RESUMO
In this study we examined whether and to what extent oxidative stress is induced in seedling leaves of Pisum sativum L. cv. Cysterski in response to pea aphid (Acyrthosiphon pisum Harris) infestation. A. pisum caused oxidative stress conditions in pea leaves through enhanced production of the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and superoxide anion radical (O2(·-)). Early, strong generation of H2O2 was observed at 24h in aphid-infested leaves. The highest level of H2O2 at this time point may be related to the functioning of H2O2 as a signaling molecule, triggering defense mechanisms in pea leaves against A. pisum. Additionally, the strong generation and continuous increase of O2(·-) production in aphid-infested leaves from 0 to 96 h enhanced the defense potential to protect against aphid herbivory. Also in the study cytochemical localization of H2O2 and O2(·-) in pea leaves after aphid infestation was determined using the confocal microscope. Relative release of H2O2 and O2(·-) was estimated by staining leaves with specific fluorochromes, i.e. dichlorodihydro-fluorescein diacetate (DCFH-DA) and dihydroethidium (DHE), respectively. DCFH-DA and DHE derived fluorescence was observed to cover a much larger tissue area in aphid-infested leaves, whereas little or no fluorescence was observed in the control leaves. Enhanced activity of the antioxidant enzymes superoxide dismutase (SOD, 1.15.1.1) and catalase (CAT, 1.11.1.6) is one of the most essential elements of defense responses in pea seedling leaves to oxidative stress. Additionally, generation of semiquinones, stable free radicals with g-values of 2.0020 and 2.0035, detected by electron paramagnetic resonance spectroscopy (EPR), was suggested as a protective action of pea that may contribute to build-up of a defensive barrier or activate other defense mechanisms. Concentrations of semiquinone radicals in aphid-infested seedling leaves not only were generally higher than in the control plants but also significantly increased with cultivation time. On the other hand, the small increase in content of thiobarbituric acid reactive substances (TBARS), a product of lipid peroxidation, and the percentage of injury (3-8%) indicated that the cellular damage was caused by oxidative stress. The induced changes in levels of H2O2, O2(·-) and semiquinone radicals as well as activities of antioxidant enzymes in the pea defense responses were proportional to the population size of A. pisum. These findings indicate that the defensive strategies against A. pisum infestation were stimulated in seedling leaves of P. sativum L. cv. Cysterski. Our observations of the enhanced defense responses of P. sativum to infestation by A. pisum reveal some aspects and contribute to current knowledge of regulatory mechanisms in plant-aphid interactions.
