Effects Of Exogenous Paraquat On Antioxidative Enzymes And DNA Methylation In Salt Stressed Cucumis Sativus

Salt tolerance of plants is of great significance as soil salinization is one of the most important global environmental and ecological problems. Cucumber (Cucumis sativus L.) cv. Chunguang no. 2 was cultivated to study whether exogenous paraquat (PQ) could protect plants from salt 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 salt stress mitigated by PQ on the sides of antioxidant enzymes and DNA methylation. It also could lay the foundations for applying methylation polymorphic genes and exogenous PQ to salt-resisting cultivation of cucumbers and give references to studying the mechanisms of stress mitigated by other substances.At the two-leaf stage, cucumber seedlings were selected and pretreated with 10μM PQ under moderate light (100μmol m-2 s-1) for 1 h. After rinsed 6 times with water and nutrient solution, cucumber plants were kept in darkness for 24 h. Then, the cucumbers were watered with 100 mM salt for 2 d. Samples of the second leaf was used to determine the activities of antioxidants such as superoxide dismutase (SOD, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7), ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6), dehydrateascorbate reducatase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), glutathione reductase (GR, EC 1.6.4.2), ascorbic acid (AsA) and reduced glutathione (GSH). The leaf samples were also used to assay the cntents of malondialdehyde (MDA), hydrogen peroxide (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 of this study were listed as the following:1. Effects of paraquat on antioxidant enzymes in salt-stressed cucumber leavesThe growth of cucumber seedlings was inhibited observably by salt stress. The dry weights of shoot and root were significantly decreased under salt stress, while they were increased in PQ-pretreated stressed plants. So PQ pretreatment alleviated the inhibition of salt stress on the growth of cucumber seedlings. Under salt conditions, PQ pretreatment relieved oxidative stress as observed by the decreases in levels of MDA, H₂O₂ and O₂.-, which correlated with the increase in antioxidant defenses. Compared to the salt treatment, PQ pretreatment increased the activities of antioxidants such as SOD, GPX, CAT, APX, GSH-Px, DHAR, MDHAR, GR, ASA and GSH and also enhanced the ratios of reduced ASA/oxidized ASA and GSH/GSSG. Therefore, we proposed that the PQ pretreatment increased the activities of antioxidant enzymes under conditions of salt and thereby decreased the levels of ROS and improved the salt tolerance of cucumber seedlings.2. Effects of paraquat on DNA methylation in salt stressed cucumber leavesThirty-two pairs of primers were used for MSAP analysis. Total of 1324 fragments were amplified, and the level of global DNA methylation in four treatments (control, PQ pretreatment, salt and PQ+salt treatments) were 19.8%, 19.9%, 21.5% and 20.4%, respectively. The levels of DNA methylation in cucumber plants were obviously changed under drought stress, while PQ pretreatment directionally induced the demethylation. After part of the methylated DNA fragments were cloned and sequenced, six fragments were found to be homologous to the functional protein, indicating that the fragments were involved in the process where PQ improved the salt tolerance of cucumber seedlings.