Study On Chromatin Accessibility Changes And Transcriptional Regulatory Networks Of The Rice PTGMS Line Wuxiang S

A novel PTGMS(Photoperiod-thermo-sensitive genic male sterile)male-sterile line Wuxiang S(WXS),generated by our laboratory and derived from a mutant tms5 locus in indica rice,was used as an experimental material in the present study.To determine the critical period of WXS fertility conversion,we performed cytological observations.And then,the changes in chromatin accessibility along with histone modifications were assessed to conduct a global search for both epigenetic and transcriptional variation caused by temperature fluctuation.In addition,the differential cis-regulatory elements occupied by specific TFs were combined with our RNA sequencing gene expression data to identify new TF control modules and to predict the downstream target genes of these TF networks.The main results of this study are as follows:1.To investigate the H3K9ac and H3K4me2 in the male-fertile(WXS-F)and the male-sterile(WXS-S)during the meiosis period(P3)and the uninucleate period(P4),the ChIP-seq of genome-wide histone modifications was performed.The results revealed that the levels of both antibodies across five panicle developmental stages in WXS-F showed significantly higher than in WXS-S.H3K9ac is present exclusively on active genes,while H3K4me2 occurs on both active and inactive genes;in addition,the former mainly occurs at the initiation of transcription,and the latter is involved in both transcription initiation and the early stage of transcription elongation.Therefore,the high H3K4me2 levels within the gene body potentially reflect the presence and activity of intragenic accessible chromatin elements,and the potentially active genes carrying the H3K4me2 mark under the permissive state of chromatin can easily be regulated via binding of various TFs.2.The global transcriptome profiles were examined to investigate the underlying molecular mechanisms that regulate fertility conversion.Pairwise comparisons of WXS-S-P3,WXS-S-P4,WXS-F-P3 and WXS-F-P4 were used to identify 7,870 differentially expressed genes(DEGs).Six clusters with specific expression patterns were identified by analysis of all 7,870 DEGs.To elucidate the functional implications of the clustered DEGs,the GO terms of gene suggest WXS-S mainly participates in some basic cellular developmental activities to ensure normal growth.However,WXS-F exhibits the activity of transcription factor,carbohydrate metabolic process,cell wall and stress response.The KEGG analysis of DEGs in WXS-S versus WXS-F revealed a principal enrichment of phenolic metabolites such as phenylpropanoid biosynthesis and phenylalanine metabolism,which are parts of the chemical composition of sporopollenin,a major component of the tough exine walls of microspores and pollens.To identify the key candidate transcriptional regulators involved in fertility conversion,the expression profiles of TFs were analysed in detail.A total of 687 TFs belonging to 46 families were differentially expressed with distinct patterns.Most of these transcription factors have been proven to mainly participate in the pollen development and cell wall synthesis,which might play an essential role in fertility conversion.3.We performed ATAC-seq to identify accessible chromatin regions of WXS-F and WXS-S at P3 and P4,and identified 59,787 THSs covering approximately 40 Mb(～10%of the genome),which were distributed throughout the genome in genic and intergenic regions.The genomic distribution of the THSs showed a very similar pattern among the four samples,with the up2k region occupying a major part,followed by the down2k and intergenic regions.Genes with higher levels of expression displayed higher levels of Tn5 transposase sensitivity within this region.The numbers,locations,and levels of Tn5 sensitivity of THS sites can vary significantly among different sample types and may be induced by temperature fluctuations.Thus,the identification of key regulatory modules under different temperature will be an essential future effort to achieve a comprehensive understanding of the transcriptional regulation of gene expression during fertility conversion.When compared with DEGs,the expression of these target genes might be activated or repressed due to the function of TFs that bind to the THSs.Most of the target genes associated with WXS-S-enriched-dTHSs were mainly repressed,and they might be activated or not repressed in response to temperature changes in WXS-F.GO analysis of these genes revealed known functions in response to the altered environment,which indicated that WXS-F suffering from temperature alterations activated temperature-responsive mechanisms to re-establish homeostasis and protect normal anther development.4.The tms5 gene kept mutant in both WXS-F and WXS-S with defective RNaseZS1 protein,while the expression level of UbL40S were much higher in WXS-S than in WXS-F.Our experiment data confirmed that the ERF65 and GATA10 could directly bind on the promoter of UbL40,and suggested that ERF65 repressed UbL40 mRNA expression,and GATA10 might act as a mediator to coordinate with other transcription factors and chromatin remodelling complex to affect UbL40 mRNA expression.These modules related with other TFs may directly or indirectly affect various metabolic pathway-related genes to coordinate plant development and growth with proper anther development.In the present study,the firstly successful application of ATAC-seq in the PTGMS two-line rice were performed to obtain the accessible chromatin regions.Most of the key transcription factors regulating the fertility conversion were discovered by their unique motifs in the differentially accessible chromatin regions.Our results uncovered the ERF65/GATA10 module worked as a key component directly regulating downstream UbL40 mRNA expression,which partly explained the reason why the UbL40 mRNA expression could over accumulate in high temperature,and complemented the transcriptional regulatory mechanism of UbL40 mRNA regulation directly causing defective pollen production and male sterility.We outline herein a widely applicable approach for combining accessible chromatin profiling with gene expression analyses and histone modifications to uncover transcriptional regulation and new regulatory modules in response to temperature fluctuation,which can provide important evidences regarding the transcriptional regulatory mechanisms of fertility conversion.