Effects Of Exogenous Cinnamic Acid On DNA Methylation In Leaves And Roots Of Cucumis Sativus Under Heat Stress

Heat is a familiar intimidatory factor during the growth of plants, so it is of greatsignificance to investigate the heat tolerance of plants. In this study, Cucumber (Cucumissativus L) cv. Jinchun no.4(a common variety in North China) were cultivated to studywhether exogenous cinnamic acid (CA) could protect plants from heat stress and whether theprotection was associated with the increasing of antioxidant enzymes activities and thechanges of DNA methylation. Then we cloned the methylation polymorphic fragments andanalyzed their sequences by using bioinformatics. Our work might help to elucidate themechanisms of heat stress mitigated by CA on the sides of antioxidant enzymes and DNAmethylation. It also could lay the foundations for applying methylation polymorphic genesand give references to studying the mechanisms of stress mitigated by other substances.At the two-leaf stage, cucumber seedlings were selected and pretreated with50μM CA atnormal temperature (25/18°C) for2d. Then after rinsed6times with water and nutrientsolution, the cucumbers were exposed to heat for3d. Samples of the second leaf was used todetermine the activities and contents of the antioxidants. After extracting genomic DNA fromthe second leaves, DNA methylation was surveyed by using the methylation-sensitiveamplified polymorphism (MSAP) method. Then DNA methylation polymorphic fragmentswere isolated, cloned and sequenced. Subsequently, homology search and sequence analysiswere performed in the Cucurbit Genomics Database. The main results of this study were listedas the following:(1) Effects exogenous of CA on antioxidant enzymes in heat-stressed cucumber leavesThe growth of cucumber seedlings was observably inhibited by heat stress. The freshweights of shoot and root, the fresh and dry weights of the second leaves were allsignificantly decreased under heat stress, while all the weights above were increased inheat-pretreated plants. So CA pretreatment alleviated the inhibition of heat stress on thegrowth of cucumber seedlings. Under heat conditions, CA pretreatment relieved oxidativestress as observed by the decreases in levels of MDA, H2O2and O2.-, which correlated withthe increase in antioxidant defenses. Compared to the heat treatment, CA pretreatmentincreased the activities of antioxidants such as SOD, GPX, CAT, APX, GSH-Px, DHAR,MDHAR, GR, ASA and GSH, increased the contents of praline, soluble sugars, vanillic acid and the level of relative water content, and also enhanced the ratios of reduced ASA/oxidizedASA and GSH/GSSG. Therefore, we proposed that the CA pretreatment increased theactivities of antioxidant enzymes under conditions of heat and thereby decreased the levels ofROS and improved the heat tolerance of cucumber seedlings.(2) Effects of exogenous CA on DNA methylation of cucumber leaves and roots under heatstressAccording to the results of MSAP, the levels of DNA methylation in cucumber plantswere obviously changed under heat stress. CA pretreatment directionally induced thedemethylation. The diversity of DNA methylation was correlated with the changes in leafmorphology and antioxidant enzyme activities under heat stress in cucumber seedlings. Theanalyzing results of methylation polymorphism sequences showed that DNA methylationinduced by heat stress and CA pretreatment were mainly distributed in the area of codingregions, indicating that cucumber could adjust the state of DNA methylation in the scope ofgenome to respond to stress environments and CA pretreatment.