Oxidative stress responses in transgenic cotton that overexpresses superoxide dismutase



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Texas Tech University


Transgenic cotton plants that express a chimeric gene encoding chloroplast-localized Mn superoxide dismutase (Chi MnSOD) from tobacco have been analyzed to determine whether they exhibit improved resistance to oxidative stress. The Chi MnSOD activity for the transgenic plants was several fold greater than that for wild-type plants as deduced from non-denaturing PAGE, and the total SOD activity was three-fold greater according to spectrophotometric enzyme assays. Also, transgenic plants exhibited ~30% greater ascorbate peroxidase activity and ~20% greater glutathione reductase activity. The immediate recovery of photosynthesis for attached leaves for the Chi MnSOD transgenic line K 10 exposed to 4°C and 1700 µmol/m^2/s was slightly better than that for wild-type leaves, especially after a 35 min exposure (significant to 0.05%). However, the recovery of photosynthesis for longer exposure times was comparable for Chi MnSOD transgenic and wild-type plants, including exposures of leaf discs in the lab to 1200 µmol/m^2/s and 7°C for 1 to 3 h. Resistance to light-mediated, paraquat-induced cellular damage was also slightly better for transgenic plants, but primarily at 0.3 µM paraquat. The oxidized/reduced ascorbate in unstressed, transgenic plants was similar to that in wild-type plants exposed for 1 h to high light and 8°C. While this stress nearly doubled the total glutathione and the percentage oxidized in wild-type plants, there was no effect of stress on these parameters in Chi MnSOD plants. However, their pre-stress levels were similar to those for stressed wild-type leaves. The activation state of stromal fructose 1,6-bisphosphatase (FBPase) was used as an indicator of the oxidative state of the chloroplast. The enzyme in wild-type and transgenic plants deactivated similarly following severe chilling stress (4°C and 1700 µmol/m^2/s). However, under milder stress conditions (10°C and 1200 |jmol/m^/s for 10 min), the activation state of FBPase in MnSOD plants was unaffected while it was significantly decreased in wild-type C-312 plants. It appears that the increase in MnSOD activity in chloroplasts is enough to provide some resistance to oxidative stress caused by chilling in high light. Furthermore, the pool sizes of ascorbate and glutathione may be factors restricting the oxidative stress defense response during severe chilling/high light stress of cotton plants that over-express MnSOD in their chloroplasts by limiting the ability of the Halliwell-Asada enzymes to compensate for the increased activity of SOD. This research was supported by USDA Plant Response to the Environment and the Texas Advanced Technology Program.