Synergistic Effects of <em>GhSOD1</em> and <em>GhCAT1</em> Overexpression in Cotton Chloroplasts on Enhancing Tolerance to Methyl Viologen and Salt Stresses

<div><p>In plants, CuZn superoxide dismutase (CuZnSOD, EC l.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), and catalase (CAT, EC l.11.1.6) are important scavengers of reactive oxygen species (ROS) to protect the cell from damage. In the present study, we isolated three homologous genes (<em>GhSOD1</em>, <em>GhAPX1</em>, and <em>GhCAT1</em>) from <em>Gossypium hirsutum</em>. Overexpressing cassettes containing chimeric <em>GhSOD1</em>, <em>GhAPX1</em>, or <em>GhCAT1</em> were introduced into cotton plants by <em>Agrobacterium</em> transformation, and overexpressed products of these genes were transported into the chloroplasts by transit peptide, as expected. The five types of transgenic cotton plants that overexpressed <em>GhSOD1</em>, <em>GhAPX1</em>, <em>GhCAT1</em>, <em>GhSOD1</em> and <em>GhAPX1</em> stack (SAT), and <em>GhSOD1</em> and <em>GhCAT1</em> stack (SCT) were developed. Analyses in the greenhouse showed that the transgenic plants had higher tolerance to methyl viologen (MV) and salinity than WT plants. Interestingly, SCT plants suffered no damage under stress conditions. Based on analyses of enzyme activities, electrolyte leakage, chlorophyll content, photochemical yield (<em>Fv/Fm</em>), and biomass accumulation under stresses, the SCT plants that simultaneously overexpressed <em>GhSOD1</em> and <em>GhCAT1</em> appeared to benefit from synergistic effects of two genes and exhibited the highest tolerance to MV and salt stress among the transgenic lines, while the SAT plants simultaneously overexpressing <em>GhSOD1</em> and <em>GhAPX1</em> did not. In addition, transgenic plants overexpressing antioxidant enzymes in their chloroplasts had higher tolerance to salt stress than those expressing the genes in their cytoplasms, although overall enzyme activities were almost the same. Therefore, the synergistic effects of <em>GhSOD1</em> and <em>GhCAT1</em> in chloroplasts provide a new strategy for enhancing stress tolerance to avoid yield loss.</p> </div

Enhanced MV-mediated oxidative stress tolerance in whole plants of transgenic and WT cotton.

<p>(A) Visible damage in leaves with 300 µM MV for 5 d. (B) Quantitative estimate of visible damage on leaves. (C) Total chlorophyll contents in MV-treated transgenic plants. Means ± standard deviation labeled with different letters are significantly different at the 0.05 level.</p

Activities of antioxidant enzymes in leaves of transgenic and WT cotton plants.

<p>(A) SOD activity. (B) APX activity. (C) CAT activity. Means ± standard deviation labeled with different letters are significantly different at the 0.05 level.</p

Agronomic traits of transgenic and WT cotton plants treated with 200 mM NaCl in the greenhouse.

<p>Three lines each of five types of transgenic cotton (six plants per line, 18 WT plants) were evaluated; the experiment was repeated three times. Values are given as means ± standard deviation (n = 54). Means within a column followed by different letters are significantly different at <i>P</i><0.05.</p

Schematic diagrams of the T-DNA structure of the plant expression vectors.

<p><i>CaMV35S::GhSOD1</i> (top), <i>CaMV35S::GhAPX1</i> (middle), and <i>CaMV35S::GhCAT1</i> (bottom) constructs. NPT II, neomycin phosphotransferase II; DE-35SP, CaMV35S promoter with double-enhancer sequence; TP, transit signal peptide; Nos T, transcriptional termination sequence of nopaline synthase gene; LB, left border of T-DNA; RB, right border of T-DNA.</p

Activities of antioxidant enzymes in chloroplasts of the transgenic and WT cotton plants.

<p>(A) SOD activity. (B) APX activity. (C) CAT activity. Means ± standard deviation labeled with different letters are significantly different at the 0.05 level.</p

Southern blot and qRT-PCR analysis of transgenic cotton overexpressing <i>GhSOD1</i>, <i>GhAPX1</i>, or <i>GhCAT1</i>.

<p>(A) Southern blot. M, DNA marker; P, pBin438 plasmid; 1–6, representative transgenic plants with <i>CaMV35S::GhSOD1</i> (1, 2, and 4 are listed as ST1, ST2, and ST4 below); 7–12, representative transgenic plants with <i>CaMV35S::GhAPX1</i> (1, 5, and 6 are listed as AT1, AT5, and AT6 below); 13–18, representative transgenic plants with <i>CaMV35S::GhCAT1</i> (2, 3, and 4 are listed as CT2, CT3, and CT4 below). DNA of the pBin438 plasmid and transgenic and WT plants was digested with <i>Bam</i>HI (only one <i>Bam</i>HI restriction site is present within the T-DNA region). The <i>npt II</i> gene-specific probe was PCR-amplified from pBin438 vector. (B) qRT-PCR analysis. Values are given as means ± standard deviation.</p

Increased tolerance of salt stress in transgenic cotton plants.

<p>(A) Phenotypes of WT and transgenic plants 21 d after treatment with 200 mM NaCl. Plants shown were 9 wks old. (B) Photosynthetic <i>Fv/Fm</i> of leaves of transgenic and WT plants 2 d after treatment with 200 mM NaCl. (C and D) Fresh shoot (C) and root (D) biomasses of transgenic and WT plants after treatment with 200 mM NaCl for 4 wks. Means ± standard deviation labeled with different letters are significantly different at the 0.05 level.</p

Enhanced MV-mediated oxidative stress tolerance in leaf discs of transgenic cotton lines.

<p>(A) Visible damage in leaf discs of WT and transgenic plants 36 h after 5 µM MV treatment. (B) Relative electrolyte leakage assay. Means ± standard deviation labeled with different letters are significantly different at the 0.05 level.</p