Fundamental Studies On The Physiological And Biochemical Mechanism For Inducing Seedlings Resistance In Populus Simonii×Populus Pyramidalis 'Opera 8277'

In response to herbivore wounding, plants mount a range of signal transduction leading to activation of direct and indirect defense response. Populus simonii×Populus pyramidalis 'Opera 8277' seedlings were used in this study. Three treatments including mechanical damage, Clostera anachoreta larvae wounding and exposure to volatiles were conducted on the seedlings. Thermo-desorption cold trap transfer-gas chromatography/mass spectrometry (TCT-GC-MS) technique, high pressure liquid chromatography (HPLC), affymetrix genechip microarray technique, histochemical localization of hydrogen peroxide (H2O2) and measurement of enzyme activity were applied to investigate (1) the differential expressions of defense genes induced by herbivore wounding; (2) the oxidative burst after herbivore wounding; (3) the function and interrelation of H₂O₂, jasmonic acid (JA) and abscisic acid(ABA) in defense response; (4) the emissions of aldehydes induced by mechanical damage; (5) the effective airborne signal molecules between poplar plants; (6) the action mode of volatile signal molecules. In order to reveal the relationship between emissions of short-chain aldedydes and photosynthesis furthmore that has been found in P. simonii ×P. pyramidalis 'Opera 8277' seedlings, the Acer negundo seedlings were unsed to investigate the emissions of C5-C10 aldehydes at undamaged condition. Based on the results, the conclusions showed as follows:1. Using affymetrix genechip microarray technique, differential expressions of abundant defense genes were observed in poplar leaves after herbivore wounding. The genes involved in Ca²⁺ signal and oxidative burst including calmodulin-like protein, APX, NADPH oxidase, the genes involved in JA and ABA pathway including LOX, HPL, ABA-responsive protein, the genes involved in defense proteins includingpathogenesis-related protein, chitinase, POD, PPO, PAL were all significantly upregulated.2. Herbivore wounding induced an array of oxidative responses in poplar leaves. H₂O₂ accumulation was observed at the edges of wounds and veins after herbivore wounding, and the oxidases enzyme activities of peroxidase (POD) and polyphenol oxidase (PPO) were also enhanced. The increase in oxidases activities together with the increase in H₂O₂ might form an important front line in poplar against herbivores. At the same time, the activities of catalase (CAT) and ascorbic acid peroxidase (APX) were enhanced to reduce the oxidative stress of H₂O₂. The dynamic balance between antioxidative enzymes and reactive oxygen specie (ROS) not only maintained the defense function of H₂O₂, but also decreased its toxicity.3. The enzyme activities of POD, PPO, APX and CAT also increased in systemic leaves of poplar seedlings after herbivore wounding, showing the oxidative responses were induced in unwounded leaves, which indicated that systemic resistance was triggered.4. Ca²⁺ signal played a key role in early steps of signal transduction in plants after herbivore wounding. The Ca²⁺ chelator EGTA and Ca²⁺ channel inhibitor LaCl₃ suppressed the increase in activities of APX, CAT, POD and PPO induced by herbivore wounding, which indicated that Ca²⁺ signal located in upstream of these oxidative responses. Compared to EGTA, LaCl₃ showed stronger inhibited effect.5. H₂O₂, JA and ABA were all involved in wound signal transduction in plants. The enhancement in contents of H₂O₂, JA and ABA after herbivore wounding and exposure to methyl jasmonic acid (MeJA) indicated these signal molecules played important roles in defense response of poplar against herbivores. The results showed that H₂O₂ contents increased earlier than JA and ABA in wounded leaves, which implied H₂O₂ acted in the upstream of JA and ABA. In the upper unwounded leaves near to the wounded leaves these three signal molecules were also systemically induced, which revealed the generation of systemic induced resistance. A signal transduction mode of H₂O₂, JA and ABA was proposed: on one hand, H₂O₂ accumulation induced by herbivore wounding and exposure to MeJA activated JA and ABA signal system triggering genes expression downstream including NADPH oxidase, leading to H₂O₂ production again; on the other hand, H₂O₂ and JA were transmitted to unwounded leaves resulting in the systemic induced resistance in poplar.6. Aldehydes were important components of volatiles emitted from plants Eleven linear saturated aldehydes including acetaldehyde, butanal, pentanal, hexanal, heptanal,octanal, nonanal, decanal, undecanal, dodecanal and tetradecanal, three linear unsaturated aldehydes including (Z)-3-hexenal, (E)-2-hexenal, (E)-2-nonenal, and two nonlinear aldehydes including benzaldehyde, furfural were found to emit from wounded poplar. Their release rates showed similar change pattern: sharply increased at 2h, then dropped, and strongly rose at 12h again. This result suggested that there were two different mechanisms involved in production and emissions of aldedhydes after wound: (1) aldehydes emissions from storage pools after damage resulted in the first release peak; (2) the biosynthesis pathways were activated to produce aldehydes leading to the second peak.7. The release rates of pentanal, hexanal, heptanal, octanal, nonanal, decanal from intact ashleaf maple leaves showed obvious positive correlation with light intensity and O₂ contens, and negative correlation with CO₂ content. The subsequent results of histochemical localization of H₂O₂ and superoxide anion in maple leaves at different light intensity and O₂ contents indicated that production of these aldehydes was closely related with the ROS generated in photosynthesis. ROS produced in electron transport chain of photosynthesis might generate aldehydes through two pathways: (1) ROS caused the peroxidation of membrane lipid leading to the direct production of aldehydes; (2) the peroxide products of membrane lipid caused by ROS provided substrates for lipoxygenase/ hydroperoxide lyase pathway generating aldehydes.8. Communication between plants mediated by airborne signals has been revealed. It was found that the release rates of MeJA, methyl salicylic acid (MeSA) and benzothiazole were elevated from poplar leaves after mechanical damage, moreover, exposure to exogenous MeJA, MeSA and benzothiazole caused the content variety of phenolics. So it could be concluded that these three volatiles could function as airborne signals between poplars.9. The increase in activity of POD, PPO and PAL in intact poplar induced by wound-induced volatiles was inhibited by pretreatment of wounded leaves with EGTA and LaCl₃. This result implied that Ca²⁺ signal mediated synthesis and emissions of wound-induced airborne signal molecules. Furthermore, Ca²⁺ signal also was involved in the activation of defense response in intact poplar after receiving the airborne signals.10. The whole information transfer between plants was mediated by "compound signal" composed of different airborne signals. Four mixed volatiles including MeJA and MeSA, MeJA and trans-hexenal, MeJA and cis-hexenal, MeJA and benzothiazole were created in experiment. All of four mixed volatiles induced higher activity of POD, PPO and PAL than only single volatile in intact poplar leaves, which showed that mixedvolatiles possessed stronger inducing capability. Moreover the inducing effect of mixed volatiles was not just the addition of single volatile. Among the four mixed volatiles, mixture of MeJA and cis-hexenal exhibited the highest inducing capability.In this study, using the P.simonii×P.pyramidalis 'Opera 8277' seedlings after Clostera anachoreta larvae wounding, basesd on differential expressions of full length genome, from the view of molecular physiology, the physiological and biochemical processes in relation to genes induced by herbivore woundting were investigated systemically, and the results elementarily revealed the physiological and biochemical mechanism for induced resistance in P.simonii×P.pyramidalis 'Opera 8277' seedlings in response to Clostera anachoreta larvae wounding. MeJA was detected in wound-induced volatiles emitted from poplar for the first time, and the first exploration in action mode of "compound signal" between plants was conducted in this study.