Light control of three-dimensional chromatin organization in soybean.

Higher-order chromatin structure is critical for regulation of gene expression. In plants, light profoundly affects the morphogenesis of emerging seedlings as well as global gene expression to ensure optimal adaptation to environmental conditions. However, the changes and functional significance of chromatin organization in response to light during seedling development are not well documented. We constructed Hi-C contact maps for the cotyledon, apical hook and hypocotyl of soybean subjected to dark and light conditions. The resulting high-resolution Hi-C contact maps identified chromosome territories, A/B compartments, A/B sub-compartments, TADs (Topologically Associated Domains) and chromatin loops in each organ. We observed increased chromatin compaction under light and we found that domains that switched from B sub-compartments in darkness to A sub-compartments under light contained genes that were activated during photomorphogenesis. At the local scale, we identified a group of TADs constructed by gene clusters consisting of different numbers of Small Auxin-Upregulated RNAs (SAURs), which exhibited strict co-expression in the hook and hypocotyl in response to light stimulation. In the hypocotyl, RNA polymerase II (RNAPII) regulated the transcription of a SAURs cluster under light via TAD condensation. Our results suggest that the 3D genome is involved in the regulation of light-related gene expression in a tissue-specific manner.

Increased long-distance and homo-trans interactions related to H3K27me3 in Arabidopsis hybrids.

In plants, the genome structure of hybrids changes compared with their parents, but the effects of these changes in hybrids remain elusive. Comparing reciprocal crosses between Col × C24 and C24 × Col in Arabidopsis using high-throughput chromosome conformation capture assay (Hi-C) analysis, we found that hybrid three-dimensional (3D) chromatin organization had more long-distance interactions relative to parents, and this was mainly located in promoter regions and enriched in genes with heterosis-related pathways. The interactions between euchromatin and heterochromatin were increased, and the compartment strength decreased in hybrids. In compartment domain (CD) boundaries, the distal interactions were more in hybrids than their parents. In the hybrids of CURLY LEAF (clf) mutants clfCol × clfC24 and clfC24 × clfCol , the heterosis phenotype was damaged, and the long-distance interactions in hybrids were fewer than in their parents with lower H3K27me3. ChIP-seq data revealed higher levels of H3K27me3 in the region adjacent to the CD boundary and the same interactional homo-trans sites in the wild-type (WT) hybrids, which may have led to more long-distance interactions. In addition, the differentially expressed genes (DEGs) located in the boundaries of CDs and loop regions changed obviously in WT, and the functional enrichment for DEGs was different between WT and clf in the long-distance interactions and loop regions. Our findings may therefore propose a new epigenetic explanation of heterosis in the Arabidopsis hybrids and provide new insights into crop breeding and yield increase.

Mapping nucleosome-resolution chromatin organization and enhancer-promoter loops in plants using Micro-C-XL.

Although chromatin organizations in plants have been dissected at the scales of compartments and topologically associating domain (TAD)-like domains, there remains a gap in resolving fine-scale structures. Here, we use Micro-C-XL, a high-throughput chromosome conformation capture (Hi-C)-based technology that involves micrococcal nuclease (instead of restriction enzymes) and long cross-linkers, to dissect single nucleosome-resolution chromatin organization in Arabidopsis. Insulation analysis reveals more than 14,000 boundaries, which mostly include chromatin accessibility, epigenetic modifications, and transcription factors. Micro-C-XL reveals associations between RNA Pols and local chromatin organizations, suggesting that gene transcription substantially contributes to the establishment of local chromatin domains. By perturbing Pol II both genetically and chemically at the gene level, we confirm its function in regulating chromatin organization. Visible loops and stripes are assigned to super-enhancers and their targeted genes, thus providing direct insights for the identification and mechanistic analysis of distal CREs and their working modes in plants. We further investigate possible factors regulating these chromatin loops. Subsequently, we expand Micro-C-XL to soybean and rice. In summary, we use Micro-C-XL for analyses of plants, which reveal fine-scale chromatin organization and enhancer-promoter loops and provide insights regarding three-dimensional genomes in plants.

