The Role Of Hydrogen Peroxide In Induced Defense Response Of Populus Simonii×P.pyramidalis Cv. 'Opera 8277' Cuttings

Three treaments including Clostera anachoreta feeding, mechanical wounding and exogenous inhibitor on Populus simonii×P. pyramidalis cv.'Opera 8277'cuttings were used. Differential expressions of defense genes were investigated in poplar leaves after herbivore feeding; hydrogen peroxide (H2O2) level and the activity of antioxidant enzymes was observed in the treated leaves and the leaves above and below the treated leaves after treatment. The mechanism of plant defense response induced by wounding in poplar cuttings was discussed. More attentions on the correlation of H2O2 as signal molecule and other signal molecule in signal transduction process. The study had more important meanings on the mechanism of the induced resistance in woody plant and improved integrated pathogen-pest management in forestry. The main research results were as follows:1. Affymetrix genechip microarray technique was used to analyze gene expression profiles in response to insect feeding. The gene involved in oxidative burst including NADPH oxidase, peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD); the gene involved in signal transduction including lipoxgenase (LOX), hydroperoxide lyase (HPL), phenylalanine ammonia-lyase (PAL) were all up regulated. However, the ethylene-related genes including 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) and S-adenosylmethionine-dependent methyltransferase were significantly depressed.2. Both mechanical wounding and insect feeding treatments could induce the accumulation of H2O2 in the treated leaves of poplar. The increasing of H2O2 was also measured in the leaves above and below the treated leaves in the same plant. DAB staining of H2O2 accumulated in the damaged and undamaged leaves of poplar. H2O2 was mainly localized in the damaged zone, and then spread throughout the veins and tissues. The results showed that H2O2 could be systemically induced by mechanical wounding and insect feeding treatments.3. The accumulation of O2-. and H2O2 in the treated leaves and the leaves above and below the treated leaves of poplar was induced by mechanical wounding and insect feeding. Accordingly, malondialdehyde (MDA) also enhanced. The results showed that both treatments could induce membrane lipid peroxidation to a certain extent in poplar leaves. At the same time, the activity of antioxidant enzymes (SOD, CAT, APX and POD) and defense enzymes (PAL, PPO) was also significantly increased in different leaves of poplar. The results suggested that H2O2 acted as a signal molecule to involve the defense response which is induced by wounding in poplar, thus the membrane lipid peroxidation decreased and the oxidative damage also decreased, and maintained the dynamic balance between reactive oxygen species (ROS) and antioxidant enzymes. We used diphenylene iodonium (DPI) and sodium azide (NaN3) to pretreat the poplar leaves, the results showed two inhibitors could significantly inhibit O2-. and H2O2 level and antioxidant enzymes activity induced by mechanical wounding. DPI showed stronger inhibited effect on ROS and antioxidant enzymes (except POD) than NaN3.Thus, the plasma membrane NADPH oxidase perhaps was the critical enzyme leading to H2O2 accumulation in poplar defense response.4. When poplar cuttings were treated by SA, the level of O2-. and H2O2 was increased in damaged leaves, and SA could inhibited the activity of antioxidant enzymes (SOD, CAT, APX, POD). Therefore, SA might be induced the accumulation of H2O2 by suppressed the activity of CAT in poplar defense response. H2O2 used as signal molecule to pass wound information and induce poplar defense response, which indicated that H2O2 may play a role in the downstream of SA.5. Ca2+ signal played a key role in signal transduction of plant cell. The Ca2+ chelator EGTA and Ca2+ channel inhibitor LaCl3 suppressed the accumulation of O2-. and H2O2 in damaged leaves, in the meantime the activity of SOD, APX, CAT and POD was obviously decreased. It indicated that Ca2+ might be involved in the accumulation of O2-. and H2O2 and the change of antioxidant enzymes activity induced by mechanical wounding. The results suggested that Ca2+ signal may play a role in the upstream of the active oxygen burst. Compared to LaCl3, EGTA showed stronger inhibited effect. As indicated above, the differential expressions of defense genes in poplar leaves induced by insect feeding. H2O2 could be systemically induced and acted as signal molecule involve in the defense response of poplar to wounding. At the same time, there may be a complicated interaction among the three signal molecules H2O2, SA and Ca2+. The interaction played important function in the defense response of poplar.