Exogenous Hydrogen Supply Regulate The Occurrence Of Programmed Cell Death In Rice Aleurone Layer Mediated By H₂O₂

Programmed cell death（PCD）is an active and ordered cell death controlled by genes,which occurs at various stages of plant growth and development.The aleurone layer PCD plays a key role in the germination of cereal seeds.Studies have shown that exogenous H₂promotes seed germination and seedling growth,and gibberellin（GA）plays an inducing role in the germination of cereal seeds and regulates the process of PCD in the aleurone layers mediated by H₂O₂.In this study,using the seeds of Rice（Oryza sativa L.）"Teyou 128"as experimental materials,exogenous hydrogen donor（Mg H₂）,GA synthesis inhibitor Uniconazole（Uni）,exogenous hydrogen peroxide（H₂O₂）,H₂O₂ scavenger N,N’-Dimethylthiourea（DMTU）and reduced nicotinamide adenine dinucleotide phosphate（NADPH）oxidase inhibitor diphenyleneiodonium chloride（DPI）were used for single and multi-factor co-treatment.According to the methods of pharmacology and molecular biology,combined with flow cytometry and microscopy,the radicle length and embryo length of germinated rice seeds were measured.In addition,the growth potential was observed.At the same time,the contents of H₂O₂ in root tip and aleurone layers were detected.Meanwhile,the expression level of Os NOX8 gene and the activity of NADPH oxidase were determined in the aleurone layers.Moreover,the cell mortality of aleurone layers was measured.Based on the above survey,the mechanism of exogenous H₂ regulating the occurrence of PCD in rice aleurone layer was explored under the inhibition of GA synthesis.The results indicated that:（1）Where GA is inhibited or not,Mg H₂ increased the germination rate of rice seeds,namely,the exogenous hydrogen donor promoted the germination of rice seeds in a dose-dependent manner,but their optimal concentrations were different.The optimal concentrations of Mg H₂ is 0.005 mg/m L under the normal condition that GA synthesis is not inhibited.The optimal concentrations of Mg H₂ was increased to 0.02 mg/m L under the GA synthesis being inhibited.Thus,GA maintains the endogenous H₂ level of the germinated rice seeds.（2）The content of H₂O₂ in rice seeds was increased at appropriate concentrations of exogenous Mg H₂ and exogenous H₂O₂,and the production of endogenous H₂O₂ is induced by exogenous Mg H₂,that is,Mg H₂ is located in the upstream of H₂O₂.H₂O₂ content was increased by 0.005 mg/m L Mg H₂ under normal condition.The content of H₂O₂ was increased by exogenous hydrogen donor Mg H₂ and H₂O₂ when GA synthesis is insufficient,and both of them have counter effects when inducing the production of H₂O₂.The most significant concentration of Mg H₂ was still 0.02 mg/m L,and the effect of Mg H₂ could be reversed by DMTU.（3）Both Mg H₂ and H₂O₂ could up-regulate the levels of Os NOX8 gene expression and NADPH oxidase activity when GA synthesis was inhibited.It is speculated that exogenous hydrogen donor Mg H₂ induces H₂O₂ production in the aleurone layers of the germinated rice seeds through NADPH oxidase pathway.（4）When GA synthesis is insufficient,the cell mortality of aleurone layers is reduced in the germinated rice seeds,that is,GA promotes the occurrence of PCD.The cell mortality was increased by exogenous hydrogen donor Mg H₂ and exogenous H₂O₂,but their mortality was still lower than that of the control when GA production is insufficient.It is followers that exogenous hydrogen donor Mg H₂ can properly accelerate the formation of PCD in aleurone layers of rice seeds when GA synthesis is insufficient.In conclusion,GA up-regulates the endogenous H₂ level in the germinated rice seeds,and then H₂ regulates the occurrence of PCD in aleurone layers and the germination of rice seeds through H₂O₂ produced by NADPH oxidase pathway.