Exogenous Cinnamic Acid On DNA Methylation In Leaves Of Two Cucumber Cultivars Under Drought Stress

Cucumber (Cucumis sativus L.) and some other crops have shallow root systemsand large leaf areas and therefore are sensitive to inadequate water supply. So,drought tolerance of cucumber is of great significance.We elucidate the mechanism ofdrought stress mitigated by exogenous CA. We investigated the exogenous CA effecton antioxidant enzymes activities and the changes of DNA methylation of droughtstressed cucumber. We also aimed to clone the methylation polymorphic fragmentsand analyze their sequence by using bioinformatics. Our primary work might help toelucidate the mechanisms of drought stress mitigated by exogenous CA. It also couldlay the foundations for exogenous CA-resisting cultivation of cucumbers and givereferences to studying the mechanisms of stress mitigated by other substances.Cucumber (Cucumis sativus L) of North China ecotype cv. Yuexiu no.3and cv.Zhongnong no.12were cultivated to study. Samples were used for performingtransmission electron microscopy, determining the activities of ROS, antioxidantsproline, soluble sugar, CA and VA, studying the methylation changes of level andstatus. DNA methylation was surveyed by using the methylation-sensitive amplifiedpolymorphism (MSAP) method. Then, DNA methylation polymorphic fragmentswere isolated, cloned and sequenced. The main results were showed as the following:1. Effects of exogenous CA on the growth parameters, ultrastructure, antioxidantenzymes and other related indexes of cucumber seedlings under drought stressIn the CA pretreatment experiment, drought significantly decreased the growthparameters. However, the growth parameters were significantly elevated in theCA+PEG treatment. In the different concentrations of CA, the best growth parameterswere50μM. The growth of cucumber seedlings was inhibited observably by droughtstress. CA pretreatment alleviated the inhibition of drought stress on the growth ofcucumber seedlings.Under drought stress, the chloroplasts of Yuexiu no.3were abnormal and swollen and had the disorganized non-parallel lamellae. CA pretreatment protectschloroplast ultrastructure from drought stress.In the CA pretreatment experiment, drought increased the levels of MDA andendogenous H2O2. The combination of CA pretreatment and drought stress increasedthe activities of antioxidant enzymes such as SOD, CAT, GPX, GSH-Px, APX,DHAR, MDHAR and GR,and antioxidant contents of AsA, GSH, proline, solublesuger, and thereby decreased the level of MDA and endogenous H2O2. We proposethat exogenous CA increases antioxidant enzymes activities in cucumber leaves andthereby decreases the level of ROS, thus protecting the ultrastructure of chloroplastsfrom drought stress.2. Effects of exogenous CA on DNA methylation of cucumber leaves under heatstressDNA of cucumber leaves were used for MSAP analysis. According to the resultsof MSAP, the levels of DNA methylation in two breeds of cucumber plants were14%~19%. In the CA pretreatment experiment, sequences of16MSAP loci MSAP-1,MSAP-2, MSAP-3, MSAP-9, MSAP-10, MSAP-11, MSAP-12, MSAP-14,MSAP-15, MSAP-18, MSAP-19, MSAP-20, MSAP-21, MSAP-22and MSAP-27inthe study showed were located in the chromosome of cucumber, sequences of13MSAP loci MSAP-1, MSAP-2, MSAP-6, MSAP-7, MSAP-8, MSAP-11, MSAP-15,MSAP-16, MSAP-17, MSAP-22, MSAP-23, MSAP-26å'ŒMSAP-27in the studyshowed the significant similarities to cucunber genomic coding region sequences.When comparing the drought treatment with control and CA+PEG treatment withdrought treatment the level of methylation was higher than demethylation in Yuexiuno.3and the level of demethylation was higher than methylation in Zhongnong no.12.