The Role Of NADPH Oxidase In Aluminum Toxicity-induced Lignification Of Rice Root Tips And The Regulatory Mechanism Of NO And H₂O₂

Aluminum toxicity is a major factor limiting plant growth in acidic soil,inhibiting root elongation for a short period of time.Existing studies have shown that aluminum toxicity induces lignin deposition in the root tip cell wall to induce the sclerosis of cell wall,thereby inhibiting cell elongation,and this process is closely related to reactive oxygen species（ROS）metabolism.Hydrogen peroxide（H₂O₂）is the most abundant and stable type of ROS and plays a role in many important resistance mechanisms in plants.The H₂O₂ in the cell wall is involved in the oxidative polymerization of sinapyl alcohol,coumarin and coniferyl alcohol into lignin.At the same time,H₂O₂ is also an important signaling molecule when the content of H₂O₂ is low,and cooperates with nitric oxide（NO）to regulate the tolerance of plants to abiotic and biotic stresses.Nicotinamide adenine dinucleotide phosphate（NADPH）oxidase on the plasma membrane of plant cells is one of the main ways to generate exosomal H₂O₂ and plays an important role in various environmental stresses.Under the effect of aluminum toxicity,there is no direct evidence that H₂O₂ produced by NADPH oxidase pathway mediates cell wall lignification and NO is involved in the signal transduction pathway that regulates aluminum-induced lignification of rice root tip cell wall.Therefore,in this study,the japonica rice Nipponbare（Al-tolerant genotype）and the indica rice Zhefu 802（Al-sensitive genotype）were used as materials to investigate the activation of NADPH oxidase,the relationship between H₂O₂ production and lignification of rice root tip cell wall under aluminum toxicity,the glucose-6-phosphate dehydrogenase（G6PDH）involved NADPH oxidase-dependent ROS production and Al-induced lignification and mitogen-activated protein kinase（MAPK）signaling for NO involved in regulating Al-induced lignification of rice root tip cell walls to explore the reactive oxygen species production pathway and signal transduction mechanism in the process of aluminum-induced lignification of the root tip cell wall.The main findings obtained are as follows:（1）Hydroponics was used to study the response of exoplast reactive oxygen species production and NADPH oxidase activity in rice root tip to aluminum toxicity and the effect of exogenous H₂O₂on lignin synthesis,to clarify the effect of the activation of NADPH oxidase and the production of H₂O₂ on cell wall lignification.The results showed that after 0,50 and 100μmol/L Al treatment for 3,6,9 and 24 h,the activities of NADPH oxidase and cell wall horseradish peroxidase（c POD）in rice root tips,as well as H₂O₂ content in the cell wall were significantly increased with the increase of Al concentration,and rapidly increased in a time-dependent manner,the magnitude of the increase was consistent with Al-induced lignin deposition.The effect of H₂O₂ on lignin synthesis was analyzed by using exogenous H₂O₂ and H₂O₂ scavenger N-N dimethylthiourea（DMTU）.Compared with the single Al treatment group,the H₂O₂content of the root tip cell walls of Nipponbare and Zhefu 802 treated with Al+H₂O₂decreased by 19.71%and 38.41%,respectively,the lignin content was decreased by45.59%and 69.25%,and the total phenolic content was decreased by 53.5%and45.89%,respectively.In addition,the activities of NADPH oxidase and c POD were also significantly reduced.It was shown that exogenous H₂O₂ signaling molecules reduced the H₂O₂content of rice cell walls by regulating NADPH oxidase activity and c POD activity,thereby reducing the deposition of lignin and the accumulation of Al in rice root tips.（2）Diphenylene iodonium chloride（DPI）,an inhibitor of NADPH oxidase,and trisodium phosphate（Na₃PO₄）,an inhibitor of G6PDH,were used to investigate the involvement of G6PDH in NADPH oxidase-dependent ROS production and Al-induced lignification.Under aluminum toxicity,exogenous Na₃PO₄ reduced the lignin content,H₂O₂ content,O₂^-and malondialdehyde（MDA）content,and alleviated the lignification of rice roots.Which showed that G6PDH mediates Al-induced accumulation of lignin in rice root tips.The cell wall H₂O₂ content,lignin content,SOD activity and MDA content in root tips with exogenous addition of DPI were significantly lower than those in the Al treatment group,indicating that NADPH oxidase is involved in the generation of ROS and the Al-induced rice root tips cell wall lignification under aluminum toxicity.Al toxicity increased NADPH oxidase activity in two rice genotypes.However,exogenous addition of Na₃PO₄ significantly reversed the Al toxicity-induced increase in NADPH oxidase activity.These experimental results suggest that G6PDH is involved in aluminum toxicity-induced ROS accumulation and rice root lignification from the regulation of NADPH oxidase.In addition,exogenous addition of NO and H₂O₂ was also found to reduce the activity of G6PDH enzyme,suggesting that G6PDH and NADPH oxidase regulate the formation of rice root lignification under Al tooxicity.（3）The signal transduction pathway of NO regulation of Al tolerance in rice root tip cell wall under aluminum toxicity was explored by adding MAPKinhibitor.The results showed that under the Al+PD98059 treatment,the activity of NADPH oxidase of Nipponbare and Zhefu 802 decreased by 20.7%and 20.39%,respectively,compared with the Al treatment group,the activity of c POD was decreased by 70.57%and 33.31%,respectively,compared with the only Al treatment group,and the cell wall H₂O₂content was 53.93%and 44.8%lower than that of only Al treatment group,respectively,the lignin content was 16.40%and 18.87%lower than that of the Al single treatment group,the PAL activity was 15.08%and 15.22%lower than that of the Al single treatment group,and the Al content in the root tip was decreased by34.07%and 31.07%than that of the Al single treatment group,respectively,indicating that MAPK is located upstream of NADPH oxidase to mediate Al-induced lignification.It was found that adding NO could inhibit the activities of G6PDH and MAPK,indicating that NO was located upstream of G6PDH and MAPK and had a positive regulatory effect on their activities,while MAPK was located downstream of G6PDH and had a negative regulatory effect on their activities.