DataSheet_4_SgNramp1, a plasma membrane-localized transporter, involves in manganese uptake in Stylosanthes guianensis.doc

Transporters belonging to the natural resistance-associated macrophage protein (Nramp) family play important roles in metal uptake and homeostasis. Although Nramp members have been functionally characterized in plants, the role of Nramp in the important tropical forage legume Stylosanthes guianensis (stylo) is largely unknown. This study aimed to determine the responses of Nramp genes to metal stresses and investigate its metal transport activity in stylo. Five SgNramp genes were identified from stylo. Expression analysis showed that SgNramp genes exhibited tissue preferential expressions and diverse responses to metal stresses, especially for manganese (Mn), suggesting the involvement of SgNramps in the response of stylo to metal stresses. Of the five SgNramps, SgNramp1 displayed the highest expression in stylo roots. A close correlation between SgNramp1 expression and root Mn concentration was observed among nine stylo cultivars under Mn limited condition. The higher expression of SgNramp1 was correlated with a high Mn uptake in stylo. Subsequent subcellular localization analysis showed that SgNramp1 was localized to the plasma membrane. Furthermore, heterologous expression of SgNramp1 complemented the phenotype of the Mn uptake-defective yeast (Saccharomyces cerevisiae) mutant Δsmf1. Mn concentration in the yeast cells expressing SgNramp1 was higher than that of the empty vector control, suggesting the transport activity of SgNramp1 for Mn in yeast. Taken together, this study reveals that SgNramp1 is a plasma membrane–localized transporter responsible for Mn uptake in stylo.

天然抗性相关巨噬细胞蛋白（natural resistance-associated macrophage protein，Nramp）家族转运蛋白在金属吸收与稳态调控中发挥关键作用。尽管Nramp家族成员在植物中的功能已得到功能解析，但在重要热带饲用豆科植物柱花草（Stylosanthes guianensis，简称stylo）中，Nramp的功能尚不明晰。本研究旨在解析Nramp基因对金属胁迫的响应，并探究其在柱花草中的金属转运活性。本研究从柱花草中鉴定得到5个SgNramp基因。表达谱分析显示，SgNramp基因呈现组织偏好性表达模式，且对金属胁迫的响应各具差异，尤其对锰（Mn）的响应最为显著，提示SgNramp家族蛋白参与柱花草对金属胁迫的应答过程。在5个SgNramp基因中，SgNramp1在柱花草根系中的表达量最高。在锰限制条件下的9个柱花草品种中，SgNramp1的表达量与根系锰浓度呈显著正相关，且SgNramp1的高表达与柱花草的高锰吸收能力密切相关。后续亚细胞定位分析显示，SgNramp1定位于质膜。此外，异源表达SgNramp1可互补锰吸收缺陷型酿酒酵母（Saccharomyces cerevisiae）Δsmf1突变体的表型。表达SgNramp1的酵母细胞内锰浓度高于空载体对照，表明SgNramp1在酵母中具备锰转运活性。综上，本研究证实SgNramp1是定位于质膜的转运蛋白，负责柱花草的锰吸收过程。