Conserved H3K27me3-associated chromatin looping mediates physical interactions of gene clusters in plants.

Higher-order chromatin organization is essential for transcriptional regulation, genome stability maintenance, and other genome functions. Increasing evidence has revealed significant differences in 3D chromatin organization between plants and animals. However, the extent, pattern, and rules of chromatin organization in plants are still unclear. In this study, we systematically identified and characterized long-range chromatin loops in the Arabidopsis 3D genome. We identified hundreds of long-range cis chromatin loops and found their anchor regions are closely associated with H3K27me3 epigenetic modifications. Furthermore, we demonstrated that these chromatin loops are dependent on Polycomb group (PcG) proteins, suggesting that the Polycomb repressive complex 2 (PRC2) complex is essential for establishing and maintaining these novel loops. Although most of these PcG-medicated chromatin loops are stable, many of these loops are tissue-specific or dynamically regulated by different treatments. Interestingly, tandemly arrayed gene clusters and metabolic gene clusters are enriched in anchor regions. Long-range H3K27me3-marked chromatin interactions are associated with the coregulation of specific gene clusters. Finally, we also identified H3K27me3-associated chromatin loops associated with gene clusters in Oryza sativa and Glycine max, indicating that these long-range chromatin loops are conserved in plants. Our results provide novel insights into genome evolution and transcriptional coregulation in plants.

Nitrogen starvation induces genome-wide activation of transposable elements in Arabidopsis.

Nitrogen (N) availability is a major limiting factor for plant growth and agricultural productivity. Although the gene regulation network in response to N starvation has been extensively studied, it remains unknown whether N starvation has an impact on the activity of transposable elements (TEs). Here, we report that TEs can be transcriptionally activated in Arabidopsis under N starvation conditions. Through genetic screening of idm1-14 suppressors, we cloned GLU1, which encodes a glutamate synthase that catalyzes the synthesis of glutamate in the primary N assimilation pathway. We found that glutamate synthase 1 (GLU1) and its functional homologs GLU2 and glutamate transport 1 (GLT1) are redundantly required for TE silencing, suggesting that N metabolism can regulate TE activity. Transcriptome and methylome analyses revealed that N starvation results in genome-wide TE activation without inducing obvious alteration of DNA methylation. Genetic analysis indicated that N starvation-induced TE activation is also independent of other well-established epigenetic mechanisms, including histone methylation and heterochromatin decondensation. Our results provide new insights into the regulation of TE activity under stressful environments in planta.

The small nucleolar RNA SnoR28 regulates plant growth and development by directing rRNA maturation.

Small nucleolar RNAs (snoRNAs) are noncoding RNAs (ncRNAs) that guide chemical modifications of structural RNAs, which are essential for ribosome assembly and function in eukaryotes. Although numerous snoRNAs have been identified in plants by high-throughput sequencing, the biological functions of most of these snoRNAs remain unclear. Here, we identified box C/D SnoR28.1s as important regulators of plant growth and development by screening a CRISPR/Cas9-generated ncRNA deletion mutant library in Arabidopsis thaliana. Deletion of the SnoR28.1 locus, which contains a cluster of three genes producing SnoR28.1s, resulted in defects in root and shoot growth. SnoR28.1s guide 2'-O-ribose methylation of 25S rRNA at G2396. SnoR28.1s facilitate proper and efficient pre-rRNA processing, as the SnoR28.1 deletion mutants also showed impaired ribosome assembly and function, which may account for the growth defects. SnoR28 contains a 7-bp antisense box, which is required for 2'-O-ribose methylation of 25S rRNA at G2396, and an 8-bp extra box that is complementary to a nearby rRNA methylation site and is partially responsible for methylation of G2396. Both of these motifs are required for proper and efficient pre-rRNA processing. Finally, we show that SnoR28.1s genetically interact with HIDDEN TREASURE2 and NUCLEOLIN1. Our results advance our understanding of the roles of snoRNAs in Arabidopsis.

ALBA proteins confer thermotolerance through stabilizing HSF messenger RNAs in cytoplasmic granules.

