Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is crucial for profiling histone modifications and transcription factor binding throughout the genome. However, its application in economically important plant organs (EIPOs) such as seeds, fruits and flowers is challenging due to their sturdy cell walls and complex constituents. Here we present advanced ChIP (aChIP), an optimized method that efficiently isolates chromatin from plant tissues while simultaneously removing cell walls and cellular constituents. aChIP precisely profiles histone modifications in all 14 tested EIPOs and identifies transcription factor and chromatin-modifying enzyme binding sites. In addition, aChIP enhances ChIP efficiency, revealing numerous novel modified sites compared with previous methods in vegetative tissues. aChIP reveals the histone modification landscape for rapeseed dry seeds, highlighting the intricate roles of chromatin dynamics during seed dormancy and germination. Altogether, aChIP is a powerful, efficient and sensitive approach for comprehensive chromatin profiling in virtually all plant tissues, especially in EIPOs. This study presents an advanced ChIP-seq method, provides a valuable epigenomics resource for 14 economically important plant organs and reveals histone modification landscape dynamics and functions during rapeseed seed dormancy and germination.

Although utilization of heterosis has largely improved the yield of many crops worldwide, the underlying molecular mechanism of heterosis, particularly for allopolyploids, remains unclear. Here, we compared epigenome and transcriptome data of an elite hybrid and its parental lines in three assessed tissues (seedling, flower bud, and silique) to explore their contribution to heterosis in allopolyploid B. napus. Transcriptome analysis illustrated that a small proportion of non-additive genes in the hybrid compared with its parents, as well as parental expression level dominance, might have a significant effect on heterosis. We identified histone modification (H3K4me3 and H3K27me3) variation between the parents and hybrid, most of which resulted from the differences between parents. H3K4me3 variations were positively correlated with gene expression differences among the hybrid and its parents. Furthermore, H3K4me3 and H3K27me3 were rather stable in hybridization and were mainly inherited additively in the B. napus hybrid. Together, our data revealed that transcriptome reprogramming and histone modification remodeling in the hybrid could serve as valuable resources for better understanding heterosis in allopolyploid crops.

Additional file 10: Table S9. Two-hop interactions mediated by genes implicated in flowering-time control.

Photoperiods integrate with the circadian clock to coordinate gene expression rhythms and thus ensure plant fitness to the environment. Genome-wide characterization and comparison of rhythmic genes under different light conditions revealed delayed phase under constant darkness (DD) and reduced amplitude under constant light (LL) in rice. Interestingly, ChIP-seq and RNA-seq profiling of rhythmic genes exhibit synchronous circadian oscillation in H3K9ac modifications at their loci and long non-coding RNAs (lncRNAs) expression at proximal loci. To investigate how gene expression rhythm is regulated in rice, we profiled the open chromatin regions and transcription factor (TF) footprints by time-series ATAC-seq. Although open chromatin regions did not show circadian change, a significant number of TFs were identified to rhythmically associate with chromatin and drive gene expression in a time-dependent manner. Further transcriptional regulatory networks mapping uncovered significant correlation between core clock genes and transcription factors involved in light/temperature signaling. In situ Hi-C of ZT8-specific expressed genes displayed highly connected chromatin association at the same time, whereas this ZT8 chromatin connection network dissociates at ZT20, suggesting the circadian control of gene expression by dynamic spatial chromatin conformation. These findings together implicate the existence of a synchronization mechanism between circadian H3K9ac modifications, chromatin association of TF and gene expression, and provides insights into circadian dynamics of spatial chromatin conformation that associate with gene expression rhythms.

BACKGROUND:Although spatial organization of compartments and topologically associating domains at large scale is relatively well studied, the spatial organization of regulatory elements at fine scale is poorly understood in plants. RESULTS:Here we perform high-resolution chromatin interaction analysis using paired-end tag sequencing approach. We map chromatin interactions tethered with RNA polymerase II and associated with heterochromatic, transcriptionally active, and Polycomb-repressive histone modifications in Arabidopsis. Analysis of the regulatory repertoire shows that distal active cis-regulatory elements are linked to their target genes through long-range chromatin interactions with increased expression of the target genes, while poised cis-regulatory elements are linked to their target genes through long-range chromatin interactions with depressed expression of the target genes. Furthermore, we demonstrate that transcription factor MYC2 is critical for chromatin spatial organization, and propose that MYC2 occupancy and MYC2-mediated chromatin interactions coordinately facilitate transcription within the framework of 3D chromatin architecture. Analysis of functionally related gene-defined chromatin connectivity networks reveals that genes implicated in flowering-time control are functionally compartmentalized into separate subdomains via their spatial activity in the leaf or shoot apical meristem, linking active mark- or Polycomb-repressive mark-associated chromatin conformation to coordinated gene expression. CONCLUSION:The results reveal that the regulation of gene transcription in Arabidopsis is not only by linear juxtaposition, but also by long-range chromatin interactions. Our study uncovers the fine scale genome organization of Arabidopsis and the potential roles of such organization in orchestrating transcription and development.

