Effects Of Exogenous Hydrogen Peroxide On Antioxidant Enzymes And DNA Methylation In Leaves Of Two Cucumber Ecotypes Under Drought Stress

Cucumber (Cucumis sativus L) cv. Jinchun no. 4 (a North China ecotype) and cv. Lvfeng no. 6 (a South China ecotype) were cultivated to study whether exogenous hydrogen peroxide (H₂O₂) could protect plants from drought stress and whether the protection was associated with the increasing of antioxidant enzymes activities and the changes of DNA methylation. We also aimed to clone the methylation polymorphic fragments and analyze their sequence by using bioinformatics. Our work might help to elucidate the mechanisms of heat stress mitigated by PQ on the sides of antioxidant enzymes and DNA methylation. It also could lay the foundations for applying methylation polymorphic fragments and exogenous H₂O₂ to drought-resisting cultivation of cucumbers and give references to studying the mechanisms of stress mitigated by other substances.When the second leaves were fully expanded, seedlings were selected and sprayed with 1.5 mM H₂O₂ for 8 h. And then, they were exposed to drought stress simulated by 10% polyethylene glycol (PEG) for 60 h. Samples of the second leaf were used for electron microscopy studies or were ground with liquid nitrogen to determine the activities of antioxidants such as superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), catalase (CAT), dehydrateascorbate reducatase (DHAR), monodehydroascorbate reductase (MDAR), glutathione reductase (GR), ascorbic acid (AsA) and reduced glutathione (GSH). The leaf samples were also used to assay the cntents of malondialdehyde (MDA), H₂O₂ and superoxide anion (O₂^-). After extracting genomic DNA from the second leaves, DNA methylation was surveyed by using the methylation-sensitive amplified polymorphism (MSAP) method. Then, DNA methylation polymorphic fragments were isolated, cloned and sequenced. Subsequently, Homology search and sequence analysis were performed at the public database NCBI.The main results were showed as the following:1. Effect of exogenous H₂O₂ on antioxidative enzymes of cucumber leaves under drought stress mimicked with PEGUnder drought stress induced by PEG, most of the second leaves from two cucumber ecotypes seedlings were withered. When H₂O₂ pretreatment was combined with the drought stress, cucumber leaves was normal. Under drought stress induced by PEG, the chloroplasts and mitochondria of two cucumber ecotypes were abnormal, respectively. The stress condition disintegrated the membranes of most chloroplasts and mitochondria in the leaf cells of both cucumber ecotypes, and it also increased the levels of MDA, endogenous H₂O₂ and O₂^-. The combination of H₂O₂ pretreatment and drought stress reduced the rates of abnormal chloroplasts and mitochondria occurred in the two cucumber ecotypes. It also resulted in most of the investigated membranes to be well organized, and led to the lower levels of MDA, endogenous H₂O₂ and O₂^- in cucumber leaves. Drought stress and exogenous H₂O₂ both increased the activities of antioxidants such as SOD, GPX, CAT, APX, GR, MDHAR, DHAR, AsA and GSH. But the combined effect of drought stress and exogenous H₂O₂ resulted in the highest antioxidant activities in both cucumber ecotypes. We propose that exogenous H₂O₂ increases antioxidant activity in cucumber leaves and thereby decreases lipid peroxidation, thus protecting the ultrastructure of chloroplasts and mitochondria under drought stress.₂. Effect of exogenous H₂O₂ on DNA methylation of cucumber leaves under drought stress mimicked with PEGAccording to the results of MSAP, the levels of DNA methylation in cucumber plants were obviously changed under drought stress. H₂O₂ pretreatment directionally induced the demethylation. The diversity of DNA methylation was correlated with the changes in leaf morphology, ultrastructure and antioxidant enzyme activities under drought stress in cucumber seedlings. The analyzing results of methylation polymorphism sequences showed that DNA methylation induced by drought stress and H₂O₂ pretreatment were mainly distributed in the area of coding regions, indicating that cucumber could adjust the state of DNA methylation in the scope of genome to respond to stress environments and H₂O₂ pretreatment.