High temperature is one of the major environmental stresses affecting plant growth and fitness. Heat stress transcription factors (HSFs) play critical roles in regulating the expression of heat-responsive genes. However, how HSFs are regulated remains obscure. Here, we show that ALBA4, ALBA5 and ALBA6, which phase separate into stress granules (SGs) and processing bodies (PBs) under heat stress, directly bind selected messenger RNAs, including HSF mRNAs, and recruit them into SGs and PBs to protect them from degradation under heat stress in Arabidopsis. The alba456 triple mutants, but not single and double mutants, display pleiotropic developmental defects and hypersensitivity to heat stress. Mutations in XRN4, a cytoplasmic 5' to 3' exoribonuclease, can rescue the observed developmental and heat-sensitive phenotypes of alba456 seedlings. Our study reveals a new layer of regulation for HSFs whereby HSF mRNAs are stabilized by redundant action of ALBA proteins in SGs and PBs for plant thermotolerance.

Genomic insights on the contribution of introgressions from Xian/Indica to the genetic improvement of Geng/Japonica rice cultivars.

Hybridization between Xian/indica (XI) and Geng/japonica (GJ) rice combined with utilization of plant ideotypes has greatly contributed to yield improvements in modern GJ rice in China over the past 50 years. To explore the genomic basis of improved yield and disease resistance in GJ rice, we conducted a large-scale genomic landscape analysis of 816 elite GJ cultivars representing multiple eras of germplasm from China. We detected consistently increasing introgressions from three XI subpopulations into GJ cultivars since the 1980s and found that the XI genome introgressions significantly increased the grain number per panicle (GN) and decreased the panicle number per plant. This contributed to the improvement of plant type during modern breeding, changing multi-tiller plants to moderate tiller plants with a large panicle size and increasing the blast resistance. Notably, we found that key gene haplotypes controlling plant architecture, yield components, and pest and disease resistance, including IPA1, SMG1, DEP3, Pib, Pi-d2, and Bph3, were introduced from XI rice by introgression. By GWAS analysis, we detected a GN-related gene Gnd5, which had been consistently introgressed from XI into GJ cultivars since the 1980s. Gnd5 is a GRAS transcription factor gene, and Gnd5 knockout mutants showed a significant reduction in GN. The estimated genetic effects of genes varied among different breeding locations, which explained the distinct introgression levels of XI gene haplotypes, including Gnd5, DEP3, etc., to these GJ breeding pedigrees. These findings reveal the genomic contributions of introgressions from XI to the trait improvements of GJ rice cultivars and provide new insights for future rice genomic breeding.

Chromatin remodeling complexes regulate genome architecture in Arabidopsis.

In eukaryotes, three-dimensional (3D) chromatin architecture maintains genome stability and is important in regulating gene transcription. However, little is known about the mechanisms by which diverse ATP-dependent chromatin remodeling complexes regulate the 3D chromatin structure in plants. We examined the 3D chromatin structure within the ATPase subunit of the SWI/SNF, ISWI, INO80, and CHD remodeling complexes in wild-type (WT) and mutant Arabidopsis thaliana plants by combining high-throughput sequencing with in situ Hi-C, the enrichment of histone marks, nucleosome density, and gene expression. We found that compartment regions switched and compartmental strength was significantly weakened in all four enzyme mutants. Chromatin remodeling complexes differentially regulated the nucleosome distribution pattern and density within the switching compartments. Alterations of nucleosome distribution pattern and density were associated with a reduction in H3K27me3 levels in the chromatin remodeling enzyme mutants and led to compartment switching. Our data show that chromatin remodeling complexes regulate the linear nucleosome distribution pattern and density to promote H3K27me3 deposition, which in turn regulates 3D chromatin structure.

Deciphering the synergistic and redundant roles of CG and non-CG DNA methylation in plant development and transposable element silencing.