Background It is challenging to determine the effect of DNA methylation on the epigenetic landscape and the function in higher organisms due to the lack of DNA methylation-free mutants. Results Here, the analysis of a recently generated Arabidopsis mutant completely devoid of DNA methylation reveals that DNA methylation underpins the genome-wide landscape of histone modifications. Complete loss of DNA methylation causes an upheaval of the histone modification landscape, including complete loss of H3K9me2 and widespread redistribution of active and H3K27me3 histone marks, mostly owing to the role of DNA methylation in initiating H3K9me2 deposition and excluding active marks and repressive mark H3K27me3; CG and non-CG methylation can act independently at some genomic regions while they act cooperatively at many other regions. The transcriptional reprogramming upon loss of all DNA methylation correlates with the extensive redistribution or switches of the examined histone modifications. Histone modifications retained or gained in the DNA methylation-free mutant serve as DNA methylation-independent transcriptional regulatory signals: active marks promote genome transcription, whereas the repressive mark H3K27me3 compensates for the lack of DNA hypermethylation/H3K9me2 at multiple transposon families. Conclusions Our results show that an intact DNA methylome constitutes the scaffolding of the epigenomic landscape in Arabidopsis and is critical for controlled genome transcription and ultimately for proper growth and development.

Additional file 2: Table S1. Summary of transcriptome and epigenome data in this study.

RICE INDETERMINATE 1 (RID1) plays a critical role in controlling floral transition in rice (Oryza sativa). However, the molecular basis for this effect, particularly the target genes and regulatory specificity, remains largely unclear. Here, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) in young leaves at the pre-floral-transition stage to identify the target genes of RID1, identifying 2,680 genes associated with RID1 binding sites genome-wide. RID1 binding peaks were highly enriched for TTTGTC, the direct binding motif of the INDETERMINATE DOMAIN protein family that includes RID1. Interestingly, CACGTG and GTGGGCCC, two previously uncharacterized indirect binding motifs, were enriched through the interactions of RID1 with the novel flowering-promoting proteins OsPIL12 and OsTCP11, respectively. Moreover, the ChIP-seq data demonstrated that RID1 bound to numerous rice heading-date genes, such as HEADING DATE 1 (HD1) and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (OsFKF1). Notably, transcriptome sequencing (RNA-seq) analysis revealed roles of RID1 in diverse developmental pathways. Genetic analysis combined with genome-wide ChIP-seq and RNA-seq results showed that RID1 directly binds to the promoter of OsERF#136 (a repressor of rice flowering) and negatively regulates its expression. Overall, our findings provide new insights into the molecular and genetic mechanisms underlying rice floral transition and characterize OsERF#136 as a previously unrecognized direct target of RID1.

Background The daily cycling of plant physiological processes is speculated to arise from the coordinated rhythms of gene expression. However, the dynamics of diurnal 3D genome architecture and their potential functions underlying the rhythmic gene expression remain unclear. Results Here, we reveal the genome-wide rhythmic occupancy of RNA polymerase II (RNAPII), which precedes mRNA accumulation by approximately 2 h. Rhythmic RNAPII binding dynamically correlates with RNAPII-mediated chromatin architecture remodeling at the genomic level of chromatin interactions, spatial clusters, and chromatin connectivity maps, which are associated with the circadian rhythm of gene expression. Rhythmically expressed genes within the same peak phases of expression are preferentially tethered by RNAPII for coordinated transcription. RNAPII-associated chromatin spatial clusters (CSCs) show high plasticity during the circadian cycle, and rhythmically expressed genes in the morning phase and non-rhythmically expressed genes in the evening phase tend to be enriched in RNAPII-associated CSCs to orchestrate expression. Core circadian clock genes are associated with RNAPII-mediated highly connected chromatin connectivity networks in the morning in contrast to the scattered, sporadic spatial chromatin connectivity in the evening; this indicates that they are transcribed within physical proximity to each other during the AM circadian window and are located in discrete “transcriptional factory” foci in the evening, linking chromatin architecture to coordinated transcription outputs. Conclusion Our findings uncover fundamental diurnal genome folding principles in plants and reveal a distinct higher-order chromosome organization that is crucial for coordinating diurnal dynamics of transcriptional regulation.

