Mechanism Of Polyamine-Induced Resistance Against Pseudoperonospora Cubensis In Cucumber

Cucumber （Cucumis sativus L.） is one of the most important vegetables all over the world. It’s always seriously damaged by downy mildew during production with heavy loss of yield. Although agricultural complicated method, ecological method and fungicide can provide some level of disease control, low efficiency and heavy pesticide residue in cucumber remain. In green or organic production in cucumber, induced resistance, a kind of resistance system in plant, plays a key role. However, little information is available in the induced resistance of cucumber. To elucidate the inducer and the mechinasm of induced resistance in cucumber, we used polymine （PA）, universal in organism of plants, as an inducer. After the leaves of cucumber were treated with the inducer and Pseudoperonospora cubensis, the disease index of the susceptible inbred line 112 and the resistant inbred line HNAU-0023 was calculated, furthermore, accumulation of hydrogen peroxide （H2O2）, antioxidase activity and some resistance-related genes expression profiling were investigated. The main results were as follows:1. After the leaves were treated with spermine （Spm）, spermidine （Spd） and putrescine （Put）, Spd inhibited disease development and induced the resistance against P. cubensis remarkably. An inhibitor of Spd biosynthesis, Methylglyoxalbis （MGBG）, Diphenyleneiodonium （DPI）, a potent inhibitor of NADPH oxidase, and Dimethylthiourea （DMTU）, an H2O2 scavenger, reduced the resistance.2. The histochemical staining results suggest that after 12 h treatment, Spd could induce the increase of H2O2 coupled with hypersensitive response （HR）. Wheras, the content of H2O2 were no significant changed after Spm and Put treatment. Moreover, after 12 h treatment with MGBG, the contents of H2O2 reduced. After Spd treated again, the level of H2O2 recovered to the control’s via cytochemistry. In detail, the content of H2O2 reduced by 4.63% compared with the control, and yet reduced by 23.76% compared with the treatment with exogenous H2O2. DPI and DMTU were treated 12 h and reduced H2O2 contents evidently, After Spd treated again, the level of H2O2 recovered to the control.3. Assaying the changes of H₂O₂ contents after Spd and MGBG treatment, Spd induced H2O2 production in a high level during 0 to 96 h （except 48 h）. Meanwhile MGBG prohabited H2O2 production rapidly after 3 h treatment. After 96 h treatment, the content of H2O2 was approximately same as in the treatments with Spd and water. It is conclude, therefore, Spd and MGBG increased or reduced H2O2 accumulation in the treated leaves of cucumber during 0 to 72 h, respectively.H₂O₂ content was lower after 3 to 48 h DPI treatment than Spd treatment. While after treated with Spd again at, H2O2 content increased during 6 to 24 h. The results remained simaliar with the DMTU treatment, suggesting that DPI and DMTU inhibited H2O2 accumulation at a certain timepoints.4. The result of H₂O₂ subcellular localization showed that H2O2 accumulated at least in chloroplast, mitochondria and plasma membrane after Spd induced, compared with no H2O2 accumulation after water and MGBG treatment.5. Ascorbate peroxidase （APX） activity increased steadily after Spd induced, and reached to maximum at 96 h. Superoxide dismutase （SOD） activity decreaed rapidly from 0 to 3 h, and then increased rapidly after 3 h and reached to maximum at 12 h. In overall, the activity of SOD was higher than the control’s. Catalase （CAT） activity was higher than the control at 24 h treatment, but in the other timepoints, the CAT activity was simaliar with the control’s. The results suggested that activity of antioxidant enzymes such as SOD^ CAT and APX were all increased after Spd induced.6. EFla and UBI-ep were as reference genes for the normalization of target gene expression. To investigate the relationshiop between resistance gene expression and the treatments, the expression profiles of some resistace-related genes were monitored by real-time quantitative RT-PCR. As a result, Spd induced the antioxidant genes cAPX、CAT、MDAR, defense genes PAL、Dnaj, transcription factor B1B2、F8R8、 WRKY30、MYB, signal transduction gene RBOH and PA synthesis related gene F12R12 significant up-regulation, DPI surpressed significanttly expression of cAPX, CAT、MDAR、PAL、Dnaj、WRKY30, F12R12, while Spd could reverse the both inhibition.