Studies On The Mechanism Of Regulations Of G6PDH And MAPKK9-MAPK3/MAPK6in Plant Adaptation To Tolerance

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in supplying reduced nicotine amide cofactors for biochemical reactions and modulating the redox state in cells. The article includes two parts. In the first part of study, different cultivar soybeans (Glycine max L. Merr. cv Del Soy) of JINDOU21(JD-21, tolerance to drought stress) and WDD00172(WDD-172, sensitive to drought stress) were used as material to study their physiological responses to drought stress. And the regulative roles of G6PDH and the signal-transduction mechanism were further investigated. The main results were summarized as follows:1. The relative electrolyte leakage and H2O2content increased with the time extension of PEG6000treatment in both JD-21and WDD-172. However, they were higher in WDD-172root. With the concentration and time of PEG6000treatment increased, the primary root length and the lateral root number were lower than control in both JD-21and WDD-172. And they were much more in JD-21than in WDD-172. These results suggested that JD-21had stronger drought tolerance.2. Under9%PEG6000treatment for72h, JD-21showed higher tolerance than WDD-172not only in higher activities of ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR), but also in higher contents of glutathione (GSH) and ascorbate (Asc). These results indicated that the enhanced antioxidant enzyme activities and higher GSH and Asc content might be responsible for the higher tolerance to drought stress in JD-21than WDD-1723. The G6PDH activity markedly induced during PEG6000treatments in both JD-21and WDD-172root. After G6PDH activity in both soybeans was inhibited by Glucosamine (Glucm, a G6PDH inhibitor) under PEG6000treatment, the primary root length and the lateral root number were markedly reduced in both JD-21and WDD-172. And the activities of GR, DHAR and MDHAR were decreased much more in WDD-172than in JD-21by Glucm and PEG6000 treatment compared to PEG6000treatment alone. These results demonstrated that G6PDH might play an important role to maintain the dynamic balance of oxidation-reduction under drought stress.4. DPI (a plasma membrane NADPH oxidase inhibitor) could counteract PEG6000-induced H2O2accumulation and decrease the enzyme activities of G6PDH, GR, DHAR and MDHAR as well as GSH and Asc contents. Furthermore, exogenous application of H2O2increased the activities of GR, DHAR and MDHAR that were decreased by Glucm under drought stress in both soybeans. These results showed that H2O2might involve in regulation of G6PDH under drought stress.5. Western-blot analysis showed that the G6PDH expression was stimulated by PEG6000and buthionine sulfoximine (BSO, an inhibitor of glutathione biosynthesis), and blocked by Glucm, DPI and N-acetyl-L-cysteine (NAC, a GSH precursor) in both soybeans. These results demonstrated that GSH and NADPH oxidase maybe involved in the regulation of G6PDH in tolerance of soybean to drought stress.Taken together, our evidence indicates that G6PDH plays a central role in the process of H2O2regulated GR, DHAR and MDHAR activities to maintain higher levels of GSH and Asc under drought stress.Mitogen-activated protein kinase (MAPK) cascades play important roles in regulating plant growth, development, and responses to various environmental stimuli. In the part two of study, we used the calli from wild-type Arabidopsis(Arabidopsis thaliana, Col-0) and Mitogen-activated protein kinase kinase9(MAPKK9) mutants (MKK9KR, MKK9DD, MKK9DDmapk3, MKK9DDmapk6and mkk7mkk9) to study their physiological responses to salt stress, and further investigated the possible regulation and physiological function of alternative pathway (AP) and MAPKK7/MAPKK9-MAPK3/MAPK6cascades under salt stress. The main results were summarized as follows:1. The total respiration rate (Vt), the capacity of alternative pathway (Valt) and cytochrome pathway (Vcyt) in MKK9DD callus was induced and relieved the inhibition of NaCl to Vt、Vcyt and Valt in MKK9DD callus by10μM dexamethasone (DEX). However,the same phenomenon was not observed in calli of Col and MKKPKR. The application of DEX could induce the expression of AOX1a, AOX1d and AOX2genes and the increase of AOX protein expression in MKK9DD callus under salt stress. These results suggested that the regulation of MAPKK9to alternative pathway in MKK9DD callus under NaCl stress might by increasing the expression of AOX in both transcriptional and translational level. 2. There was no release the reduction of Vt, Vcyt and Valt which were induced by NaCl in MKK9DD mapk3callus under DEX treatment. Meanwhile, the expressions of AOX1α, AOX1d and AOX2genes and AOX protein were not induced by DEX under salt stress in MKK9DD mapk3callus. These results demonstrated that MAPKK9-MAPK3cascade might participate in the regulation of alternative pathway and might be an important role in the stress responses of Arabidopsis calli. Moreover, MAPKK7and ethylene might be involved in regulation of respiration in Arabidopsis calli under salt stress.