HvHMA2, a P1B-ATPase from Barley, Is Highly Conserved among Cereals and Functions in Zn and Cd Transport

Manipulation of crops to improve their nutritional value (biofortification) and optimisation of plants for removal of toxic metals from contaminated soils (phytoremediation) are major goals. Identification of membrane transporters with roles in zinc and cadmium transport would be useful for both aspects. The P1B-ATPases play important roles in heavy metal allocation and detoxification in Arabidopsis and it is now important to elucidate their roles in monocots. We identified nine P1B-ATPases in barley and this study focuses on the functional characterization of HvHMA2, providing evidence for its role in heavy metal transport. HvHMA2 was cloned using information from EST analysis and 5′ RACE. It possesses the conserved aspartate that is phosphorylated during the reaction cycle of P-type pumps and has motifs and key residues characteristic of P1B-ATPases, falling into the P1B-2 subclass. Homologous sequences occur in three major sub-families of the Poaceae (Gramineae). Heterologous expression in Saccharomyces cerevisiae demonstrates that HvHMA2 functions as a Zn and Cd pump. Mutagenesis studies show that proposed cation coordination sites of the P1B-2 pumps are crucial for the metal responses conferred by HvHMA2 in yeast. HvHMA2 expression suppresses the Zn-deficient phenotype of the Arabidopsis hma2hma4 mutant indicating that HvHMA2 functions as a Zn pump in planta and could play a role in root to shoot Zn transport. When expressed in Arabidopsis, HvHMA2 localises predominantly to the plasma membrane.

通过作物遗传改良提升其营养价值（生物强化，biofortification），以及优化植物以清除污染土壤中的有毒金属（植物修复，phytoremediation），是当前两大核心研究目标。鉴定参与锌（Zn）与镉（Cd）转运的膜转运蛋白，对这两个研究方向均具有重要价值。P1B-ATP酶（P1B-ATPases）在拟南芥（Arabidopsis）的重金属分配与解毒过程中发挥关键作用，而阐明其在单子叶植物中的功能则成为当前亟需解决的科学问题。本研究在大麦中鉴定出9种P1B-ATP酶，并以HvHMA2的功能表征为核心，为其参与重金属转运提供实验证据。研究团队通过表达序列标签（EST）分析与5'末端快速扩增技术（5' RACE）克隆得到HvHMA2基因。该基因编码的蛋白具有P型泵反应循环中被磷酸化的保守天冬氨酸残基，同时具备P1B-ATP酶的特征性基序与关键氨基酸残基，归类于P1B-2亚型。其同源序列广泛存在于禾本科（Poaceae，Gramineae）的三大主要亚科中。在酿酒酵母（Saccharomyces cerevisiae）中的异源表达实验证实，HvHMA2可作为锌与镉转运泵发挥功能。诱变实验结果表明，P1B-2型泵所预测的阳离子结合位点，对于HvHMA2在酵母中赋予的金属抗性响应至关重要。在拟南芥hma2hma4双突变体中异源表达HvHMA2，可恢复其锌缺乏表型，这表明HvHMA2在植物体内可作为锌转运泵发挥功能，并可能参与锌从根系向地上部的转运过程。在拟南芥中表达HvHMA2时，该蛋白主要定位于质膜。