R-loops act as regulatory switches modulating transcription of COLD-responsive genes in rice.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance.

Characterization of functional relationships of R-loops with gene transcription and epigenetic modifications in rice.

We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expression and epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-loop peak levels was dependent on the positions of R-loops within gene structures (hereafter named "genic position"). Both antisense only (ASO)-R-loops and sense/antisense (S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peak levels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generally spread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. In addition, integrative analyses of R-loop data with existing RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitive sites sequencing (DNase-seq), and whole-genome bisulfite sequencing (WGBS or BS-seq) data revealed interrelationships and intricate connections among R-loops, gene expression, and epigenetic signatures. Experimental validation provided evidence that the demethylation of both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants that reveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptional regulation.

Salt-Responsive Genes are Differentially Regulated at the Chromatin Levels Between Seedlings and Roots in Rice.

The elucidation of epigenetic responses of salt-responsive genes facilitates understanding of the underlying mechanisms that confer salt tolerance in rice. However, it is still largely unknown how epigenetic mechanisms are associated with the expression of salt-responsive genes in rice and other crops. In this study, we reported tissue-specific gene expression and tissue-specific changes in chromatin modifications or signatures between seedlings and roots in response to salt treatment. Our study indicated that among six of individual mark examined (H3K4me3, H3K27me3, H4K12ac, H3K9ac, H3K27ac and H3K36me3), a positive association between salt-related changes in histone marks and the expression of differentially expressed genes (DEGs) was observed only for H3K9ac and H4K12ac in seedlings and H3K36me3 in roots. In contrast, chromatin states (CSs) with combinations of six histone modification marks played crucial roles in the differential expression of salt-responsive genes between seedlings and roots. Most importantly, CS7 containing the bivalent marks H3K4me3 and H3K27me3, with a mutual exclusion of functions with each other, displayed distinct functions in the expression of DEGs in both tissues. Specifically, H3K27me3 in CS7 mainly suppressed the expression of DEGs in roots, while H3K4me3 affected the expression of down- and up-regulated genes, possibly by antagonizing the repressive role of H3K27me3 in seedlings. Our findings indicate distinct impacts of the CSs on the differential expression of salt-responsive genes between seedlings and roots in rice, which provides an important background for understanding chromatin-based epigenetic mechanisms that might confer salt tolerance in plants.

Chromatin states responsible for the regulation of differentially expressed genes under 60Co~γ ray radiation in rice.

BACKGROUND: The role of histone modifications in the DNA damage response has been extensively studied in non-plant systems, including mammals and yeast. However, there is a lack of detailed evidence showing how chromatin dynamics, either an individual mark or combined chromatin states, participate in regulating differentially expressed genes in the plant DNA damage response. RESULTS: In this study, we used RNA-seq and ChIP-seq to show that differentially expressed genes (DEGs), in response to ionizing radiation (IR), might be involved in different pathways responsible for the DNA damage response. Moreover, chromatin structures associated with promoters, exons and intergenic regions are significantly affected by IR. Most importantly, either an individual mark or a certain chromatin state was found to be highly correlated with the expression of up-regulated genes. In contrast, only the chromatin states, as opposed to any individual marks tested, are related to the expression of the down-regulated genes. CONCLUSIONS: Our findings demonstrate that IR-related DEGs are modulated by distinct epigenetic mechanisms. Either chromatin states or distinct histone dynamics may act sequentially or in combination in regulating up-regulated genes, but the complex chromatin structure is mainly responsible for the expression of down-regulated genes. Thus, this study provides new insights into how up- and down-regulated genes are epigenetically regulated at the chromatin levels, thereby helping us to understand distinct epigenetic mechanisms that function in the plant DNA damage response.

Histone modifications facilitate the coexpression of bidirectional promoters in rice.

BACKGROUND: Bidirectional gene pairs are highly abundant and mostly co-regulated in eukaryotic genomes. The structural features of bidirectional promoters (BDPs) have been well studied in yeast, humans and plants. However, the underlying mechanisms responsible for the coexpression of BDPs remain understudied, especially in plants. RESULTS: Here, we characterized chromatin features associated with rice BDPs. Several unique chromatin features were present in rice BDPs but were missing from unidirectional promoters (UDPs), including overrepresented active histone marks, canonical nucleosomes and underrepresented H3K27me3. In particular, overrepresented active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me2 and H3K36me3) were truly overrepresented in type I BDPs but not in the other two BDPs, based on a Kolmogorov-Smirnov test. CONCLUSIONS: Our analyses indicate that active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me3, H3K9ac and H3K27ac) may coordinate with repressive marks (H3K27me3 and H3K9me1/3) to build a unique chromatin structure that favors the coregulation of bidirectional gene pairs. Thus, our findings help to enhance the understanding of unique epigenetic mechanisms that regulate bidirectional gene pairs and may improve the manipulation of gene pairs for crop bioengineering.

Functional characterization of chloroplast transit peptide in the small subunit of Rubisco in maize.

Functions of domains or motifs, which are encoded by the transit peptide (TP) of the precursor of the small subunit of Rubisco (prSSU), have been investigated intensively in dicots. Functional characterization of the prSSU TP, however, is still understudied in maize. In this study, we found that the TP of maize prSSU1 did not function fully in chloroplast targeting in Arabidopsis or vice versa, indicating the divergent function of TPs in chloroplast targeting between maize and Arabidopsis. Through deletion or substitution assays, we found that the N-terminal region of maize or Arabidopsis prSSU1 was necessary and sufficient for importing specifically the fused-green fluorescent protein (GFP) into each corresponding chloroplast. Finally, we found that the first-five amino acids and MM motif in the N-terminal domain of the maize TP played an essential role in maize chloroplast targeting. Thus, our analyses demonstrate that the N-terminal domain of the prSSU1 TP is the key determinant in chloroplast targeting between maize and Arabidopsis. Our study highlights the unique properties of the maize prSSU1 TP in chloroplast targeting, thus helping to understand the role of N-terminal domain in chloroplast targeting across species. It will help to manipulate chloroplast transit peptides (cTPs) for crop bioengineering.

Functional characterization of open chromatin in bidirectional promoters of rice.

Bidirectional gene pairs tend to be highly coregulated and function in similar biological processes in eukaryotic genomes. Structural features and functional consequences of bidirectional promoters (BDPs) have received considerable attention among diverse species. However, the underlying mechanisms responsible for the bidirectional transcription and coexpression of BDPs remain poorly understood in plants. In this study, we integrated DNase-seq, RNA-seq, ChIP-seq and MNase-seq data and investigated the effect of physical DNase I hypersensitive site (DHS) positions on the transcription of rice BDPs. We found that the physical position of a DHS relative to the TSS of bidirectional gene pairs can affect the expression of the corresponding genes: the closer a DHS is to the TSS, the higher is the expression level of the genes. Most importantly, we observed that the distribution of DHSs plays a significant role in the regulation of transcription and the coexpression of gene pairs, which are possibly mediated by orchestrating the positioning of histone marks and canonical nucleosomes around BDPs. Our results demonstrate that the combined actions of chromatin structures with DHSs, which contain functional cis-elements for interaction with transcriptional machinery, may play an important role in the regulation of the bidirectional transcription or coexpression in rice BDPs. Our findings may help to enhance the understanding of DHSs in the regulation of bidirectional gene pairs.