| R-loop is a three-strand nucleic acid structure that exists widely in the genome of an organism.It consists of a heterozygous double strand of DNA:RNA and a single strand of DNA that has been replaced.R-loop structure can affect a series of biological processes in plant and animal genomes.For example,it causes the instability of the human genome,increases the risk of cancer in humans,causes DNA double-strand breaks,affects gene transcription,and regulates plant flowering and root growth.At present,compared with the studies on humans and animals,the research on R-loop in plants is very limited and needs to be further developed.The existing research results show that cold or low temperature stress will have a serious impact on the normal growth and development of crops,and even lead to crop yield,thus threatening the safety of food production.Therefore,analyzing the epigenetic molecular mechanism of plant response to cold stress will provide a theoretical reference for molecular breeding of cold resistance in crops.Previous studies in our laboratory showed that the R-loop structure in rice genome has a dual regulatory effect on gene expression,but whether or how the R-loop structure affects gene expression and how it participates in the response of rice to cold stress has not been reported yet.Therefore,the study of the molecular mechanism of R-loop mediated cold stress is beneficial to deepen the understanding of the molecular mechanism of plant response to cold stress,and to enrich the biological functions of R-loop in plants,providing theoretical support for stress-resistant molecular breeding of crops based on R-loop.In this study,rice Nipponharp growing at room temperature was used as the control and the seedlings of the same period were treated at 4℃ for one day as the research object.Firstly,we identified the effects of cold treatment on the distribution and sequence characteristics of R-loop sites in rice genome.Secondly,the relationship between differential R-loop sites and differentially expressed genes induced by cold treatment in rice was analyzed.Finally,cold-induced gene co-expression regulatory network and cold-induced R-loop related differential transcription factor regulatory network were constructed and weighted gene co-expression network analysis(WGCNA)was performed.The main findings of this study are as follows:1.Compared with the control,cold treatment resulted in the appearance of 17,103 new R-loop sites and the disappearance of 6,809 R-loop sites in rice genome.In addition,the number of R-loop sites in rice seedlings was significantly increased after cold treatment.In addition,the distribution ratio of the number of sites in the promoter and exon regions was significantly reduced in the cases of R-loop(SO-R-loop)containing RNA strand and gene transcription in the same direction on a single gene and R-loop(ASO-R-loop)containing RNA strand and gene transcription in the opposite direction on a single gene.Among the variation R-loop sites,the number of R-loop sites in the control group with increased number of R-loop sites compared to the cold treatment group(CK-biased R-loop)was 4,847.Compared to the control group,the cold-treated group that has an increased number of R-loops(Cold-biased R-loop)was 9009,which is approximately 45% more than the CK-biased R-loop number.Cold-biased R-loops represent a higher proportion of promoter and exon regions than CK-biased R-loops.These results indicated that cold treatment affected the stability and dynamic distribution of R-loop in rice genome,and suggested that genomic R-loop changes caused by cold stress.2.The correlation analysis results of ASO-R-loop,SO-R-loop and R-loop(S/AS-R-loop)with reverse direction and same direction of RNA strand and gene transcription showed that SO-R-loop was mainly distributed in gene region to inhibit the expression of genes;ASO-R-loop can activate gene expression in promoter region.S/AS-R-loop simultaneously promotes and inhibits gene expression: it activates gene expression in the promoter region and inhibits gene expression in the terminator region and the promoter region.3.Compared with the control,cold treatment increased GC content,GC-skew and AT-skew overall levels in R-loop region,and such sequence differences mainly came from cold-induced R-loops.Moreover,cold treatment resulted in changes in R-loop sequence characteristics and motif sequences contained in the R-loop,which could lead to differences in binding of related functional proteins.4.After cold treatment,the GO enrichment degree of R-loop related genes changed,which was mainly related to biological,molecular and cellular life processes,suggesting that R-loop may be involved in the response of rice to cold stress by regulating the expression of these genes.5.The chromatin status of rice genome was changed dynamically after cold treatment.Cold treatment increased the abundance of H3K27 ac and H4K12 ac,and decreased the abundance of H3K36me3,H3K4me3,and H3K9 ac.In addition,cold treatment increased the probability of histone modification co-distributing with R-loop by 20%.In particular,Cold treatment increased the enrichment of histones H3K27 ac,H3K36me3,and H4K12 ac in the R-loop region and decreased the enrichment of histones H3K4me3 and H3K9ac.These enrichment changes are related to the Cold-biased R-loop.6.WGCNA results indicates that R-loop direct affects the number of cold induced related geneis 22.7%(1,342/5,889),and by influencing the expression of R-loop related TF,there by indirect adjustment cold induced genes differentially expressed.Among the 11co-expression modules,differential TF regulated 3,006 differential genes.Therefore,the number of cold-induced differential expression genes directly and indirectly regulated by R-loop was 3,604,accounting for 61%(3,604/5,889)of the total differential genes.In particular,five key TF genes,Os WBKY59,ERF,HB-other,Os ERF101 and Os ZOU-1,have been identified through transcriptional regulatory networks,which may play an important role in mediating cold stress response in rice.In conclusion,our study results show that R-loop by influencing the chromatin state and directly or indirectly regulating the expression of cold-induced genes,thereby involved in cold stress response of the rice.Our study enriched the plant R-loop biological function research,strengthened the understanding of the molecular mechanisms of rice cold stress,and provided theoretical reference based on R-loop cold resistance of rice molecular breeding. |
