The Molecular Mechanism Of Maize Aleurone Layer PCD Regulated By GA

Aleurone layer is the ultimate differentiated nondissociative tissues,which store large amounts of phophates and mineral cations such as K⁺and Mg²⁺for seed germination,between endosperm and seed coat in cereal plants.Evidence from extensive research suggests that plant hormone gibberellin（GA）produced by the embryo and abscisic acid（ABA）from the endosperm regulate gene expression,secretion and cell death cooperatively in aleurone layer.Aleurone cells help mobilize the nutrients stored in the seed by synthesizing and secreting a variety of amylase and hydrolytic enzymes that participate in the breakdown of endosperm starch,protein,lipid and cell walls during plant germination.And then,aleurone cells eventually die a few days after germination.There are many researches about programmed cell death（PCD）in plants.The PCD of aleurone cells induced by GA is necessary for plant germination.The PCD process of aleurone layers is a complex biological process involving a variety of signal transduction.Epigenetic modification is a reversible,heritable change in gene function that occurs without a change in the nucleotide DNA sequence and plays a key role during plants germination and development.DNA methylation and histone acetylation are two of the most common chromatin modifications.In this article,the maize aleurone layers were taken as the research object to explore the phenomenon of rDNA chromatin decondensation and PCD process of aleurone cells during germination.In an attempt to reveal the molecular mechanism of maize aleurone layers regulated by phytohormone GA during seedlings germination.The research contents of this paper are as follows:1.Histone acetylation is involved in GA-mediated 45S rDNA decondensation in maize aleurone layersIn this study,we reported that the expression level of 45S rDNA in maize aleurone layers was increased,accompanied by the rDNA decondensation,which was inhibited by the phytohormone ABA,during the seed expanded with water and entered into the germination stage.The aleurone layers were gently peeled away from maize seeds with a razor blade and cultured it in vitro.After treating the isolated aleurone layers with GA for 24 h,we found that the expression of 45S rDNA was increased accompanied by the decondensation of rDNA,and the level of histone acetylation was increased.To determine if histone acetylation/deacetylation involved in rDNA condensation or decondensation,we examined the enzyme activity of HDACs and HATs in GA-treated aleurone layers.The results showed that the enzyme activity of HATs and HDACs was gradually increased in dissected aleurone layers after GA treatment compared with in untreated dissected aleurone layers.However,the increased rate of enzyme activity of HDACs seems slower than that of HATs activity,eventually resulting in lower relative activity of HDACs compared with HATs.Therefore,we hypothesized that histone acetylation plays a key role in the GA-induced decondensation of rDNA in aleurone layers.Next,we further treated the isolated aleurone layers with two HDACs inhibitors（TSA and CUDC-101）to explore the role of histone acetylation.The treatment of TSA and CUDC-101 resulted in the increased level of acetylation in aleurone layers and decondensation of rDNA chromatin.Further chromatin immunoprecipitation（Ch IP）results showed that GA promoted the level acetylation of related gene promoter regions of rDNA and HAT/HDAC.Based on the above data,we believe that in the early germination stage of maize seeds,histone acetylation mediates the GA-regulated decondensation of 45S rDNA chromatin in aleurone layer.2.DNA methylation and lipid signaling pathways are involved in PCD in maize aleurone layerRecent studies have been reported that epigenetic modification involved in PCD in mammals and plants.In previous explores,we have shown that histone deacetylase is necessary for GA-induced PCD in maize aleurone layers.In this study,we found that DNA methylation modification and lipid metabolism were involved in PCD in maize aleurone layers induced by GA and HATs inhibitor.Aleurone layers were isolated from maize seeds.And experimental group aleurone layers were treated with GA and HATs inhibitor respectively for 1-8 d,control group aleurone layers were treated with diluted DMSO.Tissues of each group were mixed and total RNA was extracted from 1-5 d,a total of 45 RNA libraries were constructed for RNA sequencing（RNA-seq）.Finally,we collected a lot of differentially expressed genes（DEGs）,and the DEGs were involved in many biological processes,mainly in epigenetic modification and lipid metabolism.Further research suggested that DNA methyltransferase inhibitor,which lead to genome-wide DNA hypomethylation,can prevented the maize aleurone layers PCD induced by GA and HATs inhibitor.The results showed that DNA methylation play a role in aleurone layers PCD regulated by GA.In addition,GA and HATs inhibitor treatment can increase the expression of lipoxygenase genes and promote the degradation of lipid in aleurone cells,and with the production of reactive oxygen species（ROS）.Otherwise,DNA methylation inhibitor could regulated the expression of ROS-related genes,and inhibited the production of H₂O₂ induced by GA.Overall,the linking of DNA methylation,lipoxygenase,and ROS,together with our previously reported data on H₂O₂,may indicate that GA-induced higher HDACs activity in aleurones caused breakdown of lipids via regulating lipoxygenase gene expression,and increased DNA methylation positively mediated H₂O₂ production;thus,DNA methylation and lipid metabolism pathways may represent an important and complex signaling network in PCD in maize aleurone layers.