Functional analysis of CAX2-like transporters isolated from two ecotypes of Sedum alfredii

Two genes encoding CAX2-like proteins were isolated from a Zn/Cd hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance, and they were named SaCAX2h and SaCAX2n, respectively. Both SaCAX2h:eGFP and SaCAX2n:eGFP proteins were localized to the vacuolar membrane of tobacco epidermal cells and yeast mutants. Heterologous expression of SaCAX2h or SaCAX2n in the Delta zrc1 yeast mutant increased Cd content in yeast cells. Yeast complementary assay also revealed that both the transporters could suppress Ca and Mn hypersensitivity and enhance Ca and Mn accumulation in a K667 yeast mutant. The expression patterns of the two genes were different under the Cd treatment. Transcription of SaCAX2h was down-regulated in roots and up-regulated in shoots whereas transcription of SaCAX2n was down-regulated in shoots after the exposition to Cd. Furthermore, over-expression of SaCAX2h enhanced metal accumulation in the tobacco plants. The Cd content increased by 17-19 % in shoots and 31-36 % in roots; the Ca content of the transgenic plants increased by 31-32 % in shoots, and the Mn content increased by 60-79 % in shoots and 22-29 % in roots. These results indicate that SaCAX2h was responsible for Ca and Mn sequestration into vacuoles, and over-expression of SaCAX2h enhanced Cd accumulation in the transgenic tobacco.