Functional Characterization Of ZxHKT1;2 Transporters In The Zygophyllum Xanthoxylum

Drought is one of the main abiotic stresses leading to crop loss,so improving crop drought resistance is the key to solve this problem.The most direct and effective method is to explore the mechanism of drought resistance in desert xerophytes,so as to discover the key genes and make use of them.Zygophyllum xanthoxylum is a kind of xerophytic shrub of the family Zygophyllaceae.It has strong drought resistance and a complex and unique drought resistance mechanism.In previous studies,the transport mechanism of Na~+has been studied,and found that the Na~+/H~+antiporter Zx SOS1 is located on the plasma membrane and mediates the longer distance transport of Na~+from root to leaf.In addition,the tonoplast Na~+/H~+antiporter Zx NHX1 can mediate the sequestration of Na~+from the cytosol into vacuoles,and improve the osmotic regulation ability to resist drought stress.However,the molecular mechanism of Na~+uptake in roots of Z.xanthoxylum remained unclear.Essentially,the above processe is the absorption of Na~+by cells.It has been shown that the HKT transporter mediates Na~+uptake at the cellular level.Our laboratory has cloned ZxHKT1;1,ZxHKT1;2 and ZxHKT1;3,and found that ZxHKT1;2 located in plasma membrane and was preferentially expressed in root epidermis detected by in situ PCR.The expression of ZxHKT1;2 was up-regulated under 50 mM Na Cl,heterologous expression of ZxHKT1;2 in Saccharomyces cerevisiae and Xenopus laevis oocytes showed that ZxHKT1;2 specifically mediated Na~+absorption.Therefore,we speculate that ZxHKT1;2 mediated Na~+uptake in root of Z.xanthoxylum.In this study,based on previous research results,ZxHKT1;2 was controlled by the native promoter and 35S promoter were transferred into wild-type Arabidopsis thaliana to analyze the function of ZxHKT1;2 transporter.The results are as follows:1.The ZxHKT1;2 promoter(p ZxHKT1;2),about 911 bp,containing common cis-acting elements,as well as tissue-specific regulatory elements related to dehydration stress response,osmotic stress response,and gibberellin synthesis.2.GUS staining showed that p ZxHKT1;2 was specifically localized in the epidermis and cortex of plant roots,which was consistent with the tissue localization of ZxHKT1;2,indicating that the promoter can drive the expression of downstream gene in the heterologous system.3.After adding 75 mM Na Cl to the solid medium(2.5 mM K~+),both tissue-specific and whole plant overexpression of ZxHKT1;2 increased salt sensitivity of A.thaliana.After 5 mM Na Cl was added to the solid medium with low concentration of K~+(0.02mM),the growth of the p ZxHKT1;2::ZxHKT1;2 lines recovered,but the growth of the35S::ZxHKT1;2 lines was weaker,these results indicate that ZxHKT1;2 is expressed in different parts of A.thaliana,and its functions are different.4.Under the treatment of 5 and 75 mM Na Cl,the p ZxHKT1;2::ZxHKT1;2 lines treated with Na Cl had an enhanced ability to selectively absorb Na~+,and the content of Na~+in vivo,leading to the decrease of K~+and thus affecting their growth.Suggesting that ZxHKT1;2 might mediate the absorption of Na~+by roots.5.After adding low concentration of Na Cl under low potassium condition,the content of Na~+in p ZxHKT1;2::ZxHKT1;2 lines was increased,and its growth was significantly better than that of wild type,these further indicate that the Na~+absorption capacity of the tissue-specific expression of ZxHKT1;2 is stronger than that of the wild type,and it can alleviate the damage caused by potassium deficiency by absorbing low concentration of Na~+.In conclusion,this study confirmed the results of previous studies in A.thaliana:ZxHKT1;2 mediates the uptake of Na~+in roots and may mediate the absorption of Na~+under the condition of low K~+to alleviate the damage caused by K~+deficiency.