MeGLYI-13, a Glyoxalase I Gene in Cassava, Enhances the Tolerance of Yeast and Arabidopsis to Zinc and Copper Stresses

Abstract

Although zinc and copper are the two essential nutrients necessary for plant growth, their excessive accumulation in soil not only causes environmental pollution but also seriously threatens human health and inhibits plant growth. The breeding of plants with novel zinc or copper toxicity tolerance capacities represents one strategy to address this problem. Glyoxalase I (GLYI) family genes have previously been suggested to be involved in the resistance to a wide range of abiotic stresses, including those invoked by heavy metals. Here, a MeGLYI-13 gene cloned from a cassava SC8 cultivar was characterized with regard to its potential ability in resistance to zinc or copper stresses. Sequence alignment indicated that MeGLYI-13 exhibits sequence differences between genotypes. Transient expression analysis revealed the nuclear localization of MeGLYI-13. A nuclear localization signal (NLS) was found in its C-terminal region. There are 12 Zn²⁺ binding sites and 14 Cu²⁺ binding sites predicted by the MIB tool, of which six binding sites were shared by Zn²⁺ and Cu²⁺. The overexpression of MeGLYI-13 enhanced both the zinc and copper toxicity tolerances of transformed yeast cells and Arabidopsis seedlings. Taken together, our study shows the ability of the MeGLYI-13 gene to resist zinc and copper toxicity, which provides genetic resources for the future breeding of plants resistant to zinc and copper and potentially other heavy metals.

Keywords: Arabidopsis; cassava; copper; glyoxalase; heavy metal; overexpression; subcellular localization; yeast; zinc.

Figure 1

The Zn²⁺ and Cu²⁺ ion binding site analysis in the MeGLYI-13 amino acid sequence. (A) The Zn²⁺ binding sites in MeGLYI-13. (B) The Cu²⁺ binding sites in MeGLYI-13.

Figure 2

Subcellular localization of MeGLYI-13 in onion epidermal cells.

Figure 3

Overexpression of MeGLYI-13 enhanced the yeast resistance to Zn²⁺ and Cu²⁺ toxicity. (A) Growth activity of yeast after different concentrations of Zn²⁺ treatments. (B) The growth of MeGLYI-13 transgenic yeast and empty yeast cells after the 30 mmol/L Zn²⁺ treatment. (C) Growth activity of yeast after different concentrations of Cu²⁺ treatments. (D) The growth of MeGLYI-13 transgenic yeast and empty yeast cells after the 3 mmol/L Cu²⁺ treatment. The error lines represent ±SD, n = 3; * signals a significant difference of p ≤ 0.05 and ** p ≤ 0.01.

Figure 4

Generation of MeGLYI-13 overexpressing Arabidopsis. (A) PCR analysis of transgenic Arabidopsis. M, DL2000 DNA Marker; 1~6, the transgenic Arabidopsis lines; +, the positive control (pCambia1300-MeGLYI-13-GFP vector plasmid); −, the negative control (wild type Arabidopsis). (B) Expression analysis of the different transgenic lines. WT, wild type Arabidopsis; OE1~OE6, the transgenic Arabidopsis lines. The error lines represent ±SD, n = 3; the character on the top of each bar represents a significant difference. (C) Fluorescence detection of the different transgenic lines. WT, wild type Arabidopsis; OE1~OE6, the transgenic Arabidopsis lines.

Figure 5

Zinc and copper tolerance analysis of MeGLYI-13 transgenic Arabidopsis. (A) Phenotypes of Arabidopsis grown on normal 1/2 MS medium. (B) Statistics of the taproot length of Arabidopsis grown on normal 1/2 MS medium. (C) Phenotypes of Arabidopsis under different Zn²⁺ concentrations. (D) Statistics of the taproot length of Arabidopsis under different Zn²⁺ concentration treatments. (E) Phenotypes of Arabidopsis under different Cu²⁺ concentration stresses. (F) Statistics of the taproot length of Arabidopsis under different Cu²⁺ concentration treatments. The error lines represent ±SD, n = 3; ** indicates significant differences of p ≤ 0.01, respectively.