The Regulatory Roles Of Glucose-6-phosphate Dehydrogenase And ABA In Aluminum-induced Programmed Cell Death Of Soybean Root Tips

Aluminum（Al）toxicity is the main factor limiting crop yield in acidic soil.Programmed cell death（PCD）induced by Al stress in root tips is considered to be an important cause of Al toxicity.However,the signal process of how Al induces PCD occurrence in the root tips of plant remains largely unknown.Glucose-6-phosphate dehydrogenase（G6PDH）is the key enzyme involved in pentose phosphate pathway,which can supply reduced power NADPH for biosynthesis.Although G6PDH has been involved in plant responses to various environmental stresses,the role of G6PDH in response to Al stress is still elusive.Abscisic acid（ABA）,a key signal molecule,participates in plant response to Al stress.However,whether ABA is involved in the regulation of Al-induced PCD remains unclear.In this paper,the roles of G6PDH and ABA in the regulation of Al-induced PCD occurrence were investigated in root tips of soybean,and the relationship between G6PDH and ABA in this process was also explored.The main results are summarized as follows:1.Treatment with different Al concentrations and times showed that high Al concentration increased G6PDH activity and cytosolic G6PDH gene expression in soybean root tips.Compared with wild type（WT）plants,transgenic soybean hairy roots overexpressing cytosolic G6PDH enhanced G6PDH activity under Al stress,indicating that Al stress may enhance G6PDH activity through transcriptional regulation,and cytosolic G6PDH contributes to G6PDH activation induced by Al stress.2.Al treatment increased the ratio of cell death,expression of apoptosis marker gene Hsr203J,activity of caspase-3-like protease and relase of cytochrome c（Cyt c）from mitochondria in root tips of soybean,suggesting that Al stress induced PCD occurrence.Application of a G6PDH inhibitor alleviated Al-induced PCD occurrence in root tips.Furthermore,overexpression of cytosolic G6PDH significantly aggravated the cell death in root tips under Al stress.These results suggest that G6PDH mediates PCD occurrence in soybean root tips under Al stress.3.Al stress increased reactive species oxygen（ROS）generation in root tips,whereas addition of G6PDH inhibitor decreased Al-triggered ROS accumulation.Exogenous H₂O₂treatment further increased PCD occurrence in Al-treated root tips.Overexpression of cytosolic G6PDH significantly enhanced ROS production in root tips under Al stress.Furthermore,application of NADPH oxidase inhibitor eliminated Al-triggered ROS accumulation,indicating that the source of ROS generation induced by Al originates from NADPH oxidase pathway.Further investigation showed that addition of G6PDH inhibitor decreased NADPH content and NADPH oxidase activity in the root tips of soybean.Overexpression of cytosolic G6PDH significantly enhanced NADPH content and NADPH oxidase activity under Al stress.These results indicate that G6PDH mediates Al-triggered PCD occurrence through the regulation of ROS generation resulting from NADPH oxidase activation.4.Al stress induced the production of ABA in root tips and showed very similar changes as did in G6PDH activity.Addition of ABA inhibitor decreased cell death ratio,Hsr203J expression and ROS production in Al-treated root tips,suggesting that ABA mediates PCD occurrence induced by Al.Furthermore,exogenous ABA treatment could increase G6PDH activity and cytosolic G6PDH expression in Al-treated root tips,whereas addition of G6PDH inhibitor has no significant effect on ABA production under Al stress.Overexpression of cytosolic G6PDH has little effect on ABA generation in the absence or presence of Al stress.These results suggest that ABA mediates PCD occurrence induced by Al through the positive regulation of G6PDH activity.To sum up,our results demonstrated that ABA enhanced the G6PDH activity by regulating the expression of cytosolic G6PDH gene under Al stress.G6PDH positively modulated NADPH oxidase activity through supplying NADPH and subsequently triggered ROS generation in root tips,which promoted mitochondrial Cyt c release and caspase activity,thus resulting in PCD occurrence under Al stress.