Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

Abstract

Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide and hydroxyl radicals are generated through normal biochemical processes, but their production is increased by abiotic stresses. The prospects for enhancing ROS scavenging, and hence stress tolerance, by direct gene expression in a vulnerable cell compartment, the chloroplast, have been explored in tobacco. Several plastid transformants were generated which contained either a Nicotiana mitochondrial superoxide dismutase (MnSOD) or an Escherichia coli glutathione reductase (gor) gene. MnSOD lines had a three-fold increase in MnSOD activity, but interestingly a five to nine-fold increase in total chloroplast SOD activity. Gor transgenic lines had up to 6 times higher GR activity and up to 8 times total glutathione levels compared to wild type tobacco. Photosynthetic capacity of transplastomic plants, as measured by chlorophyll content and variable fluorescence of PSII was equivalent to non-transformed plants.The response of these transplastomic lines to several applied stresses was examined. In a number of cases improved stress tolerance was observed. Examples include enhanced methyl viologen (Paraquat)-induced oxidative stress tolerance in Mn-superoxidase dismutase overexpressing plants, improved heavy metal tolerance in glutathione reductase expressing lines, and improved tolerance to UV-B radiation in both sets of plants.

Impact of Enhanced Capacity to Scavenge Reactive Oxygen Species on Cold Tolerance of Tobacco

“…These combinations were of interest since DHAR and GR catalyze consecutive steps in the ascorbate-glutathione cycle in chloroplasts, and combining expression of GR and GST together could significantly influence glutathione homeostasis, further affecting the ROS-scavenging capacity of the chloroplast. As has already been described in detail (Le Poage et al 2011), Southern blot analysis confirmed integration of the foreign genes, and Western blot analysis confirmed expression of the relevant transgenes in leaf tissue. The transplastomic lines showed the predicted altered activity of the relevant enzymes (threefold increase in SOD activity, up to threefold increase in DHAR activity, up to sixfold increase in GR activity, twofold increase in both GST and GPX as a result of insertion of the GST gene, up to fourfold increase in DHAR/GST, and fivefold increase in GR in the double transformants) and of the redox state of ascorbate and glutathione.…”

“…This laboratory has introduced genes encoding manganese SOD (MnSOD), DHAR, GR, and GST (with GPX activity) into the tobacco chloroplast genome (plastome) to obtain transplastomic lines, as has been previously described Poage et al 2011). Double transplastomic lines expressing DHAR and GR (DHARϩGR) or GR and GST (GRϩGST) were also obtained (Le .…”

“…Several studies on different enzymes have found that overexpression of only some of those enzymes enhanced performance under a particular stress (Foyer et al 1995;Payton et al 2001;Faize et al 2011;Le Martret et al 2011;Poage et al 2011). Photosynthetic processes are often the first physiological/metabolic processes to be inhibited by chilling temperature (Berry and Bjö rkman 1980).…”

“…Introduction of GR, DHAR, GST, DHARϩGR, and GRϩGST genes into tobacco led to the production of several lines from each transformation Poage et al 2011). In transplastomic plants, targeted integration should result in different lines responding in a similar manner, because targeted integration eliminates the position effects associated with nuclear transformants.…”

Elevated ROS-scavenging enzymes contribute to acclimation to UV-B exposure in transplastomic tobacco plants, reducing the role of plastid peroxidases

“…integration and expression of transgenes into the chloroplast genome) can directly increase the activity of several ROS scavenging enzymes in this organelle, resulting in both altered levels and redox states of metabolites (ascorbate and glutathione) and modified response to abiotic stresses. Poage et al (2011), also showed a diminished impact of UV-B radiation on relative variable fluorescence in transformants expressing either superoxide dismutase (SOD) or glutathione reductase (GR) in their chloroplasts. However, the most pronounced effects of abiotic stress (chilling, salt and methyl viologen-induced oxidative stress) were observed in double transformants ) expressing either dehydroascorbate reductase (DHAR) and glutathione reductase (GR), or gluathione reductase and glutathione-S-transferase (GST).…”