Physiological Response And The Regulative Mechanism In Plant Calli Under High Temperature Stress

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In this study, the calli from Przewalskia tangutica (Przewalskia tangutica, Maxim) and tobacco (Nicotiana tabacum, L) were used as material to study their physiological responses to high temperature stress. And the regulative roles of G6PDH and the signal-transduction mechanism were further investigated. The main results were summarized as follows:The relative growth rate and the relative cell viability decreased, the relative electrolyte leakage and MDA content increased with the time extension of high temperature (40℃) treatment in both calli of Przewalskia tangutica and tobacco. The contents of H2O2and total soluble sugar, as well as the activities of antioxidant enzymes (APX, CAT, POD and SOD) in both calli were also induced, although two calli exhibited a difference in the degree and timing of response to high temperature treatment. Exposure to the same time of high temperature stress, the relative electrolyte leakage and MAD content in Przewalskia tangutica callus were higher than that in tobacco callus, and relative cell viability and ralative growth rate were lower. Meanwhile, higher activities of antioxidant enzymes (APX, CAT, POD and SOD) in tobacco callus were higher than that in Przewalskia tangutica callus. These results showed that Przewalskia tangutica callus was more sensitive to high temperature stress than tobacco callus, which might be determined by their antioxidant enzymes activities.Under high temperature stress, G6PDH activity was induced in both calli from Przewalskia tangutica and tobacco. When G6PDH was partially inhibited by glucosamine pretreatment, the relative electrolyte leakage increased, and the ralative growth rate decreased in both calli under high temperature stress comparied with that under high temperature treatment alone. These results demonstrated that G6PDH maybe involved in the tolerance of high temperature stress in both calli from Przewalskia tangutica and tobacco.The high temperature stress induced activities of antioxidant enzymes (APX, CAT, POD and SOD) significantly decreased when G6PDH activity was inhibited in both calli from Przewalskia tangutica and tobacco. Simultaneously, the high temperature stress induced H2O2accumulation and the plasma membrane (PM) NADPH oxidase activity were also reduced. Exogenous application of H2O2increased the activities of G6PDH and antioxidant enzymes in both calli. Diphenylene iodonium (DPI, a PM NADPH oxidase inhibitor) counteracted heat-induced H2O2accumulation and reduced the activities of heat-induced G6PDH and antioxidant enzymes, suggesting NADPH oxidase involved in heat-induced H2O2accumulation. Moreover, exogenous H2O2was effective in restoring the activities of G6PDH and antioxidant enzymes after glucosamine pretreatment.Western-blotting analyses showed that G6PDH protein level in both Przewalskia tangutica and tobacco calli increased under high temperature stress. Glucosamine and DPI significantly inhibited the high temperature stress induced expression of G6PDH in both calli, whereas exogenous application of H2O2induced G6PDH expression and restored the decrease of G6PDH expression after glucosamine treatment. These results demonstrated that the change of G6PDH activity under high temperature stress and other treatments were consistent with its protein abundance.Taken together, the reason that Przewalskia tangutica callus had less thermotolerance than that of tobacco callus was the higher activities of antioxidant enzymes in tobacco callus than that in Przewalskia tangutica callus under high temperature stress. G6PDH was involved in high temperature stress. Under high temperature stress, G6PDH promoted H2O2accumulation via NADPH oxidase and the elevated H2O2was involved in regulating the activities of antioxidant enzymes, which in turn facilitate to maintain steady-state H2O2level and protect plants from the oxidative damage.