Functional Characterization Of A Potassium Transporter Gene OsHAK22 In Rice Salt Tolerance

Potassium(K)is the most abundant essential cation for plant growth and development.It plays important roles in various physiological processes,including maintaining charge balance for other nutrients uptake and translocation,turgor pressure establishment and photosynthesis,as well as improving salt tolerance.Soil salinization is a worldwide problem for agriculture production,China confronts the same problem as well.The toxicity effect of high salts(mainly sodium,Na)on plant growth includes two phases,an immediate osmotic stress which may rapidly inhibit plant growth,and a delayed ionic stress which may even kill plant.To cope with high-salts in soil and irrigation water,plants have evolved variety of salt-tolerant mechanisms.Enhancing K uptake and root to shoot translocation and thus maintaining high K Na ratio,particular in the shoot,is an import strategy of plant salt tolerance.In plants,several families of K channels and transporters have been identified for their functions in K absorption and translocation,of which HAK/KUP/KT family is the largest K transport family which is further divided into four or five clusters.Until today,all the characterized HAK/KUT/KT transporters are belonging to cluster Ⅰ and cluster Ⅱ and mostly in Arabidopsis and Rice.In Arabidopsis,there is only one member,AtHAK5,in cluster Ⅰ,while there are eight members in rice,implicating their functional diversity and possible redundancy.In this study,we focused on the expression and function of OsHAK22,one of the members in cluster Ⅰ of HAK/KUP/KT gene family in rice.We generated the knockout mutant of OsHAK22(oshak22 or OsHAK22-KO)by Crispr-Cas9 technique and OsHAK22-overexpression(OsHAK22-OX)lines by using a constitutive promoter in the genetic background of the cultivar Nipponbare.The major results were obtained as follows.1.Different from other HAK/KUP/KT members with knowing functions,OsHAK22 is not expressed at rice root,but at the root-shoot junction,vascular bundles of leaf sheath,leaf pillow,flower,and glume shell.Its subcellular location is also not exact the same as those characterized members in cluster I from different plant species which are identified at plasma membrane.OsHAK22 shows only a small fraction at plasma membrane,and the most remaining is at endomembrane system.2.In hydroponic culture of normal K supply(1 mM),K content and distributions in root and shoot among Nipponbare wild type(WT),oshak22 mutant and OsHAK22-OX lines were similar.At low K(0.1 mM)condition,in comparison to WT,overexpression of OsHAK22 increased K distribution in the shoot,particular in the leaf sheath,while knockout of OsHAK22 did not show significant effect on the K distribution.2.In comparison to WT,OsHAK22-KO resulted in decrease of K but increasing of Na concentration in the leaf sheath.Os HA K2 2-knockout also increased K but reduced Na concentration in the phloem sap under salt stressed condition.In addition,both K and Na concentrations in the shoot and K in the phloem sap of OsHAK22-OX lines were similar to those of WT,differently,while Na concentration in the phloem sap of OsHAK22-OX lines increased significantly,implicating that OsHAK22 enhances rice salt tolerance by increasing K uptake and translocation but reducing Na retention in the shoot.3.Results of field experiments demonstrated that OsHAK22-OX increased rice yield due to the increasing effective tiller numbers while OsHAK22-KO reduced rice yield due to less seed-setting ratio in well fertilized condition.Notably,K concentrations in both the root and shoot of WT,OX and KO lines were all similar grown at K supply sufficient field,implicating that the effect of OsHAK22 expression on rice growth and yield is not simply due to the improved K nutrition at least under K-rich condition.The further physiological and genetical evidences need to be provided for the function and regulation of OsHAK22 in rice at limited K supply and salt stress conditions.