Drought Stress and High Temperature Affect the Antioxidant Metabolism of Cotton (<i>Gossypium hirsutum</i> L.) Anthers and Reduce Pollen Fertility

Both drought and high temperature can influence the antioxidant metabolism of crop reproductive organs in different ways, affecting the fertility of reproductive organs and yield formation. However, the combined effects of drought stress and high temperature on the crop reproductive physiology have not yet been widely considered. In order to broaden our understanding of this mechanism of influence, a pond experiment was conducted using a cotton variety Yuzaomian 9110 divided into four treatment groups: control (CK), drought stress (DS), high temperature (HT), and drought stress coupled with high temperature (DS+HT). Results showed a significant negative correlation between pollen viability and superoxide anion (O₂⁻) content, as well as hydrogen peroxide (H₂O₂). Compared with CK, DS did not alter O₂⁻ content in anthers, but HT treatment resulted in higher anther O₂⁻. Compared with single-stress groups, DS+HT further promoted the formation of O₂⁻ in anthers, leading to more malondialdehyde in anthers. Moreover, a higher H₂O₂ content in anthers was found in DS and HT than in CK. DS+HT did not show altered H₂O₂ content relative to HT treatment, although its H₂O₂ was higher than in DS. Further analyses of the antioxidant enzyme system showed that DS had no significant effect on superoxide dismutase gene (<i>GhCu/ZnSOD</i>) expression, but HT and DS+HT significantly downregulated its expression. The expression of <i>GhCu/ZnSOD</i> was lower under DS+HT than HT, which might be why O₂⁻ content was not altered under DS treatment compared with CK and was higher in DS+HT than HT. DS and HT significantly downregulated the expression of the peroxidase gene (<i>GhPOD</i>) and catalase gene (<i>GhCAT</i>), which should be the main reason for the larger accumulation of H₂O₂ under drought stress and high-temperature conditions. Compared with single-stress groups, DS+HT had lower expression of <i>GhCAT,</i> resulting in a larger H₂O₂ content. Regarding the ascorbic acid–glutathione (AsA–GSH) cycle, DS and HT significantly downregulated the expression of monodehydroascorbate reductase gene (<i>GhMDHAR</i>) to hinder the production of AsA and upregulated the expression of ascorbate oxidase gene (<i>GhAAO</i>) to promote the oxidation of AsA, which was theoretically detrimental to AsA accumulation. However, HT downregulated the expression of the ascorbate peroxidase gene (<i>GhAPX</i>), hindering the reduction of H₂O₂ by AsA, which was the reason for AsA and H₂O₂ accumulation. Moreover, DS also significantly upregulated the expression of the dehydroascorbate reductase gene (<i>GhDHAR2</i>) to enhance the reduction of dehydroascorbate to form AsA, leading to a higher content of AsA under DS than HT. The combined stress significantly downregulated the expression of <i>GhAAO</i> to inhibit the oxidation of AsA but significantly upregulated the expression of <i>GhMDHAR</i> and <i>GhDHAR2</i>, promoting the AsA production, and downregulated the expression of <i>GhAPX</i>, hindering the reduction of H₂O₂ by AsA. All these resulted in increased AsA content under combined stresses. In addition, HT significantly downregulated the glutathione reductase gene (<i>GhGR</i>) expression, hindering the reduction of oxidized glutathione (GSSG), which led to the reduction of GSH. However, DS and DS+HT significantly downregulated the glutathione peroxidase gene (<i>GhGPX</i>) expression, resulting in the accumulation of GSH. Overall, compared with single-stress treatments, the effects of DS+HT on cotton pollen fertility and peroxide accumulation were more significant. The effects of DS+HT on the antioxidant enzyme system were mainly caused by high temperature, while the mechanism of abnormal accumulation of AsA and GSH caused by DS+HT was different from those of single-stress groups.