Chilling Tolerance Induced By MeJA And NO In Cool-stored Cucumber In Relation To Antioxidant Defence System

Cucumber（Cucumis sativus L.）is a chilling sensitive subtroptic vegetable,which is susceptible to chilling injury when stored in the suboptimal low temperature,thus affecting the shelf life and quality of cucumber.Previous studies have shown that MeJAand NO can reduce the occurrence of chilling injury,but the role of the two substances inducing chilling resistance and the relationship with each other remains unclear.This paper addressed the effects of MeJA and NO on antioxidant defense system relation to chilling tolerance,analyzed the function of CsCAT3 gene by means of Ectopic expression,the main results were as follows:1.Among the different temperatures cucumbers stored in 10℃was suitable for cucumber storage,5℃caused chilling injury.The longer time cucumber was stored in 5℃,the shorter shelf-life after transferring to 20℃.Solutions of MeJA and SNP at 0.1,1 or 10μmol·L^-1were applied before the cucumber was stored in 5℃,the results showed that1μmol·L^-1 of SNP,10μmol·L^-1 of MeJA showed the best effect in chilling-resistance induction.2.MeJA and SNP treatments reduced the accumulation of MDA,slowed down the rate of O₂^·-production,decreased the content of H₂O₂,enhanced the expression of CsCAT1,CsCAT3 genes and CsCAT3 protein,and increased CAT,SOD,PPO and PAL activities,suggesting that that MeJA and SNP activate the antioxidant defense system of cool-stored cucumber,reduced accumulation of redicals,and maintained the integrity of cell membrane.3.The results showed that chilling resistance induced by DPI plus MeJA or SNP treatment was not significantly different from MeJA and SNP treatment,suggesting that MeJA or NO did not require H₂O₂ for chilling tolerance induction.Chilling resistance induced by DPI was equal to that induced by MeJA and SNP treatment,suggesting that inhibiting endogenous H₂O₂ could enchence chilling tolerance.4.The results showed that chilling tolerance induced by MeJA plus cPTIO treatment was weaker than MeJA,and SNP plus SHAM treatment was not significant different from SNP,suggesting that chilling resistance induced by MeJA was dependent on NO,but that induced by NO was not dependent on MeJA.It could be supposed that in induction signal transduction pathway mediating chilling-resistance,MeJA is upstream of NO.5.MeJA plus cPTIO treatment showed more O₂^·-production and H₂O₂ content than MeJA treatment,while SNP plus SHAM resulted in the similar or less O₂^·-and H₂O₂ relative to SNP treatment.c PTIO plus SHAM treatment showed higher O₂^·-production and H₂O₂content than MeJA and SNP treatment.These results suggest that MeJA is dependent on NO in inhibiting generation of O₂^·-and H₂O₂,that NO plays a major role in inhibiting production of O₂^·-and H₂O₂.MeJA plus cPTIO treatment displayed lower activities of CAT,SOD,PPO and PAL relative to MeJA treatment,while SNP plus SHAM treatment showed higher enzymes activities.In addition,cPTIO plus SHAM treatment resulted in lower activities of these four enzymes than MeJA and SNP treatment.These results suggested that MeJA required the involvement of NO in enhancing activities of the four enzymes,and NO as an endogenous signaling molecular,was necessary for increasing activities of these enzymes.Compared with control,MeJA and SNP treatment did not increase POD activity steadily.Meanwhile,MeJA plus cPTIO treatment did not result in obvious difference from MeJA treatment,while SNP plus SHAM treatment was largerly higher than SNP treatment.Furthermore,cPTIO plus SHAM treatment was lower than all the other treatments.This indicated that MeJA and SNP treatment did not show obvious influence on POD activity,and therefor,the level of activities of POD was not colsely related to MeJA and SNP induced cold resistance in cucumber.6.The analysis of the gene expression profile of cucumber tissue sampled 24 h following treatment with MeJA and SNP by transcriptome sequencing showed,4627 new transcripts among the 23248 genes identified.Of the 425 differencial expression genes induced by MeJA,131 were up-regulated and 294 down-regulated.Among the 57genes involved in oxidordeuctases,31were up-regulated,and of the 7 genes involved in response to oxidative stress,all were up-regulated.Of the 330 differencial expression genes induced by SNP,133were up-regulated and 197 down-regulated.Among the 43genes involved in oxidordeuctase activity,28 were up-regulated,and among the 6 genes involved in response to oxidative stress,all were up-regulated.This indicated that the antioxidant defense system plays an important role in MeJA and NO induced chilling resistance.7.CsCAT3 overexpression vector was constructed and introduced into Arabidopsis and stable transgenic plants were obtained.Compared with the wild-type Arabidopsis plants,under the condiction of low temperature,the transgenic plants over-expressing CsCAT3showed lower relative electric conductivity,less accumulation of O₂^·-and H₂O₂,and less number of dead cells,suggesting that the transgenic plant had better cell membrane integrity and stronger chilling tolerance.Simultaneously,higher activity of CAT and enhanced expression of AtCAT1,AtCAT2,AtCAT3 gene were observed in transgenic plant.These results further demonstrated upregulated the expression of CsCAT3 and higher activity of CAT could enhance cold resistance of cucumber.In conclusion,the present study showed that the antioxidant defense system activated by MeJA and NO of cool-stored cucumber is closely related to the enhanced chilling tolerance.In addition,in the signaling pathway mediated by MeJA and NO,MeJA is located upstream of NO,NO is the necessary endogenous signal substance,and MeJA is dependent on NO.