The complexity of varied modifications of chromatin composition is integrated in archetypal combinations called chromatin states that predict the local potential for transcription. The degree of conservation of chromatin states has not been established amongst plants, and how they interact with transcription factors is unknown. Here we identify and characterize chromatin states in the flowering plant Arabidopsis thaliana and the bryophyte Marchantia polymorpha, showing a large degree of functional conservation over more than 450 million years of land plant evolution. We used this new resource of conserved plant chromatin states to understand the influence of chromatin states on gene regulation. We established the preferential association of chromatin states with binding sites and activity of transcription factors. These associations define three main groups of transcription factors that bind upstream of the transcription start site, at the + 1 nucleosome or further downstream of the transcription start site and broadly associate with distinct biological functions including a list of potential candidate pioneer factors we know little about in plants, compared to their important roles in animal stem cells and early development.

Histone variants are key regulators of chromatin function. The H3 variants H3.1 and H3.3 evolved independently in animals and plants and differ at amino acid position 31, where H3.1 contains alanine (A), whereas H3.3 carries serine (S) in animals or threonine (T) in plants. Although S and T can both be phosphorylated, the biological significance of plants having selectively adopted T over S remains unclear. Here, we report that H3.3T31 plays a critical role in plant development and stress responses by promoting H3K36me3 on H3.3. T31 prevents plant-specific H3K27 methyltransferases ATXR5 and ATXR6 from depositing H3K27me1, which otherwise inhibits the H3K36 methyltransferase EFS. The substitution of H3.3T31 with S or A increases ATXR5/6 activity and elevates H3K27me1, leading to reduced H3K36me3. Together, these findings suggest co-selection of the plant-specific H3.3T31 residue and ATXR5/6 to ensure the preferential accumulation of H3K27me1 on H3.1 and H3K36me3 on H3.3, thereby supporting chromatin function in plants.

Maintaining transcriptional fidelity is essential for precise gene regulation and genome stability. Despite this, cryptic antisense transcription, occurring opposite to canonical coding sequences, is a pervasive feature across all domains of life. How such potentially harmful cryptic sites are regulated remains incompletely understood. Here, we show that nucleosome arrays within gene bodies play a key role in suppressing cryptic transcription. Using the fission yeast Schizosaccharomyces pombe as a model, we demonstrate that the CHD-family chromatin remodeler Hrp3 coordinates with the transcription elongation machinery, via the transcriptional regulator Prf1/RTF1, to position nucleosomes at sites of cryptic transcription initiation within gene bodies. In the absence of Hrp3, AT-rich sequences within gene bodies lose nucleosome occupancy, exposing promoter-like sequences that drive cryptic initiation. While cryptic transcription is generally detrimental, we identify a subset of antisense transcripts that encode critical meiotic genes, suggesting that cryptic transcription can also serve as a source of regulatory innovation. These findings define an elongation‑coupled chromatin pathway that preserves transcriptional fidelity and reveal how nucleosome remodeling shapes antisense transcription, cellular homeostasis, and adaptive potential.

The liverwort Marchantia polymorpha is a key model organism for understanding land plant evolution, development, and gene regulation. To support the growing demand for high-quality genomic resources, we present MarpolBase, a comprehensive and integrated genome database that hosts newly assembled, high-accuracy reference genomes for both the male Tak-1 and female Tak-2 accessions, designated as ver. 7.1 reference genomes. These new assemblies, generated using PacBio HiFi long-read sequencing, represent nearly telomere-to-telomere chromosome-level genomes, with improvements in assembly continuity, annotation accuracy, and structural resolution-especially for repeat-rich regions and sex chromosomes. MarpolBase offers not only access to genome sequences and gene annotations but also provides a unified platform for data exploration, comparative analysis, and community-driven gene nomenclature for M. polymorpha. It includes keyword-searchable gene pages with structural and functional annotations, expression data integration, genome browser visualization, and online analytical and utility tools. By unifying genome assembly, annotation, nomenclature, and analysis tools in a single platform, MarpolBase serves as a central resource for functional genomics and evolutionary studies in M. polymorpha, and a model for future plant genome databases. The genomic resources of MarpolBase are freely available at https://marchantia.info.

The unicellular red algae, Cyanidiophyceae, that diverged early during Archaeplastida (algal and plant) evolution, occupy a variety of extreme habitats that are inhospitable for most other eukaryotes. With the use of modern genomics and genetics methods, Cyanidiophyceae show a remarkable taxonomic diversity, share haplodiplophasic life cycles, and are engaged in complex trophic interactions with microbes that occupy geothermal niches. Amenable to molecular engineering, Cyanidiophyceae are excellent models for understanding evolutionary mechanisms that underpin their extremophilic lifestyles. Their unique growth conditions make these choice red algae of high interest for biotechnological exploitation in environments unsuitable for crops.

