Comparative Analysis Of The Differences In Amino Acid Sites Between Zygophyllum Xanthoxylum ZxNHX1 And Arabidopsis Thaliana AtNHX1 And Their Effects On Na~+ And K~+ Selectivity

Drought and soil salinization have become the two major abiotic stresses restricting the development of agriculture and animal husbandry in China.Zygophyllum xanthoxylum,a succulent xerophyte,has developed unique salt-and drought-resistant mechanisms to cope with these harsh habitats:It could absorb large amounts of Na⁺from low-salinity soil and store it in leaves as a kind of beneficial and inexpensive osmotic regulate substance to against osmotic stress.The compartmentalization of Na⁺from cytoplasm to vacuole was mainly mediated by the tonoplast Na⁺/H⁺antiporter(NHX1),so ZxNHX1 plays a crucial role in the response to salt stress of Z.xanthoxylum.While in Arabidopsis thaliana,a typical glycophyte,the updated research has shown that the different vacuolar AtNHXs have different affinities to Na⁺and K⁺.The previous study showed that the growth of Na⁺sensitive yeast strain AXT3K transformed with ZxNHX1 was significantly better than that of AXT3K transformed with AtNHX1 under different NaCl treatment,and the results of Na⁺(K⁺)/H⁺exchange activities had shown that the ability of ZxNHX1 to compartmentalize Na⁺was significantly higher than that of AtNHX1,while the ability of ZxNHX1 to compartmentalize K⁺was significantly lower than AtNHX1,these results indicated that there were differences in Na⁺and K⁺selectivity between ZxNHX1 and AtNHX1.As we known,the function of protein are closely related to the structure,thus we speculate that the difference in the ion selectivity between ZxNHX1 and AtNHX1 might be closely related to the diversities of their NHXs structure.In view of this,our study analyzed the differences in amino acid sequences and3D structures of ZxNHX1 and AtNHX1,selected three key amino acid sites which might be related to the ion selective of them.After mutations,we used the yeast heterologous expression systems AXT3K to analysis the difference in growth of yeast cells and their Na⁺and K⁺accumulation among ZxNHX1 and AtNHX1 before and after mutations,respectively.At the same time,we used the vacuolar protease-deficient yeast strain TY001 to further determined the Na⁺/H⁺and K⁺/H⁺exchange activity of them.The overall results can be summarized as follows:1.Under 50 mM NaCl,the mutation of amino acid residues at 265 of ZxNHX1(T265F)inhibited the growth of yeast,significantly reduced its Na⁺concentration by16.4%and increased K⁺concentration by 10.8%.However,after the mutation of the268^thh amino acid residue of AtNHX1,the growth,ion accumulation,Na⁺/H⁺and K⁺/H⁺exchange activity of AXT3K had no significant variation compared with the AXT3K transformed with AtNHX1 under salt treatment.Based on the above results,it is speculated that the 265^thh amino acid of ZxNHX1 plays a very important role in affecting its Na⁺and K⁺selectivity,while the 268^thamino acid of AtNHX1 has no effect on its Na⁺and K⁺selectivity.2.In comparison with the yeast strain AXT3K transforming ZxNHX1,the mutation of the 341^stamino acid residue in ZxNHX1 had no difference in the growth,Na⁺and K⁺concentration and their ion exchange activity,these results indicating that the 341^stt amino acid residue of ZxNHX1 might not be essential for regularly the ion selectivity of ZxNHX1.However,under 50 mM NaCl,the mutation of this site in AtNHX1(I344V)can significantly increase the Na⁺concentration by 8.2%,decreased K⁺concentration by 18.8%,and Na⁺/H⁺exchange activity significantly increased while K⁺/H⁺exchange activity significantly decreased compared with the AXT3K transformed with AtNHX1.It is speculated that this site(I344)may be one of the key amino acid residues affecting Na⁺and K⁺selectivity of AtNHX1 in A.thaliana.3.The mutation of the 419^thamino acid of ZxNHX1(S419T)can significantly inhibit the ability of Na⁺compartmentalization of mutation yeast strain AXT3K,decreased the Na⁺accumulation by 21.2%and reduced also the Na⁺/H⁺exchange activity,while it can increased the K⁺accumulation by 21.0%and enhanced the exchange activity of K⁺/H⁺as well.Based on these results,we speculate that the 419^thh amino acid residue of ZxNHX1 may be one of the key sites which affected its Na⁺and K⁺selectivity.However,after the mutation of this site in AtNHX1(T422S),the growth of AXT3K transformed with AtNHX1 T422S significantly decreased by 7.8%and the accumulation of Na⁺decreased by 10.0%,while K⁺concentration and the K⁺/H⁺exchange activity had no significant change under 50 mM NaCl,the results suggesting that the 422^edamino acid residue of AtNHX1 might only be a vital site affecting its Na⁺selectivity.4.The double mutated of 265^th,419^thh amino acid residues of ZxNHX1 can inhibit its growth by 12.1%,the Na⁺concentration was significantly decreased by29.4%,and K⁺concentration was significantly increased by 34.5%.In addition,the Na⁺/H⁺exchange activity of the double mutation yeast was significantly inhibited,while the K⁺/H⁺exchange activity was significantly increased.And this effect of double mutation yeast is more distinct than single mutation yeast.The above results further indicated that the 265^th,419^thh amino acid residues of ZxNHX1 have important influence to the ion selective,and it could be speculated that there is a kind of superposition between their roles.In summary,all above results revealed the important reason that caused the difference in Na⁺and K⁺selectivity between ZxNHX1 and AtNHX1,which provided a theoretical basis for further research on the mechanism of Z.xanthoxylum to response salt stress.