ZmHMA3, a Member of the Heavy-Metal-Transporting ATPase Family, Regulates Cd and Zn Tolerance in Maize

Abstract

The pollution of heavy metals is extremely serious in China, including zinc (Zn), copper (Cu), lead (Pb), and cadmium (Cd). Heavy-metal-transporting ATPase (HMA) belongs to a subfamily of the P-ATPase family, which absorbs and transports Zn, Cu, Pb, and Cd in plants. Here, we describe a ZmHMA-encoding HMA family protein that positively regulates Cd and Zn tolerance. The real-time fluorescence quantification (RT-PCR) results revealed that ZmHMA3 had a high expression in B73, and the expression of ZmHMA3 was sensitive to Cd in yeast cells, which was related to Cd accumulation in yeast. Additionally, the Arabidopsis thaliana homologous mutants of AtHMA2 showed Cd sensitivity compared with WT. The overexpressing ZmHMA3 plants showed higher tolerance under Cd and Zn stresses than the wild type. The overexpression of ZmHMA3 led to higher Cd and Zn accumulation in tissues based on the subcellular distribution analysis. We propose that ZmHMA3 improves maize tolerance to Cd and Zn stresses by absorbing and transporting Cd and Zn ions. This study elucidates the gene function of the ZmHMA3 response to Cd and Zn stress and provides a reference for improving the characteristics of heavy metals enrichment in existing maize varieties and the plant remediation technology of heavy-metal-contaminated soil.

Keywords: HMA3; cadmium; functional analysis; maize; zinc.

Figure 1

Phylogenetic construction and conservative sequence analysis of HMA family genes: (A) phylogenetic analysis of the HMA family in maize, rice, and Arabidopsis; (B) conservative sequence analysis of ZmHMA3 and OsHMA2, 3 in rice. Note: (A), the pink font represents the ZmHMA family, the green font represents the OsHMA family, and the blue font represents the AtHMA family. The red triangle refers to ZmHMA3; (B), the height of individual amino acids in the stack shows the relative frequency of such amino acids in this position, and the amino acid residues within black boxes are conserved functional domains in the HMA family.

Figure 2

Expression pattern analysis of ZmHMA3 in different tissues, subcellular localization of ZmHMA3 in tobacco leaves, and heterologous expression analysis of the ZmHMA3 in yeast cells: (A) analysis of expression patterns of ZmHMA3 in different tissues; (B) localization observation of ZmHMA3 in tobacco leaves; (C) heterologous expression stress of ZmHMA3 in yeasts under stress; * means p ≤ 0.05; ** means p ≤ 0.01; Student’s t-test. Note: (A), the figure shows the relative expression of the ZmHMA3 gene in roots, stems, and leaves of maize B73 after 12, 24, 48, 72, and 96 h of Cd stress at 200 μmol/L concentration; (B), the GFP server indicates the green fluorescent protein, the RFP server indicates the red fluorescent protein, the bright color indicates the cell morphology image, and the merged pattern indicates a combined image of dark filed fluorescence and cell morphology images. The scale bar is 30 μm; (C), the X-axis direction shows the 10 gradients of the diluted yeast liquid from left to right, and the Y-axis direction indicates the transformation of yeast strains with different target vectors.

Figure 3

Phenotype identification and root trait analysis of AtHMA2 homozygous in mutants: (A–D) phenotypic identification of Arabidopsis AtHMA2 homozygous mutants under different concentrations of Cd stress; (E,F) statistical plots of root length and fresh weight were measured under Cd stress; * means p ≤ 0.05; ** means p ≤ 0.01; Student’s t-test.

Figure 4

Comparison of agronomic traits between overexpressed transgenic ZmHMA3 and wild-type plants at different times and under different treatments: (A) the seedling grown under 24 h of heavy metal treatment; (B) the seedling grown under 48 h of heavy metal treatment; the scale bar is 5 cm; (C) seedling plant height (SHL); (D) seedling fresh weight (SFW); (E) root fresh weight (RFW); (F) seedling dry weight (SDW); (G) root dry weight (RDW); (H) total water content; * means p ≤ 0.05; ** means p ≤ 0.01; Student’s t-test.

Figure 5

Zn content, Cd content, and transport coefficients determined in ZmHMA3-overexpressed and wild-type plants under hydroponic conditions: (A) above-ground Cd content; (B) below-ground Cd content; (C) Cd transport coefficient; (D) above-ground Zn content; (E) below-ground Zn content; (F) Zn transport coefficient; * means p ≤ 0.05; ** means p ≤ 0.01; Student’s t-test.

Figure 6

The accumulation of heavy metals in ZmHMA3-overexpressed and wild-type plants was investigated in above- and below-ground parts of the plants. Subcellular distribution in Cd treatment: (A) shoots; (B) roots. Subcellular distribution in Zn treatment: (C) shoots; (D) roots.