DNA methylation plays key roles in transposable element (TE) silencing and gene expression regulation. DNA methylation occurs at CG, CHG and CHH sequence contexts in plants. However, the synergistic and redundant roles of CG and non-CG methylation are poorly understood. By introducing CRISPR/Cas9-induced met1 mutation into the ddcc (drm1 drm2 cmt2 cmt3) mutant, we attempted to knock out all five DNA methyltransferases in Arabidopsis and then investigate the synergistic and redundant roles of CG and non-CG DNA methylation. We found that the homozygous ddcc met1 quintuple mutants are embryonically lethal, although met1 and ddcc mutants only display some developmental abnormalities. Unexpectedly, the ddcc met1 quintuple mutations only reduce transmission through the male gametophytes. The ddcc met1+/- mutants show apparent size divergence, which is not associated with difference in DNA methylation patterns, but associated with the difference in the levels of DNA damage. Finally, we show that a group of TEs are specifically activated in the ddcc met1+/- mutants. This work reveals that CG and non-CG DNA methylation synergistically and redundantly regulate plant reproductive development, vegetative development and TE silencing in Arabidopsis. Our findings provide insights into the roles of DNA methylation in plant development.

TCP transcription factors suppress cotyledon trichomes by impeding a cell differentiation-regulating complex.

Trichomes are specialized epidermal cells that act as barriers against biotic and abiotic stresses. Although the formation of trichomes on hairy organs is well studied, the molecular mechanisms of trichome inhibition on smooth organs are still largely unknown. Here, we demonstrate that the CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors inhibit the formation of trichomes on cotyledons in Arabidopsis (Arabidopsis thaliana). The tcp2/3/4/5/10/13/17 septuple mutant produces cotyledons with ectopic trichomes on the adaxial sides. The expression patterns of TCP genes are developmentally regulated during cotyledon development. TCP proteins directly interact with GLABRA3 (GL3), a key component of the MYB transcription factor/basic helix-loop-helix domain protein/WD40-repeat proteins (MYB-bHLH-WD40, MBW) complex essential for trichome formation, to interfere with the transactivation activity of the MBW complex in cotyledons. TCPs also disrupt the MBW complex-R3 MYB negative feedback loop by directly promoting the expression of R3 MYB genes, which enhance the repression of the MBW complex. Our findings reveal a molecular framework in which TCPs suppress trichome formation on adaxial sides of cotyledons by repressing the activity of the MBW complex at the protein level and the transcripts of R3 MYB genes at the transcriptional level.

Phylogenomic Insights into Deep Phylogeny of Angiosperms Based on Broad Nuclear Gene Sampling.

Angiosperms (flowering plants) are the most diverse and species-rich group of plants. The vast majority (∼99.95%) of angiosperms form a clade called Mesangiospermae, which is subdivided into five major groups: eudicots, monocots, magnoliids, Chloranthales, and Ceratophyllales. The relationships among these Mesangiospermae groups have been the subject of long debate. In this study, we assembled a phylogenomic dataset of 1594 genes from 151 angiosperm taxa, including representatives of all five lineages, to investigate the phylogeny of major angiosperm lineages under both coalescent- and concatenation-based methods. We dissected the phylogenetic signal and found that more than half of the genes lack phylogenetic information for the backbone of angiosperm phylogeny. We further removed the genes with weak phylogenetic signal and showed that eudicots, Ceratophyllales, and Chloranthales form a clade, with magnoliids and monocots being the next successive sister lineages. Similar frequencies of gene tree conflict are suggestive of incomplete lineage sorting along the backbone of the angiosperm phylogeny. Our analyses suggest that a fully bifurcating species tree may not be the best way to represent the early radiation of angiosperms. Meanwhile, we inferred that the crown-group angiosperms originated approximately between 255.1 and 222.2 million years ago, and Mesangiospermae diversified into the five extant groups in a short time span (∼27 million years) at the Early to Late Jurassic.

Active DNA demethylation regulates tracheary element differentiation in Arabidopsis.

DNA demethylation is important for the erasure of DNA methylation. The role of DNA demethylation in plant development remains poorly understood. Here, we found extensive DNA demethylation in the CHH context around pericentromeric regions and DNA demethylation in the CG, CHG, and CHH contexts at discrete genomic regions during ectopic xylem tracheary element (TE) differentiation. While loss of pericentromeric methylation occurs passively, DNA demethylation at a subset of regions relies on active DNA demethylation initiated by DNA glycosylases ROS1, DML2, and DML3. The ros1 and rdd mutations impair ectopic TE differentiation and xylem development in the young roots of Arabidopsis seedlings. Active DNA demethylation targets and regulates many genes for TE differentiation. The defect of xylem development in rdd is proposed to be caused by dysregulation of multiple genes. Our study identifies a role of active DNA demethylation in vascular development and reveals an epigenetic mechanism for TE differentiation.

