Functional Characterization Of HKT Transporters In The Succulent Xerophyte Zygophyllum Xanthoxylum

Drought is one of the major abiotic stresses that threaten agricultural productivity all over the world.About 25%of the world's agricultural land is affected by drought stress.Approximately 50.8%of land throughout the China is affected by drought,which is mainly distributed in the northwest of China.The succulent xerophytes have developed a wide range of specialized stress-resistance mechanisms to survive in harsh conditions during a long-term evolutionary process.Our previous investigations have demonstrated that Zygophyllum xanthoxylum,a dominant species of desert vegetation in the northwest of China.To cope with these harsh conditions,Z.xanthoxylum can absorb large quantity of Na~+from low-salinity soil and transport a large quantity of Na~+efficiently from roots to leaves mediated by ZxSOS1,and eventually,ZxNHX1 compartmentalize it into the vacuoles for osmotic adjustment.These findings suggested that Z.xanthoxylum exhibited a unique Na~+transport mechanism.However,the molecular mechanisms of Na~+uptake by the root,Na~+retrieval from the root xylem and Na~+loading into mesophyll tissue from the leaves vein have not been explored.The HKT transporter is a candidate for mediating Na~+influx on the plasma membrane of higher plants and plays an important role in Na~+homeostasis.However,the function of HKT transporters in the succulent xerophyte species Z.xanthoxylum had not been well explored.In this study,to reveal the function of HKT transporters in the Z.xanthoxylum,the following experiments were based on transcriptome and expression profile of HKT transporter genes.The goals of the present work were cloning and functionally characterize the full length of HKT genes;analyzing the ion-selective characteristics by using heterologous expression systems;revealing the expression patterns in Z.xanthoxylum species;the cell-specific expression was performed by in situ PCR;and the subcellular localize by the transient expression of fluorescence fusion protein in tobacco.The overall results can be summarized as follows:1.Based on our transcriptomic data of Z.xanthoxylum,there were 9-Unigenes of HKT transporters were found,among them,the expression level of Unigene11474_All in roots was increased by salt treatment and osmotic stress,and the transcription level of CL4173.Contig2_All in leaves was decreased by osmotic stress.2.The gene encoding ZxHKT1;1 was cloned from Z.xanthoxylum based on the sequence of Unigene13502_All,and phylogenetic tree showed that ZxHKT1;1belongs to the higher plant HKT subfamily I and is closer to the McHKT1;1.ZxHKT1;1 was located on the plasma membrane.ZxHKT1;1 functioned as a selective Na~+uptake transporter in Saccharomyces cerevisiae and Xenopus laevis oocytes.ZxHKT1;1 was preferentially expressed in root xylem parenchyma by in situ PCR.Constitutively expressing ZxHKT1;1 in the athkt1;1 mutant driven by the cauliflower mosaic virus 35S promoter could not rescue the salt-sensitive phenotype of seedlings under 75 mM NaCl.In contrast,under control of the Arabidopsis AtHKT1;1 native promoter,specific overexpression of ZxHKT1;1 in wild type enhanced the salt tolerance;the ZxHKT1;1 complementation the athkt1;1 mutant not only rescued the Na~+-sensitive phenotype of seedlings,but also grow better than wild type under 75mM NaCl.These results showed that the function of ZxHKT1;1 is similar to AtHKT1;1 which unloads Na~+from the xylem sap to root xylem parenchyma cells.However,moderate concentration of Na~+(50 mM NaCl)significantly decreased the transcripts of ZxHKT1;1 in roots of Z.xanthoxylum,resulting less Na~+unloading from the xylem sap.Furthermore,the expression of ZxHKT1;1 in roots of Z.xanthoxylum was up-regulated under 150 mM NaCl which was perceived as salt stress for Z.xanthoxylum,which can effectively avoid the accumulation of Na~+in photosynthetic tissues.3.Depended on the sequence of Unigene11474_All,the gene encoding ZxHKT1;2 was cloned from Z.xanthoxylum,and phylogenetic tree showed that it belongs to the higher plant HKT subfamily I and has high homology with EcHKT1;2.ZxHKT1;2 was located on the plasma membrane.ZxHKT1;2 acted as a selective Na~+uptake transporter in S.cerevisiae and X.laevis oocytes.ZxHKT1;2 was mainly expressed in roots of Z.xanthoxylum,and up-regulated by salt treatments.Moreover,50 mM NaCl induced upregulation of ZxHKT1;2 in roots much higher than 150 mM NaCl.ZxHKT1;2 was preferentially expressed in root epidermis and cortex detected by in situ PCR,indicating that ZxHKT1;2 might be mediated Na~+uptake into the roots of Z.xanthoxylum.4.The gene encoding ZxHKT1;3 was cloned from Z.xanthoxylum based on the sequence of CL4173.Contig2_All,and phylogenetic tree showed that it belongs to the higher plant HKT subfamily I and shares 78%homology with ZxHKT1;2.ZxHKT1;3was located on the plasma membrane.ZxHKT1;3 was unable to mediate Na~+and K~+uptake in S.cerevisiae.The ZxHKT1;3 functioned as a selective Na~+uptake transporter in X.laevis oocytes.The ZxHKT1;3 was mainly expressed in leaves of Z.xanthoxylum and up-regulated spontaneously by salt treatments.Promoter studies with the GUS reporter gene confirmed preferential expression of ZxHKT1;3 in the leaf areas surrounding the free endings of minor veins,speculating that ZxHKT1;3mediates the loading of Na~+into Z.xanthoxylum mesophyll cells from the leaves vein.In summary,all above results revealed that the function of HKT transporters mainly involved in Na~+uptake and transport in the succulent xerophyte Z.xanthoxylum,which deepened the understanding of the unique Na~+transport mechanism in Z.xanthoxylum,providing a theoretical basis for genetic improvement in grasses and other important crops species.