Strategies of ROS regulation and antioxidant defense during transition from C₃ to C₄ photosynthesis in the genus Flaveria under PEG-induced osmotic stress.

In the present study, we aimed to elucidate how strategies of reactive oxygen species (ROS) regulation and the antioxidant defense system changed during transition from C₃ to C₄ photosynthesis, by using the model genus Flaveria, which contains species belonging to different steps in C₄ evolution. For this reason, four Flaveria species that have different carboxylation mechanisms, Flaveria robusta (C₃), Flaveria anomala (C₃-C₄), Flaveria brownii (C₄-like) and Flaveria bidentis (C₄), were used. Physiological (growth, relative water content (RWC), osmotic potential), and photosynthetical parameters (stomatal conductance (g(s)), assimilation rate (A), electron transport rate (ETR)), antioxidant defense enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductases(GR)) and their isoenzymes, non-enzymatic antioxidant contents (ascorbate, glutathione), NADPH oxidase (NOX) activity, hydrogen peroxide (H₂O₂) content and lipid peroxidation levels (TBARS) were measured comparatively under polyethylene glycol (PEG 6000) induced osmotic stress. Under non-stressed conditions, there was a correlation only between CAT (decreasing), APX and GR (both increasing) and the type of carboxylation pathways through C₃ to C₄ in Flaveria species. However, they responded differently to PEG-induced osmotic stress in regards to antioxidant defense. The greatest increase in H₂O₂ and TBARS content was observed in C₃ F. robusta, while the least substantial increase was detected in C₄-like F. brownii and C₄ F. bidentis, suggesting that oxidative stress is more effectively countered in C₄-like and C₄ species. This was achieved by a better induced enzymatic defense in F. bidentis (increased SOD, CAT, POX, and APX activity) and non-enzymatic antioxidants in F. brownii. As a response to PEG-induced oxidative stress, changes in activities of isoenzymes and also isoenzymatic patterns were observed in all Flaveria species, which might be related to ROS produced in different compartments of cells.