The Copper Chaperone Protein Gene GmATX1 Promotes Seed Vigor and Seedling Tolerance under Heavy Metal and High Temperature and Humidity Stresses in Transgenic Arabidopsis

Abstract

Abiotic stresses such as high temperature, high humidity, and heavy metals are important factors that affect seed development and quality, and restrict yield in soybean. The ATX1-type copper chaperones are an important type of proteins that are used for maintaining intracellular copper ion homeostasis. In our previous study, a copper chaperone protein GmATX1 was identified in developing seeds of soybean under high temperature and humidity (HTH) stresses. In this study, the GmATX1 gene was isolated, and multiple alignment analysis showed that its encoding protein shared high sequence identities with other plant orthologues of copper chaperone proteins containing the HMA domain, and a conserved metal ion-binding site, CXXC. A subcellular localization assay indicated that GmATX1 was localized in the cell membrane and nucleus. An expression analysis indicated that GmATX1 was involved in seed development, and in response to HTH and heavy metal stresses in soybean. GmATX1-silent soybean seedlings were found to be more severely damaged than the control under HTH stress. Moreover, the silencing of GmATX1 reduced antioxidase activity and reactive oxygen species (ROS) scavenging ability in the seedling leaves. The overexpression of GmATX1 in Arabidopsis improved seed vigor and seedling tolerance, and enhanced antioxidase activity and ROS scavenging ability under HTH and heavy metal stresses. Our results indicated that GmATX1 could promote seed vigor and seedling tolerance to HTH and heavy metal stresses in transgenic Arabidopsis, and this promotion could be achieved by enhancing the antioxidase activity and ROS scavenging ability.

Keywords: GmATX1; heavy metal; high temperature and high humidity stress; seed development; seed vigor; tolerance.

Figure 1

Sequence analysis and subcellular localization of GmATX1. (A) Multiple alignment of the amino acid sequences of GmATX1 and other plant homologues. CXXC motif, heavy metal binding core motif. Gs, Glycine soja; Gm, Glycine max; Vr, Vigna radiata; Va, Vigna angularis; Vu, Vigna unguiculata; La, Lupinus angustifolius. (B) Subcellular localization of the GmATX1 protein in tobacco leaf cells. pA7GmATX1::GFP, a fusion protein; pA7GmATX1::GFP, a null-loaded protein; BF, Brightfield. Fluorescence images of protoplasts expressing GFP fusion proteins were obtained using the LSM780 confocal microscopy imaging system.

Figure 2

Expression patterns of GmATX1 in soybean. (A,B) Expression patterns of GmATX1 in the developing seeds of soybean cvs. Xiangdou No. 3 and Ningzhen No. 1 at the R7 stage, treated with the HTH stress for 0, 24, 96, and 168 h. (C,D) Expression of GmATX1 in various tissues in soybean cvs. Xiangdou No. 3 and Ningzhen No. 1 under normal growth and development conditions. Young seed, developing seed at the R5 stage (beginning of bulge); mature seed, seed at the R7 stage (maturity). ** Indicates significant differences between the control and the treatment at p < 0.01; different letters within a cultivar indicate significant differences at p < 0.05. Values are mean ± SD from three biological replicates.

Figure 3

Effects of HT, HH, and HTH stresses on the GmATX1-silent soybean line. (A) Illustration of the 5-week-old seedlings of the GmATX1-silent soybean line (pTRV2-GmATX1) and control (pTRV2-00) treated under HT (40/30 °C, 10 h/14 h (light/dark) and 70% relative humidity (RH)), HH (30/20 °C, 10 h/14 h (light/dark) and 100% RH), and HTH (40/30 °C, 10 h/14 h (light/dark) and 100% RH) condition, respectively, for 2 d. (B–E) Activities of SOD, CAT, and POD and the content of MDA in the leaves of 5-week-old seedlings of the gene-silenced soybean line (pTRV2-GmATX1) and control (pTRV2-00) treated under HT, HH, and HTH conditions, respectively, for 2 d. (F) Staining of H₂O₂ in the leaves of 5-week-old seedlings of the gene-silenced soybean line (pTRV2-GmATX1) and the control (pTRV2-00) treated under the HT, HH, and HTH conditions, respectively, for 2 d. The values shown are mean ± SD from three biological replicates. ** Indicates significant differences between the GmATX1-silent soybean line (pTRV2-GmATX1) and the control (pTRV2-00) at p < 0.01.