Rice (Oryza sativa) is a major crop that feeds billions of people, and its yield is strongly influenced by flowering time (heading date). Loss of RICE INDETERMINATE1 (RID1) function causes plants not to flower; thus, RID1 is considered a master switch among flowering-related genes. However, it remains unclear whether other proteins function together with RID1 to regulate rice floral transition. Here, we revealed that the chromatin accessibility and H3K9ac, H3K4me3, and H3K36me3 levels at Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1) loci were significantly reduced in rid1 mutants. Notably, RID1 interacted with SET DOMAIN GROUP PROTEIN 722 (SDG722), a methyltransferase. We determined that SDG722 affects the global level of H3K4me2/3 and H3K36me2/3, and promotes flowering primarily through the Early heading date1-Hd3a/RFT1 pathway. We further established that rice DELLA protein SLENDER RICE1 (SLR1) interacted with RID1 to inhibit its transactivation activity, that SLR1 suppresses rice flowering, and that messenger RNA and protein levels of SLR1 gradually decrease with plant growth. Furthermore, SLR1 competed with SDG722 for interaction with RID1. Overall, our results establish that interplay between RID1, SLR1, and SDG722 feeds into rice flowering-time control.

Epigenomic modifications are instrumental for transcriptional regulation, but comprehensive reference epigenomes remain unexplored in rice. Here, we develop an enhanced chromatin immunoprecipitation (eChIP) approach for plants, and generate genome-wide profiling of five histone modifications and RNA polymerase II occupancy with it. By integrating chromatin accessibility, DNA methylation, and transcriptome datasets, we construct comprehensive epigenome landscapes across various tissues in 20 representative rice varieties. Approximately 81.8% of rice genomes are annotated with different epigenomic properties. Refinement of promoter regions using open chromatin and H3K4me3-marked regions provides insight into transcriptional regulation. We identify extensive enhancer-like promoters with potential enhancer function on transcriptional regulation through chromatin interactions. Active and repressive histone modifications and the predicted enhancers vary largely across tissues, whereas inactive chromatin states are relatively stable. Together, these datasets constitute a valuable resource for functional element annotation in rice and indicate the central role of epigenomic information in understanding transcriptional regulation.

Sulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call 'homeostatic tolerance,' is involved in amino acid signaling in Arabidopsis. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in atg5, atg7, and gcn2 single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in atg5 gcn2 and atg7 gcn2 double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced TOR RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.

The transition from vegetative to reproductive growth is a critical process in the life cycle of higher plants. Previously, we cloned Rice Indeterminate 1 (RID1), which acts as the master switch for the transition from the vegetative to reproductive phase in rice. Although the photoperiod pathway of RID1 inducing expression of the florigen genes Hd3a and RFT1 via Ehd1 has been established, the alternative pathways for the essential flowering transition need to be further examined. Here, we identified a Suppressor of rid1 (SID1), which rescues the never-flowering phenotype of rid1. SID1 encodes an INDETERMINATE DOMAIN (IDD) transcription factor. Mutation in SID1 showed the delayed flowering phenotype. Gain-of-function of SID1, OsIDD1, or OsIDD6 could restore the rid1 to flowering. Further analyses showed SID1 and RID1 directly target the promoter regions of Hd3a and RFT1, two florigen genes in rice. Taken together, our results reveal an autonomous flowering pathway might be mediated by RID1, thereby controlling the phase transition from vegetative to reproductive development in rice.

Programmed cell death (PCD) during tapetum degeneration in postmeiotic anthers is critical for the proper development of male gametophytes in flowering plants. Although several genes involved in this process have been identified recently, the molecular mechanism is still poorly understood. Here, we show that knockout of rice (Oryza sativa) APOPTOSIS INHIBITOR5 (API5), which encodes a putative homolog of antiapoptosis protein Api5 in animals, results in delayed degeneration of the tapetum due to inhibition of the tapetal PCD process leading to defects in formation of male gametophytes. Os API5 is a nuclear protein that interacts with two DEAD-box ATP-dependent RNA helicases, API5-INTERACTING PROTEIN1 (AIP1) and AIP2. AIP1 and AIP2 are homologs of yeast (Saccharomyces cerevisiae) Suppressor of Bad Response to Refrigeration1 protein 2 (SUB2p) that have critical roles in transcription elongation and pre-mRNA splicing. Os AIP1 and AIP2 can form dimers and interact directly with the promoter region of CP1, a rice cysteine protease gene. Suppression of Os AIP1/2 leads to down-regulation of CP1, resulting in sterility, which is highly similar to the effects of suppressed expression of Os CP1. Our results uncover a previously unknown pathway for regulating PCD during tapetum degeneration in rice, one that may be conserved among eukaryotic organisms.