Brassinosteroids Regulate CO₂ Assimilation Through Redox Changes In Cucumis Sativus

Cucumber (Cucumis sativus) is one of the major corps in horticultural cultivation and has key roles in agricultural production and consumption. The growth and development of cucumber are dependent on the suitable environment conditions. However, the productivity and quality of cucumber are always affected by various stresses due to the simple and crude horticultural facilities. Brassinosteroids (BRs) was applied widely in increasing yields and stress tolerance, however, the mechanism is not very clear. Hence, it is of great importance to study the mechanisms of BRs how to regulate photosynthesis. This study uses cucumber with different brassinosteroid (BRs) levels to study the involvement of redox signal in BRs and H2O2 regulate photosynthesis. Through chemical genetics, physiological and molecular biological experiments, we investigate the effects of redox signal on BRs-regulated photosynthesis. The results are as follows:1. We have examined effects of 24-epibrassinolide (EBR) and H2O2 on gas-exchange, chlorophyll fluorescence characteristics and Rubisco activity. EBR and H2O2 significantly increased the light-saturated net CO2 assimilation rate (Asat). Increased Asat in EBR and H2O2-treated leaves was accompanied by increases in the maximum carboxylation rate of Rubisco (Vc, max) and in the maximum rate of RuBP regeneration (Jmax). EBR and H2O2-treated leaves also had a higher quantum yield of PSII electron transport (ΦpsⅡ) than the controls, which was mainly due to a significant increase in the photochemical quenching (qP). EBR and H2O2 did not influence photorespiration. In addition, significant increases in the initial activity of Rubisco and in the sucrose, soluble sugars, and starch contents were observed followed by substantial increases in sucrose synthase (SS), and acid invertase (AI) activities after EBR or H2O2 treatments. It was concluded that EBR and H2O2 increase the capacity of CO2 assimilation in the Calvin cycle, which was mainly attributed to an increase in the initial activity of Rubisco. 2. We have studied the role of H2O2 signal in BRs regulate carbohydrate metabolism. To determine whether ROS plays a critical role in EBR-regulate carbohydrate, we analyzed the effects of diphenyleneodonium (DPI), an inhibitor of NADPH oxidase and dimethylthiourea (DMTU), an H2O2 scavenger, on EBR-induced changes in carbohydrate and key enzymes. However, pretreatment with DPI or DMTU completely abolished the effects of EBR on carbohydrate content and key enzymes. The results demonstrated that H2O2 plays a critical role in BRs regulate carbohydrate metabolism.3. We have examined the role of H2O2 signal in EBR-induced photosynthesis. To determine whether ROS plays a critical role in EBR-induced photosynthesis, we analyzed the effects of diphenyleneodonium (DPI), an inhibitor of NADPH oxidase and dimethylthiourea (DMTU), an H2O2 scavenger, on EBR-induced photosynthesis and calvin cycle enzymes. However, pretreatment with DPI or DMTU completely abolished the effects of EBR on photosynthesis and Rubisco activase. Immunogold-labeling and Western blotting demonstrated that BRs upregulated the expressions of RCA. The results demonstrated that H2O2 plays a critical role in BRs induced photosynthesis.4. We have examined the involvement of glutathione redox status in BRs-induced changes in photosynthesis. To determine the involvement of altered redox state in EBR-and H2O2-stimulated photosynthesis, we analyzed the effect of GSH, buthionine sulfoximine (BSO) and 6-aminonicotinamide (6-AN) on Asat with or without EBR or H2O2 treatment. In EBR-and H2O2-treated plants, the overall GSH+GSSG contents were not significantly changed but the GSH/GSSG ratios were significantly increased. Pretreatment with BSO or 6-AN blocked EBR-and H2O2-induced increase in the GSH/GSSG ratio mostly by restoring the reduced GSSG contents. However, the enzymes activities of calvin cycle were increased by exogenous GSH application. These results strongly suggested that increased GSH/GSSG ratio in EBR-and H2O2-treated plants is important for increased photosynthesis.5. We have studied the effects of EBR and Brz treatment on the accumulation of GSH and GSSG and the GSH/GSSG ratio of chloroplasts. Neither EBR nor Brz application had significant effects on the total glutathione pool (GSH+GSSG). On the other hand, the GSH/GSSH ratio of chloroplasts was substantially altered by EBR and Brz treatment. Thus, BRs-stimulated CO2 assimilation was associated with a reduced cellular redox state. GSH/GSSG ratio plays an essential role in regulation of CO2 assimilation through regulating Rubisco activase. BRs regulates the GSH/GSSG ratio, expression of chloroplastic gene and changes in Rubsico activation state is associated with amount of Rubisco activase.6. We have studied the effects of EBR on photosynthesis under ambient and elevated CO2 conditions. We found that the effects of EBR on growth mass, photosynthetica and fluorescence parameters or genes expression of Calvin-cycle were more visible under elevated CO2 condition. These results strongly suggested that EBR has effects on photosynthesis both at ambient and elevated CO2 conditions.