Theregulatory Roleofno And G6PDH Inresponse To Aluminum Stress In Soybean Seedlings

The stress of aluminum(Al)is the main constraint for plant growth and crop yield in acid soils.Inhibition of root elongation has been reported to be the most important and primary manifestation of Al toxicity.Nitric oxide(NO)serves asanimportant signaling molecule involved in responses to various biotic and abiotic stress in plants.However,the role of NO in Al stress is still largely unknown.Glucose-6-phosphate dehydrogenase(G6PDH)is the pivotal and rate-limiting enzyme of the pentose phosphate pathway,which has been implicated in supplying reduced power NADPH for biosynthesis.It has been reported that G6 PDH is involved in resistance to various environmental stresses.However,the role of G6 PDH in Al toxicity remains unclear.In this study,we aimd to investigate the responses of NO and G6 PDH toAl stress in soybean roots,and the regulatory rolesof them in Altoxicity were further explored.The main results were summarized as follows:1.The regulatory role of NO in response to low Al concentration stress.Under Al stress,root growth and cells of elongation region were inhibited,while the levels of lipid peroxidation and reactive oxygen species(ROS)(H2O2 andO2?·)increased significantly.However,the root growth inhibition and the lipid peroxidation level in Al-treated soybean seedlings was relieved by antioxidant butylated hydroxyanisole(BHA)treatment.The results showed that Al stress induced the accumulation of ROS and the level of lipid peroxidation in the roots,leading to oxidative damage,which ultimately affected the shape and size of elongation region cells,resulting in the inhibition of root growth.Application of NO donor(SNP)could alleviate root elongation inhibition,ROS accumulation and lipid peroxidation level in Al-treated soybean roots,whereas NO scavenger(PTIO)treatment aggravated the effects of the Al toxicity.Further investigartion showed that SNP treatment significantly improved the activities of antioxidant enzymes(CAT,POD)activity,the contents of antioxidants(GSH,ASA),and the activities of key enzymes in GSH-ASAcycle(APX,GR,GPX,DHAR,MDHAR),while the increase of antioxidant enzymes and the contents of antioxidants in Al-treated soybean roots was inhibited by PTIO treatment.Al stress significantly increased the NO content and nitrate reductase(NR)activity in soybean roots.Treatment with NR inhibitor abolished the Al-induced increase in NO content,but nitric oxide synthase(NOS)inhibitor had little effect on NO content under Al stress,suggesting that NO accumulation in root apexes under Al stress was mediated by NR pathway.Taken together,these results indicate that NR-mediated NO can eliminate excess ROS by regulating antioxidant enzyme activities and GSH and ASA content,thereby enhancing plant tolerance to Al toxicity.2.The regulatory role of G6 PDH in response to high Al concentration toxicity.High Al concentration(?200 ?M)treatment significantly enhanced the activities of total G6 PDH and cytosolic G6PDH(Cyt-G6PDH)in roots of soybean,and the enzyme activity increased with the increase of Al concentration.However,Al stress had no significant effect on the activity of plasmid G6 PDH,suggesting that Cyt-G6 PDH contributes to the enhanced total G6 PDH activity in soybean roots exposed to high Al concentration.Al-induced inhibition of root growth,lipid peroxidation and ROS accumulation were alleviated by a G6 PDH inhibitor(GN6P),indicating that G6 PDH was involved in the Al toxicity of soybean seedlings.Furthermore,ROS accumulation in Al-treated roots of soybean could be abolished by a NADPH oxidase inhibitor(DPI),suggesting that Al-induced ROS production resulted from NADPH oxidase.Under Al stress,application of GN6 P significantly reduced NADPH oxidase activity and NADPH content in root apexes of soybean,indicating that G6 PDH regulates the activity of NADPH oxidase.Further investigation showed that the activity of Cyt-G6 PDH and the expression level of Cyt-G6PDH(G6PDH1 and G6PDH2)were significantly intensified under Al stress.The activity and expression of Cyt-G6 PDH were further enhanced by SNP treatment,but suppressed by PTIO treatment,suggesting that NO mediates Al-induced increase of G6 PDH activityby modulating the expression of G6PDH1 and G6PDH2 insoybean roots.In addition,the content of NO was significantly increased under Al stress,and Al-induced NO generation was mainly mediated by NR-pathway.Taken together,these results indicate that NO-modulated Cyt-G6 PDH in root apexes is responsible for ROS accumulation mediated by NADPH oxidase under Al stress,subsequently suffering from oxidative stress and thus causing the inhibition of root elongation.In summary,Al-induced NO production in soybean rootsis dependent on NR pathway,and NO has two aspects of function.On the one hand,under low concentration of Al stress,it can alleviate the oxidative stress and root growth inhibition by regulating the activities of antioxidant enzymes and the contents of antioxidants.On the other hand,under high concentration of Al stress,NO enhanced Cyt-G6 PDH activity by modulating the expression of Cyt-G6 PDH genes.Thereby,the NADPH content and the activity of NADPH oxidase increased in soybean roots,thus leading to ROS accumulation and oxidative damage,eventually resulting in root growth inhibition.