Almeida Pedro M F, de Boer Gert-Jan, de Boer Albertus H
Department of Structural Biology, Faculty Earth and Life Sciences, Vrije Universiteit Amsterdam Amsterdam, Netherlands.
R&D Department, Enza Zaden Enkhuizen, Netherlands.
Front Plant Sci. 2014 Nov 4;5:600. doi: 10.3389/fpls.2014.00600. eCollection 2014.
Single Nucleotide Polymorphisms (SNPs) within the coding sequence of HKT transporters are important for the functioning of these transporters in several plant species. To unravel the functioning of HKT transporters analysis of natural variation and multiple site-directed mutations studies are crucial. Also the in vivo functioning of HKT proteins, via complementation studies performed with athkt1;1 plants, could provide essential information about these transporters. In this work, we analyzed the natural variation present in the first pore domain of the HKT1;2 coding sequence of 93 different tomato accessions, which revealed that this region was conserved among all accessions analyzed. Analysis of mutations introduced in the first pore domain of the SlHKT1;2 gene showed, when heterologous expressed in Xenopus laevis oocytes, that the replacement of S70 by a G allowed SlHKT2;1 to transport K(+), but also caused a large reduction in both Na(+) and K(+) mediated currents. The study of the transport characteristics of SlHKT1;2 revealed that Na(+)-transport by the tomato SlHKT1;2 protein was inhibited by the presence of K(+) at the outside of the membrane. GUS expression under the AtHKT1;1 promoter gave blue staining in the vascular system of transgenic Arabidopsis. athkt1;1 mutant plants transformed with AtHKT1;1, SlHKT1;2, AtHKT1;1S68G, and SlHKT1;2S70G indicated that both AtHKT1;1 and SlHKT1;2 were able to restore the accumulation of K(+) in the shoot, although the low accumulation of Na(+) as shown by WT plants was only partially restored. The inhibition of Na(+) transport by K(+), shown by the SlHKT1;2 transporter in oocytes (and not by AtHKT1;1), was not reflected in Na(+) accumulation in the plants transformed with SlHKT1;2. Both AtHKT1;1-S68G and SlHKT1;2-S70G were not able to restore the phenotype of athkt1;1 mutant plants.
HKT转运蛋白编码序列中的单核苷酸多态性（SNP）对于这些转运蛋白在多种植物物种中的功能发挥很重要。为了阐明HKT转运蛋白的功能，对自然变异的分析和多位点定向突变研究至关重要。此外，通过对athkt1;1植物进行互补研究，HKT蛋白的体内功能可以提供有关这些转运蛋白的重要信息。在这项工作中，我们分析了93种不同番茄种质的HKT1;2编码序列第一个孔结构域中的自然变异，结果表明该区域在所分析的所有种质中都是保守的。对SlHKT1;2基因第一个孔结构域中引入的突变进行分析表明，当在非洲爪蟾卵母细胞中异源表达时，用G取代S70使SlHKT2;1能够转运K⁺，但也导致Na⁺和K⁺介导的电流大幅降低。对SlHKT1;2转运特性的研究表明，膜外存在K⁺会抑制番茄SlHKT1;2蛋白的Na⁺转运。AtHKT1;1启动子驱动下的GUS表达在转基因拟南芥的维管系统中产生蓝色染色。用AtHKT1;1、SlHKT1;2、AtHKT1;1S68G和SlHKT1;2S70G转化athkt1;1突变体植物表明，AtHKT1;1和SlHKT1;2都能够恢复地上部K⁺的积累，尽管野生型植物显示的低Na⁺积累仅部分恢复。卵母细胞中SlHKT1;2转运蛋白所显示的K⁺对Na⁺转运的抑制作用（AtHKT1;1则没有）在转SlHKT1;2的植物的Na⁺积累中未体现出来。AtHKT1;1 - S68G和SlHKT1;2 - S70G都无法恢复athkt1;1突变体植物的表型。
