Cloning and Functional Characterization of SpZIP2

Abstract

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.

Keywords: Cd accumulation; Cd tolerance; S. plumbizincicola; ZIP2.

Figure 1

Expression pattern of Zinc (Zn)-regulated and iron (Fe)-regulated transporter-like proteins (ZIPs) in Sedum plumbizincicola (S. plumbizincicola) and Sedum. alfredii (S. alfredii). (A) Expression of ZIP genes in S. plumbizincicola and S. alfredii in response to Cd stress. Sp: S. plumbizincicola. Sa: S. alfredii. 0R: Roots without Cd treatment. 10R: Roots with Cd treatment. 0S: Shoots without Cd treatment. 10S: Shoots with Cd treatment. ZIP1: c33410_g1; ZIP2: c34395_g1; ZIP3: c31670_g1; ZIP4: c36833_g1. (B) FPKM values of ZIP2 in roots and shoots under normal or Cd stress conditions in S. plumbizincicola and S. alfredii. (C) Expression of SpZIP2 in roots, stems and leaves in response to Cd treatment in S. plumbizincicola. Values are means ± SD, n = 3. Statistical significance was tested by Student’s t-tests. Differences were deemed significant at p < 0.05 (*).

Figure 2

Sequence characterization and transmembrane region prediction of SpZIP2. (A) Amino acid composition analysis for SpZIP2. (B) Prediction of protein phosphorylation sites for SpZIP2. (C) Transmembrane region prediction of SpZIP2.

Figure 3

Phylogenetic analysis for SpZIP2 and ZIP members from rice and Arabidopsis thaliana (A. thaliana). SpZIP2 is shown in red color and its closely related ZIP members are shown by a green background.

Figure 4

Sequence alignment of SpZIP2 and ZIP members from rice and A. thaliana. Strictly conserved residues are highlighted in red. The corresponding positions of the predicted 8 transmembrane regions are indicated as TM1 to TM8 below the corresponding sequence.

Figure 5

Tolerance of yeast transformants expressing SpZIP2 to Cd or Zn stresses. (A) Tolerance of yeast transformants expressing SpZIP2 to Cd stress. (B) Tolerance of yeast transformants expressing SpZIP2 to Zn stress.

Figure 6

Cd accumulation in SpZIP2 expressed Δyap1 and empty vector transferred yeast Y252 and Δyap1. Values are means ± SD, n = 3. Statistical significance was tested by Student’s t-tests. Differences were deemed significant at p < 0.05 (*).

Figure 7

Subcellular localization analysis of SpZIP2. (A) Subcellular localization of SpZIP2-mRFP fusion proteins or mRFP in yeast Δyap1. (B) Tolerance of yeast transformants expressing SpZIP2-mRFP to Cd stress. (C) Subcellular localization prediction for SpZIP2 by Plant-mPLoc.