA interference lines for OsHAK21.Similar to oshak21 mutant plants,both RNA interference lines were more sensitive to salt stress than the WT.On the contrary,the complementation lines nearly rescued the salt-hypersensitive phenotype of oshak21 null mutants.Besides,the RNA interference lines of OsKx showed the similar salt-sensitive phenotype as with oskx,and the overexpression lines of OsKx showed more tolerance to salt stess than the WT.3.Compared to the wild type,oshak21 accumulated less K~+ and considerably more Na~+in both shoots and roots,and exhibited significant lower K~+ net uptake rate but higher Na~+uptake rate.Similar results were obtained in analysis of RNA interference lines,and the complementation lines were close to the WT,which were in accord with the phenotype under salt stress.4.Our analyses of expression patterns and subcellular localizations showed that OsHAK21 and OsKx were both localized in the plasma membrane.RT-PCR analysis in various organs of WT plants showed that OsHAK21 was expressed mainly in rice seedlings,and the transcripts were more abundant in roots,and expressed specifically in xylem parenchyma and individual endodermal cells(putative passage cells).Besides,OsKx was expressed mainly in shoots,the transcripts were more abundant in seedling leaves and leaf sheaths,and may specifically expressed in phloem cells.5.We further measured the Na~+ and K~+ concentrations in the xylem sap from oshak21 and WT plants.However,more Na~+ were accumulated in the xylem sap of oshak21 compared with that of WT,which indicates that disruption of OsHAK21 resulted in more Na~+ translocation to shoots via xylem,disturbing the ability of photosynthesis in oshak21 plants.6.To investigate the transporter activity of OsHAK21 and OsKx,We performed complementation tests using a K~+ uptake-deficient mutant of yeast strain CY162.When the K~+ concentration on AP medium was about 4 mM,OsHAK21 and OsKx expression exactly rescued its growth defect.The apparent affinity for OsHAK21-mediated K~+ influx in CY162 was about 3.28 mM.Besides,OsHAK21 and OsKx-mediated K~+ absorption could enhance the salt tolerance of CY162 cells.7.Expression of OsHAK21 in Arabidopsis completely rescued the low-K~+-sensitive phenotype of athak5 mutant,revealing that OsHAK21 possessed K~+ transporter activity in Arabidopsis.Overall,our results indicate that OsHAK21 and OsKx both participate in rice responses to salinity stress.OsHAK21 mediates K~+ acquisition in xylem parenchyma and individual endodermal passage cells,while OsKx may be involved in K~+ translocation in the phloem.We hypothesize that substantial salt-induced upregulation of OsHAK21 in roots and the open probability change of OsKx in shoots would result in increased rates of K~+ circulation through vascular tissue,pointing towards a long distance redistribution of K~+ between the roots and shoots.Thus,OsHAK21 and OsKx involvement in K~+ transport under salt stress may highlight the crucial roles in the maintenance of Na~+/K~+ homeostasis and salt acclimation of rice.