Characteristic Analysis Of Histone Acetylation Dynamics Under Salt Stress And Functional Analysis Of HATs In Rice

Histone modification,an important part of epigenetic regulation,including histone acetylation,methylation and phosphorylation,plays crucial roles in the growth and development of animals and plants.Histone acetylation is particularly dynamic and reversible type of histone modification,catalyzed by histone acetyltransferases and histone deacetylases.In eukaryotes,histone acetylation participates in diverse biological functions,such as transcriptional regulation,remodeling chromatin structure and so on.Rice(Oryza sativa)is one of the most important global food crops and a model organism for cereal research.However,the mechanism of histone acetylation to participate in the regulation of transcriptional activity and response to environment stress need to be further improved.Therefore,the work was focused on the study of histone acetylation and its function in plant development regulation from the following sections:large-scale analysis of the lysine acetylome in rice varietyNipponbare,the characteristics identification of histone acetylation distributed in rice genome under salt stress and the functional analysis of genes coding histone acetyltransferases p300 and GCN5 in rice,In this study,three parts of the research work had been carried out with the following results:1.Global proteome analysis links lysine acetylation(Kac)to diverse functions in rice.With the development of proteomics technology,the identification of protein function,posttranslational modification and analysis of protein structure have been further studied.High performance liquid chromatography-mass spectrometry(LC-MS/MS)is a fast-developing protein analysis technique with high throughput,high sensitivity and high accuracy,capable of identifying protein types and polypeptide modified sites,which greatly promotes the research development on covalent modification of histone in animals and plants.In this study,1353 lysine acetylated sites involving 866 proteins were identified in the leaves of seedlings grown for three weeks.Combined with previous reports,five novel histone acetylation sites were identified for the first time in rice.The findings expand our understanding of post-translational modification by Kac in rice.To further investigate the functions of proteins in the rice acetylome,all identified proteins were subjected to GO(Gene Ontology)annotation,which categorized the proteins based on their molecular function,biological process,and cellular component.Based on the biological process category,the results suggest that acetylation might occur frequently in proteins associated with responses to environmental stress and central metabolic processes.Regarding molecular function,the largest two GO categories,binding activity and catalytic activity,accounted for 45%and 40%of all acetylated proteins,respectively,indicating that proteins associated with enzyme metabolism might be acetylated to a large extent in rice.Subcellular localization analysis showed that a large number of acetylated proteins are located in the chloroplast and involved in photosynthesis.Further functional enrichment analysis indicated that lysine acetylated proteins were mainly involved in responses to stimuli,metabolic enzyme activity and cofactor binding based on GO enrichment,and carbon metabolism,carbon fixation,photosynthesis,and other central metabolic pathways based on KEGG pathway enrichment.The above results manifest that diverse cellular components are regulated by Kac,which appears to control a wide variety of biological functions and metabolic processes in rice.2.Distribution characteristics analysis of H4K5ac and H4K8ac in rice under salt stress.By analyzing ChIP-seq and RNA-seq data of rice seedling,it was found that the genes modified by novel histone acetylations,H4K5ac and H4K8ac,were widely distributed in rice genome.H4K5ac and H4K8ac modifications showed a similar genomic distribution,with the majority peaks mainly located in the intergene region,exons and introns.The two modifications were highly enriched at the transcription start site(TSS)of coding genes.By comparing the correlation between H4K5ac and H4K8ac and other active or inactive histone modifications,we found that H4K5ac and H4K8ac were highly correlated with the active histone marks,but not correlated with inactive modifications such as H3K27me3.It indicates that H4K5ac and H4K8ac were histone marks positively associated with transcriptional activity in rice.Salt stress can seriously affect the growth and development of rice.In order to study the regulatory relationship between histone modification and salt stress response genes,we comprehensively analyzed the dynamic changes of histone acetylation H4K5ac and H4K8ac and transcriptional level of rice seedlings under salt treatment.Transcriptome analysis and GO functional enrichment analysis showed that the up-regulated genes were mainly enriched in the response to stimulation and the activity of metabolic enzymes,while the down-regulated genes were mainly enriched in the metabolic pathways related to photosynthesis.ChIP-seq analysis results showed that the acetylated status of many genes were altered after salt stress.Further correlation analysis indicates that the changes of H4K5/K8ac modification level under salt stress was positively correlated with gene expression level,and H4K5/K8ac modification was inclined to regulate the expressed down-regulated genes.These results indicated that histone acetylation modification was involved in the regulation of salt-induced gene expression under salt stress in rice.3.Functional analysis of genes coding histone acetyltransferases GCN5 and p300 in rice.Through homologous sequence comparison and phylogenetic tree analysis,it was found that histone acetyltransferase genes in rice mainly include OsHAC701,OsHAC703 and OsHAC704 of p300/CBP family(collectively known as OsHACs),OsHAG702(GCN5)of GNAT family,OsHAF701 of TAFu250 family,and OsHAM701 of MYST family.In this study,the histone acetyltransferase GCN5 and p300 family were taken as research objects.The transformation vectors were constructed by using CRISPR/Cas9 system,RN A interference and other technologies,and integrated into a japonica rice variety Nipponbare to get the mutant and transgenic plants.Through molecular genetics,plant physiology and epigenetics methods,the effects of histone acetyltransferase OsGCN5 and OsHACs on the growth and development of rice were analyzed.We identified histone acetyltransferase activity of OsGCN5 using in vitro histone acetylation assay and found that OsGCN5 can catalyze histone H3 acetylation in vitro.By investigating the agronomic traits of RNAi and overexpression(OE)transgenic lines of OsGCN5,we found that the plant height,panicle length and seed setting rate of RNAi plants were significantly reduced while the expression level of OsGCN5 was decreased by RT-qPCR.In terms of grain morphology,there was no significant effect on seed width and thickness,but the seed length was reduced in RNAi plants.In OE-GCN5 lines with increased gene expression of OsGCN5,plant height,panicle length,flag leaf length and width,and tiller number were all significantly increased.These results indicate that OsGCN5 can regulate plant height,panicle length and other agronomic traits positively,with pleiotropic effects.Western blot results of histone acetylation level in wild type and transgenic lines show that only H3K18ac was significantly reduced in OsGCN5 RNAi plants,and the level of H3K18ac was significantly elevated in OE-GCN5 plants correspondingly.This indicates that GCN5 could specifically catalyze and regulate the acetylation of H3K18 in rice.We investigated various agronomic traits of OsHACs mutants and found that compared to the wild type(WT),the OsHAC701 mutants have shorter plant type,with shorter panicle length,longer flag leaf and significantly shorter internode length.However,there was no significant change in plant height of OsHAC703 and OsHAC704 mutants,while the flag leaf length and width,and the number of valid tillers increased.In terms of grain morphology,there was no significant difference between OsHACs mutants and wild type.Western blot analysis of histone acetylation level in wild type and mutants showed that H3K36ac,H4K5ac and H4K8ac modification were significantly reduced in OsHAC701 mutants,while only H3K36ac level of the OsHAC703 mutants was significantly reduced.However,there was no significant reduction of histone acetylation level in OsHAC704 mutants.The results indicated that histone acetyltransferase gene mutation changed the level of histone acetylation in rice leaves,and then affected the normal growth of rice plants.