| Mechanical wounding is an environmental stress which stimulates plants to produce the necessary signals to prepare further defenses. Oxidative burst is a common early response of plants to elicitor treatments, which induces the enhancement of plant antioxidative enzyme activity to scavenge the generated active oxide species. Wound-induced responses activate those genes involved in the production of wound-signals which then switch-on genes involved in systemic defense against stress. JA signaling can be induced by a range of abiotic stresses, including mechanical wounding. Phospholipase D (PLD), lipoxygenase (LOX) and allene oxide synthase (AOS) are key enzymes which involved in the JA synthesis.This study was to investigate the effects of salicylic acid, ethephon, or methyl jasmonate on antioxidant system and on the transcription levels of wounded-induced PLDα, LOX, AOS genes in postharvest cucumber (Cucumis sativus L. cv. Biyu-3) fruit.We identified the CsPLD gene with homology cloning. Potential orthologous PLD amino acid sequences were collected based on BLAST-P homology searches using the CsPLD gene sequence in the NCBI database. Multiple sequences alignments were performed with the ClustalX programme. A phylogenetic tree was constructed applying the neighbour-joining (NJ) method. We found that the maxisum open reading frame (ORF) of the CsPLD gene cDNA was 2,427 nt-long and encoded a predicted protein of 808 amino acids with a C2 domain related to the C2 superfamily. Phylogenetic analysis result indicated that CsPLD gene belonged to the a sub-family of PLD genes family.We found that mechanical wounding stimulated the activities of SOD, POD, APX and CAT, activities of POD and APX distinctly increased, however, activities of SOD and CAT both had slightly increase. The activities of all the four antioxidative enzymes were increased to a maxisim value and then declined. SA pre-treated inhibited activities of SOD, POD, CAT and APX in wounded cucumber. Ethephon pre-treated could slightly enhanced activites of POD and APX in several hours after wounded, but the pre-treated had no effect on activity of CAT and intensively inhibited the activity of SOD in wounded fruits. The activities of SOD and POD were slightly inhibited a couple of hours after wounding, but there were nearly no changes on activities of APX and CAT in wounded cucumber.The relative levels of expression of the PLDa gene induced by wounding following SA, ETH, or MeJA pre-treatment were detected by semi-quantitative PCR and quantitative real-time PCR analysis. Wounding increased the levels of expression of the PLDa, LOX and AOS genes. SA pre-treated slightly surpressed the relative levels of expression of PLDa and LOX genes, but strongly surpressed the relative level of expression of AOS gene. Transcript levels of PLDa and LOX genes apparently increased but transcript level of AOS gene had no change in ETh pre-treated cucumber fruits. MeJA treatment activated expression of the PLDa and AOS gene, and the stimulatory effect of ETH was greater than that of MeJA, however, this treatment surpressed the transcript level of LOX gene.Programmed cell death (PCD) is an essential physiological process occurring during plant development and in response to biotic and abiotic stresses such as pathogen, insects feeding, low oxygen concentration, chilling, draought, mechanical wounding, osmosis stress, metallic ion and so on. Our results showed that ethylene production rate, respiration rate and electrolyte leakage distinctly increased in cucumber with 9 days at 2℃chilling treatment, the hardness declined and the color tended to become yellow in cucumber after chilling treatment, DNA degraded and nucleus became deformed; when the chilling fruits were put at 20℃for 2 days, ethylene production rate, respiration rate and electrolyte leakage increased more evidently, firmness declined more quickly and the color became yellowish green; the degradation of DNA in the treatment fruits were remarkable; most of the chromatin condensed and the nucleus disappeared; the cells of chilling treatment fruits presented apparent characteristics of PCD. Ethylene production rate, respiration rate and electrolyte leakage in chilling treatment cucumber fruits with 1-MCP and EGTA pre-treated for 24 h increased slower than those in chilling treatment, the firmness declined also slowly and the color almost green in pre-treated fruits; DNA of the pre-treated fruits had a little bit degradation and the number of cell shrinkage and nucleus deformation distinctly reduced than that in chilling fruits; those indicated that 1-MCP and EGTA pre-treated could relieve the occurrence of PCD in chilling treatment cucumber fruits and the effects of 1-MCP pre-treatment was better than EGTA pre-treatment. |