Heat stress-induced transposon activation correlates with 3D chromatin organization rearrangement in Arabidopsis.

In higher eukaryotes, heterochromatin is mainly composed of transposable elements (TEs) silenced by epigenetic mechanisms. But, the silencing of certain heterochromatin-associated TEs is disrupted by heat stress. By comparing genome-wide high-resolution chromatin packing patterns under normal or heat conditions obtained through Hi-C analysis, we show here that heat stress causes global rearrangement of the 3D genome in Arabidopsis thaliana. Contacts between pericentromeric regions and distal chromosome arms, as well as proximal intra-chromosomal interactions along the chromosomes, are enhanced. However, interactions within pericentromeres and those between distal intra-chromosomal regions are decreased. Many inter-chromosomal interactions, including those within the KNOT, are also reduced. Furthermore, heat activation of TEs exhibits a high correlation with the reduction of chromosomal interactions involving pericentromeres, the KNOT, the knob, and the upstream and downstream flanking regions of the activated TEs. Together, our results provide insights into the relationship between TE activation and 3D genome reorganization.

Canonical cytosolic iron-sulfur cluster assembly and non-canonical functions of DRE2 in Arabidopsis.

As a component of the Cytosolic Iron-sulfur cluster Assembly (CIA) pathway, DRE2 is essential in organisms from yeast to mammals. However, the roles of DRE2 remain incompletely understood largely due to the lack of viable dre2 mutants. In this study, we successfully created hypomorphic dre2 mutants using the CRISPR/Cas9 technology. Like other CIA pathway mutants, the dre2 mutants have accumulation of DNA lesions and show constitutive DNA damage response. In addition, the dre2 mutants exhibit DNA hypermethylation at hundreds of loci. The mutant forms of DRE2 in the dre2 mutants, which bear deletions in the linker region of DRE2, lost interaction with GRXS17 but have stronger interaction with NBP35, resulting in the CIA-related defects of dre2. Interestingly, we find that DRE2 is also involved in auxin response that may be independent of its CIA role. DRE2 localizes in both the cytoplasm and the nucleus and nuclear DRE2 associates with euchromatin. Furthermore, DRE2 directly associates with multiple auxin responsive genes and maintains their normal expression. Our study highlights the importance of the linker region of DRE2 in coordinating CIA-related protein interactions and identifies the canonical and non-canonical roles of DRE2 in maintaining genome stability, epigenomic patterns, and auxin response.

Chloroplast Phylogenomic Inference of Green Algae Relationships.

The green algal phylum Chlorophyta has six diverse classes, but the phylogenetic relationship of the classes within Chlorophyta remains uncertain. In order to better understand the ancient Chlorophyta evolution, we have applied a site pattern sorting method to study compositional heterogeneity and the model fit in the green algal chloroplast genomic data. We show that the fastest-evolving sites are significantly correlated with among-site compositional heterogeneity, and these sites have a much poorer fit to the evolutionary model. Our phylogenomic analyses suggest that the class Chlorophyceae is a monophyletic group, and the classes Ulvophyceae, Trebouxiophyceae and Prasinophyceae are non-monophyletic groups. Our proposed phylogenetic tree of Chlorophyta will offer new insights to investigate ancient green algae evolution, and our analytical framework will provide a useful approach for evaluating and mitigating the potential errors of phylogenomic inferences.

The Origin of Land Plants: A Phylogenomic Perspective.

Land plants are a natural group, and Charophyte algae are the closest lineages of land plants and have six morphologically diverged groups. The conjugating green algae (Zygnematales) are now suggested to be the extant sister group to land plants, providing the novel understanding for character evolution and early multicellular innovations in land plants. We review recent molecular phylogenetic work on the origin of land plants and discuss some future directions in phylogenomic analyses.