Zinc (Zn)-regulated and iron (Fe)-regulated transporter-like proteins (ZIP) are key players involved in the accumulation of cadmium (Cd) and Zn in plants. Sedum plumbizincicola X.H. Guo et S.B. Zhou ex L.H. Wu (S. plumbizincicola) is a Crassulaceae Cd/Zn hyperaccumulator found in China, but the role of ZIPs in S. plumbizincicola remains largely unexplored. Here, we identified 12 members of ZIP family genes by transcriptome analysis in S. plumbizincicola and cloned the SpZIP2 gene with functional analysis. The expression of SpZIP2 in roots was higher than that in the shoots, and Cd stress significantly decreased its expression in the roots but increased its expression in leaves. Protein sequence characteristics and structural analysis showed that the content of alanine and leucine residues in the SpZIP2 sequence was higher than other residues, and several serine, threonine and tyrosine sites can be phosphorylated. Transmembrane domain analysis showed that SpZIP2 has the classic eight transmembrane regions. The evolutionary analysis found that SpZIP2 is closely related to OsZIP2, followed by AtZIP11, OsZIP1 and AtZIP2. Sequence alignment showed that most of the conserved sequences among these members were located in the transmembrane regions. A further metal sensitivity assay using yeast mutant Δyap1 showed that the expression of SpZIP2 increased the sensitivity of the transformants to Cd but failed to change the resistance to Zn. The subsequent ion content determination showed that the expression of SpZIP2 increased the accumulation of Cd in yeast. Subcellular localization showed that SpZIP2 was localized to membrane systems, including the plasma membrane and endoplasmic reticulum. The above results indicate that ZIP member SpZIP2 participates in the uptake and accumulation of Cd into cells and might contribute to Cd hyperaccumulation in S. plumbizincicola.
Isolating the genes responsible for cadmium (Cd) accumulation and tolerance in oilseed rape and uncovering their functional mechanism is of great significance for guiding genetic improvement to cope with heavy metal pollution. In this study, we screened the cDNA library of Brassica napus cv. Westar using a yeast genetic complementation system and isolated BnMT2-22a, BnMT2-22b and BnMT3b, which can mediate Cd tolerance in yeast. They all have two cysteine-rich domains in their sequence. Ectopic expression of these MTs demonstrated that all of them enhanced Cd and Cu tolerance in yeast, but had no effect on Mn and Zn tolerance. The fusion of the red fluorescent protein mRFP did not affect their function in mediating Cd tolerance, and using these functional fusion proteins we observed that they were all localized in cytosol. Meanwhile, their expression in yeast did not affect the accumulation of Cd in the yeast transformants. Gene expression analyses found that BnMT2-22a, BnMT2-22b and BnMT3b were all induced by Cd in roots, and BnMT3b was also significantly induced in shoots. These results indicate that the genes BnMT2-22a, BnMT2-22b and BnMT3b isolated with cDNA library screening can mediate Cd tolerance, and they may detoxify Cd via cytosolic chelation.
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