Figure 4

Phenotype, seed germination, and viability of WT and GmATX1-overexpressed Arabidopsis lines under HT, HH, and HTH treatments. (A) Germination percentages of seeds harvested from the WT and GmATX1-overexpressed Arabidopsis lines (L1, L2 and L3) after the HT (40/20 °C, 12 h/12 h (light/dark) and 70% RH), HH (23/20 °C, 12 h/12 h (light/dark) and 100% RH), and HTH (40/20 °C, 12 h/12 h (light/dark) and 100% RH) treatments, respectively. (B) Viability of the seeds harvested from the WT and transgenic Arabidopsis lines (L1 and L2) after the HT, HH, and HTH treatments, respectively. Dark red staining indicates viable seeds. (C) Illustration of the 4-week-old WT and transgenic Arabidopsis line (L3) after the HT, HH, and HTH treatments, respectively, for 2 d. (D,E) Stomatal morphology and stomatal aperture of the 4-week-old WT and transgenic Arabidopsis lines (L3) under the HH, HTH, and HT stresses, respectively. Values are mean ± SD from three biological replicates. ** indicates significant differences between the WT and transgenic Arabidopsis lines (L3) at p < 0.01.

Figure 5

Effects of HT, HH, and HTH stresses on antioxidase activity and ROS scavenging ability of the WT and GmATX1-overexpressed Arabidopsis lines. (A–D) Activities of SOD, CAT, and POD and the content of MDA in the leaves of the 4-week-old seedlings of the WT and transgenic Arabidopsis lines (L1, L2, and L3) after the HT, HH, and HTH treatments, respectively, for 2 d. (E) Staining of H₂O₂ in the leaves of the 4-week-old seedlings of the WT and transgenic Arabidopsis lines (L3) after the HT, HH, and HTH treatments, respectively, for 2 d. (F,G) ROS levels in the seeds harvested from the WT and transgenic Arabidopsis (L3) after the HT, HH, and HTH treatments, respectively, for 2 d. The values shown are mean ± SD from three biological replicates. * and ** indicate significant differences between the WT and transgenic Arabidopsis lines at p < 0.05 and p < 0.01, respectively.

Figure 6

Expression pattern of GmATX1 in soybean cvs. Ningzhen No. 1 and Xiangdou No. 3 under heavy metal stress. (A) The relative expression of GmATX1 under different concentrations of CuSO₄ stress (for 24 h) in roots of 2-week-old soybean seedlings. (B) The relative expression of GmATX1 under different concentrations of CdCl₂ stress (for 24 h) in roots of 2-week-old soybean seedlings. ** Indicates significant differences between the control and treatments at p < 0.01. Values are mean ± SD from three biological replicates.

Figure 7

Effects of heavy metal stresses on antioxidase activity and seedling tolerance of WT and GmATX1-overexpressed Arabidopsis. (A) Effect on the root length of the transgenic Arabidopsis seedlings after sowing on MS medium containing 50 μmol/L CuSO₄ and 50 μmol/L CdCl₂, respectively, for 10 d. (B) Effect on DW of the transgenic Arabidopsis seedlings after sowing on MS medium containing 50 μmol/L CuSO₄ and 50 μmol/L CdCl₂, respectively, for 10 d. (C) Activities of SOD, CAT, and POD, and contents of MDA in the leaves of seedlings of the transgenic Arabidopsis lines (L1, L2, and L3) and WT after sowing on MS medium containing 50 μmol/L CuSO₄ for 10 d. (D) Activities of SOD, CAT, and POD, and contents of MDA in the leaves of the seedlings of transgenic Arabidopsis lines (L1, L2, and L3) and WT after sowing on MS medium containing 50 μmol/L CdCl₂ for 10 d. Values shown are mean ± SD from three biological replicates. ** Indicates significant differences at p < 0.01.