Physiological And Molecular Mechanisms Of Brassinosteroids-regulated Photosynthesis, Stress Tolerance And Pesticide Metabolism In Cucumis Sativus

Cucumber (Cucumis sativus) is one of the major crops in greenhouse cultivation and plays important roles in agricultural production and consumption. However, the productivity and quality of cucumber are always negatively affected by chill, high temperature, salinity and pest invasion. Meanwhile, the excessive use of pesticide results in pollution to agricultural produces and has potential harmful effects on human health. For the development of sustainable agriculture, it is of great importance to study the mechanisms of plant response to environmental stresses and explore an effective strategy to regulate the tolerance, productivity, and quality of vegetable crops. This study uses cucumber with different brassinosteroid (BR) levels to study the involvement of BR in plant responses to chill, oxidative stress and CMV infection. By analyzing photosynthesis, hormone metabolism, ROS signal transduction and gene expression, we explicated the mechanisms of BR-regulated stress tolerance. We have also investigated the involvement of BR in plant response to pesticide and the pesticide degradation processes. The following are the major results:1. BR increased CO2 assimilation rate and chloroplastic electron transport rate by regulating the carboxylation velocity of Rubisco (Vc,max), initial activity of Rubisco and RuBP regeneration rate (Jmax). BR upregulated whilst Brz downregulated the expressions of rbcL, rbcS, rca and other Calvin cycle genes. The accumulation patterns of Rubisco activase (RCA) based on immunogold-labeling experiments suggested a role of RCA in BR-regulated activation state of Rubisco. Therefore, BR promotes photosynthesis and growth by positively regulating synthesis and activation of a variety of photosynthetic enzymes in cucumber.2. Exogenous application of BR significantly increased while inhibition of BR biosynthesis by Brz decreased the tolerance to photo-oxidative and cold stresses and resistance to cucumber mosaic virus (CMV). BR-induced tolerance to chill and oxidative stress was associated with enhanced antioxidant capacity whereas BR-induced resistance to CMV was not related to the SA-dependent pathway. BR treatment induced expression of both regulatory genes such as RBOH, MAPK1 and MAPK3, the transcription factors such as WRKY, MYB and MYC and genes involved in defense and antioxidant responses. However, Brz also induced expression of transcription factors to some extent. The results demonstrated that BR is directly involved in the regulation of plant stress responses.3. Exogenous BR induced H2O2 in the apoplast of mesophyll cells, whereas inhibition of BR biosynthesis by Brz reduced the H2O2 content of cucumber leave. Changes in activity of NADPH oxidase with different BR levels were consistent with that of H2O2 content, In addition, BR-induced ROS accumulation was blocked by DPI, an inhibitor of NADPH oxidase. Thus, BR induced ROS mainly through activation of NADPH oxidase. DPI and DMTU, a H2O2 scavenger, prevented the induction of gene expression, antioxidant enzyme and stress tolerance by BR. Furthermore, BR induced a periodic change of ROS and the changes of defense gene expression and stress tolerance paralleled with that of ROS levels. The results demonstrated that ROS plays a critical role in BR-induced stress tolerance.4. Local BR treatment induced systemic resistance to oxidative stress in distal untreated leaves. Induction of systemic resistance was associated with upregulation of antioxidant genes such as cAPX and MDAR. BR induced H2O2 accumulation in the vascular tissue. However, DPI and DMTU treatment in the primary or systemic leaves inhibited the systemic induction of ROS, genes expression and stress tolerance by BR. Therefore, H2O2 may mediate the movement of systemic signal and play an important role in BR-induced systemic resistance.5. Treatment of nine pesticides with different chemical structures inhibited net photosynthetic rate (Pn) by various degrees. Flusilazol had no effects on Pn, whereas paraquat (PQ) showed the most significant inhibitory effects on Pn. The effects of cuproxat and PQ were attributed to stomatal closure and damages of PSII, respectively, while the effects of fluazifop-p-butyl and chlorpyrifos were attributed to inhibition of chloroplastic electron transport. However,ΦpsII was not significantly affected by haloxyfop, cyazofamid, imidacloprid, and abamectin. Interestedly, inhibitions of Pn were alleviated by exogenous BR treatment. This effect was likely due to the enhanced CO2 assimilation and antioxidant capacity.6. The protective effects of BR against pesticides-induced toxicity were associated with reductions of pesticide residues. BR can significantly increase the activity of glutathione-S-transferase (GST), peroxidase (POD) and glutathione reductase (GR) and upregulate the expression of P450 and MRP which are involved in pesticide oxidation and transport, respectively. However, the expression of GST was lower in BR-treated plants. The stimulatory effects of BR on pesticide degradation were observed for a wide spectrum of pesticide. It can reduce the residue of cypermethrin, chlorothalonil and carbendazim by 35%,50%and 34%, respectively. Therefore, BR may be suitable for wide application in degradation of pesticides and improvement of food safety.