Comparative Analysis Of Na~+ And K~+ Selectivity Between ZxNHX1 And AtNHX1 In Atnhx1-1 And Their Differences In Amino Acid Sites

Zygophyllum xanthoxylum is the dominant species in the desert area of Northwest,China,it has strong drought resistance and salt tolerance.The previous study showed that it can absorb a large amount of Na⁺from low-salinity desert soil and store it in vacuoles for osmotic adjustment.The tonoplast Na⁺/H⁺antiporter(NHX1)plays a crucial role in the compartmentalization Na⁺from cytoplasm to vacuole.Previous research in the yeast heterologous expression system have shown that both ZxNHX1and AtNHX1 in Arabidopsis thaliana possessed transport activity of Na⁺and K⁺.The comparative analysis of the Na⁺and K⁺selectivity showed that ZxNHX1 have a stronger Na⁺transport ability than that of AtNHX1,while the ability to transport K⁺was significantly lower than AtNHX1.In addition,the 265^th and 419^th amino acid of ZxNHX1 played a crucial role in affecting its Na⁺and K⁺selectivity in the yeast heterologous expression system.However,whether ZxNHX1 and AtNHX1 have Na⁺and K⁺selectivity differences and the mutations at the T265 and S419 sites of ZxNHX1 affect its Na⁺and K⁺accumulation at the plant level is still unclear.In view of this,we transferred ZxNHX1 and AtNHX1 into atnhx1-1 to verify the difference of Na⁺and K⁺selectivity;Meanwhile,the key amino acid residues(T265and S419)that may result in the difference in Na⁺and K⁺selectivity between the ZxNHX1 and AtNHX1 were mutated to verify their influence on the accumulation of Na⁺and K⁺at the plant level.The main results are as follows:1.AtNHX1,ZxNHX1,ZxNHX1 T265F,ZxNHX1 S419T and ZxNHX1T265F&S419T were transfected into atnhx1-1.Two groups of plants with consistent expression levels of AtNHX1 and ZxNHX1 were selected(low expression lines:AtNHX1-L,ZxNHX1-L;high expression lines:AtNHX1-H,ZxNHX1-H).Besides,two groups of ZxNHX1,ZxNHX1 T265F,ZxNHX1 S419T and ZxNHX1 T265F&S419T with consistent expression levels were selected(low expression lines:ZxNHX1-L,ZxNHX1 T265F-L,ZxNHX1 S419T-L,ZxNHX1 T265F&S419T-L;high expression lines:ZxNHX1-H,ZxNHX1 T265F-H,ZxNHX1 S419T-H,ZxNHX1T265F&S419T-H).2.Under the normal potassium(2.5 mM K⁺)conditions,the overexpression of ZxNHX1 and AtNHX1 can complement the salt-sensitive phenotype of atnhx1-1,but the phenotype of the ZxNHX1 transgenic lines were better than that of the AtNHX1lines.Under 75 mM NaCl,the fresh weight,dry weight of the shoot and root in ZxNHX1-H line increased significantly by 39.5%,25.7%and 29.6%,18.0%,respectively,compared with AtNHX1-H line,the Na⁺concentration in the shoot and root also increased significantly by 8.9%and 18.5%,but the K⁺concentration in the shoot and root of AtNHX1-H line were significantly higher than that of ZxNHX1-H line by 10.7%and 12.7%,respectively.3.Under the low potassium(0.02 mM K⁺)conditions,the phenotype and biomass of ZxNHX1 transgenic lines were significantly better than AtNHX1 lines supplemented with 10 mM NaCl.In addition,the Na⁺concentration in the shoot and root of ZxNHX1-H line increased significantly by 58.7%and 73.3%compared with AtNHX1-H line,while the K⁺concentration was significantly decreased.4.Under 75 mM NaCl,the fresh weight,dry weight of the shoot and Na⁺concentration of ZxNHX1 T265F-H line was significantly decreased by 31.6%,15.8%and 16.9%compared with ZxNHX1-H line,while the K⁺concentration increased by12.0%.It showed that the 265^th amino acid of ZxNHX1 was also a key amino acid site that affects its Na⁺and K⁺selectivity at the plant level.5.Under 75 mM NaCl,the phenotype,biomass and Na⁺and K⁺concentrations of ZxNHX1 S419T transgenic lines did not change significantly compared with ZxNHX1transgenic lines.It showed the 419^th amino acid of ZxNHX1 had no significant impact on its growth and Na⁺and K⁺accumulation.6.Under 75 mM NaCl,the shoot fresh weight and dry weight of ZxNHX1T265F&S419T-H line was significantly reduced by 13.4%and 12.8%compared with ZxNHX1-H line,the Na⁺concentration was significantly reduced by 25.5%,and the K⁺concentration was significantly increased by 14.9%.However,the ZxNHX1T265F&S419T lines has no significant difference on its Na⁺and K⁺accumulation ability compared with the ZxNHX1 T265F lines.It further proved that the 419^thamino acid of ZxNHX1 may not be the key position determining its Na⁺and K⁺selectivity in the plant level.Based on the above results,it is indicated that the 265^thamino acid of ZxNHX1 plays a key role on its Na⁺and K⁺selectivity.The above results verified that there are differences in Na⁺and K⁺selectivity between ZxNHX1 and AtNHX1 at the plant level,and 265^th amino acid of ZxNHX1is also a key amino acid site that affects Na⁺and K⁺selectivity of ZxNHX1.The present research provided a theoretical basis for further research on the salt stress response mechanism of Z.xanthoxylum.