Table_3_HKT1;5 Transporter Gene Expression and Association of Amino Acid Substitutions With Salt Tolerance Across Rice Genotypes.docx

Plants need to maintain a low Na+/K+ ratio for their survival and growth when there is high sodium concentration in soil. Under these circumstances, the high affinity K+ transporter (HKT) and its homologs are known to perform a critical role with HKT1;5 as a major player in maintaining Na+ concentration. Preferential expression of HKT1;5 in roots compared to shoots was observed in rice and rice-like genotypes from real time PCR, microarray, and RNAseq experiments and data. Its expression trend was generally higher under increasing salt stress in sensitive IR29, tolerant Pokkali, both glycophytes; as well as the distant wild rice halophyte, Porteresia coarctata, indicative of its importance during salt stress. These results were supported by a low Na+/K+ ratio in Pokkali, but a much lower one in P. coarctata. HKT1;5 has functional variability among salt sensitive and tolerant varieties and multiple sequence alignment of sequences of HKT1;5 from Oryza species and P. coarctata showed 4 major amino acid substitutions (140 P/A/T/I, 184 H/R, D332H, V395L), with similarity amongst the tolerant genotypes and the halophyte but in variance with sensitive ones. The best predicted 3D structure of HKT1;5 was generated using Ktrab potassium transporter as template. Among the four substitutions, conserved presence of aspartate (332) and valine (395) in opposite faces of the membrane along the Na+/K+ channel was observed only for the tolerant and halophytic genotypes. A model based on above, as well as molecular dynamics simulation study showed that valine is unable to generate strong hydrophobic network with its surroundings in comparison to leucine due to reduced side chain length. The resultant alteration in pore rigidity increases the likelihood of Na+ transport from xylem sap to parenchyma and further to soil. The model also proposes that the presence of aspartate at the 332 position possibly leads to frequent polar interactions with the extracellular loop polar residues which may shift the loop away from the opening of the constriction at the pore and therefore permit easy efflux of the Na+. These two substitutions of the HKT1;5 transporter probably help tolerant varieties maintain better Na+/K+ ratio for survival under salt stress.

植物在土壤中钠离子浓度偏高的情况下，为了生存与生长，必须维持低Na+/K+比率。在这种条件下，高亲和力钾离子转运蛋白（HKT）及其同源物发挥着至关重要的作用，其中HKT1;5作为维持Na+浓度的主要角色。实时PCR、微阵列和RNA测序实验数据表明，HKT1;5在根系中的表达优先于茎部，这在水稻及其类似基因型中得到了观察。在敏感品种IR29和耐盐品种Pokkali，以及远缘野生盐生水稻Porteresia coarctata中，其表达趋势在增加的盐胁迫下普遍升高，这表明其在盐胁迫期间的重要性。Pokkali中的Na+/K+比率较低，而P. coarctata中的比率则更低，这些结果得到了支持。HKT1;5在耐盐和耐盐品种中存在功能性变异，Oryza物种和P. coarctata中HKT1;5序列的多序列比对显示了4个主要的氨基酸替换（140P/A/T/I，184H/R，D332H，V395L），其中耐盐基因型和盐生植物之间的相似性较高，而与敏感品种则存在差异。使用Ktrab钾离子转运蛋白作为模板，生成了HKT1;5的最佳预测三维结构。在这四个替换中，仅在耐盐和盐生基因型中观察到膜两侧Na+/K+通道上的天冬氨酸（332）和缬氨酸（395）的保守存在。基于上述发现以及分子动力学模拟研究显示，与亮氨酸相比，缬氨酸由于其侧链长度减少，无法与其周围环境形成强烈的疏水网络。孔隙刚性的变化导致Na+从木质部汁液运输到薄壁组织，再进一步运输到土壤的可能性增加。该模型还提出，在第332位置的天冬氨酸的存在可能导致与细胞外环极性残基的频繁极性相互作用，这可能导致环远离孔隙狭窄处的开口，从而允许Na+的轻松外排。HKT1;5转运蛋白的这两个替换可能有助于耐盐品种在盐胁迫下维持更好的Na+/K+比率以实现生存。