Heterochromatin formation is pivotal in many eukaryotes with repetitive sequences, such as transposable elements (TEs). However, in plants, where the known de novo DNA methylation mechanism (RdDM) targets euchromatin, how heterochromatin is formed in a region-specific manner remains unclear. We previously reported an RdDM-independent de novo establishment of H3K9me and non-CpG methylation, both of which localize in heterochromatin. Here we show that the mutually exclusive histone H2A variants, H2A.W and H2A.Z, function as guides to initiate heterochromatin formation; H2A.W and H2A.Z promotes and inhibits heterochromatin establishment, respectively, especially in chromosomal arm regions with dispersed TEs. In contrast, pericentromeric TEs demonstrate autonomous heterochromatin formation, less dependently on these H2A variants. Furthermore, H2A.Z protects protein-coding genes from ectopic heterochromatin formation, possibly by preventing its spreading. We propose that the genome indexing mechanism driven by H2A variants, as well as the autonomous formation of pericentromeric heterochromatin, shapes proper epigenomic patterns in Arabidopsis.

Cytosine methylation at CG sites is proposed to be maintained during DNA replication through a semiconservative mechanism. In mammals, this maintenance involves replication-coupled and replication-uncoupled steps; however, the underlying mechanisms and their possible conservation across eukaryotes remain unclear. Using live-cell sensors developed in this study to track CG methylation (mCG) dynamics, we uncovered that mCG maintenance extends far beyond DNA replication in the model plant Arabidopsis, with the major CG methyltransferase 1 (MET1) remaining associated with chromatin from S phase till G2 phase. Genetic dissection demonstrated that MET1, like its mammalian homolog DNA methyltransferase 1 (DNMT1), methylates nucleosome-associated DNA. Single-molecule duplex sequencing revealed that MET1/DNMT1-dependent methylation follows the rotational accessibility of nucleosomal DNA and is only partly templated by hemimethylated sites. This nucleosome-constrained de novo activity not only promotes mCG recovery after accidental loss but also shapes short- and long-term DNA methylation variation in plants and mammals.

Abstract The emergence of the eukaryotes coincided with the diversification of histone proteins and their post-translational modifications by enzymes that constitute the core of eukaryotic chromatin. Yet the evolutionary origins of this regulatory machinery are unknown. Here, we show that the key molecular components of histone-based chromatin regulation are present in the Asgard archaea, the closest prokaryotic relatives of eukaryotes. Asgard histones are abundant and have extended N-terminal tails rich in lysine residues that can be post-translationally modified, all of which are features shared with eukaryotic histones. In line with these findings, we identify enzymes from Asgard archaea that deposit or remove lysine acetylation on histone tails in vitro. Moreover, Asgard sirtuin deacetylases (SIR2 proteins) restore chromatin silencing in yeast, demonstrating the functional compatibility of Asgard enzymes with eukaryotic histone substrates. Our findings establish that the foundations of histone-based chromatin predate eukaryogenesis and place Asgard archaea as an evolutionary intermediate in the emergence of eukaryotic chromatin.

Plant galls are abnormal growing tissues induced by various parasitic organisms, exhibiting diverse and complex morphologies. Typically, these galls differ significantly in appearance from their host plants. Here, we report that larvae of a parasitic fly generate unique, rosette galls on Aster scaber, a perennial herb. These galls develop from vegetative organs after the larvae reprogram floral gene expression. To investigate the underlying mechanisms, we conducted whole-genome sequencing and transcriptome analysis. Our findings reveal that the larvae induce host organ dedifferentiation into an amorphous callus, activate floral genes, and selectively suppress genes associated with carpel development. As a result, the pseudoflowers consist solely of tepal-like leaflets and a specialized chamber, and the larvae influence pigment biosynthesis. Hijacking plants developmental gene networks by insects to sequentially mediate dedifferentiation, cytokinin regulation, and tepal-like leaflets formation provides a framework to study highly elaborate forms of parasitism and symbiosis between plants and insects.

Meiotic crossovers rearrange allele combinations and create offspring diversity. Crossovers occur nonrandomly along chromosomes, predominantly in distal euchromatin and less in pericentromeric heterochromatin marked with histone H3 lysine 9 dimethylation (H3K9me2) and the H2A variant H2A.W in Arabidopsis thaliana. Loss of H3K9me2 increases heterochromatic crossovers, but how H2A.W affects crossover formation in pericentromeric regions is unknown. Here, we report that H2A.W is required to restrict heterochromatic crossovers in Arabidopsis. Using meiosis-specific microRNA-induced gene silencing (meiMIGS) and fluorescence-tagged recombination reporters, we show that meiotic knockdown of H2A.W.6, H2A.W.7, and H2A.W.12 (meiMIGS-H2A.W.6/7/12) increases pericentromeric crossovers. High-resolution genomic maps of crossovers show that meiMIGS-H2A.W.6/7/12 enhances heterochromatic crossovers, similar to meiMIGS plants silencing the H3K9me2 pathway. Consistently, genome-wide crossover maps show that the mutants h2a.w.6, h2a.w.7, h2a.w.6 h2a.w.7, and h2a.w.6 h2a.w.7 h2a.w.12, but not h2a.w.12, exhibit a similar increase in heterochromatic crossovers to meiMIGS-H2A.W.6/7/12, demonstrating that H2A.W.6 and H2A.W.7 limit heterochromatic crossovers. Profiling of genome-wide nucleosome density using micrococcal nuclease sequencing reveals that h2a.w mutants with increased heterochromatic crossovers have increased heterochromatin accessibility, with lower H3K9me2 levels during meiosis. Our findings shed light on the role of H2A.W variants as heterochromatin compaction factors that suppress meiotic crossovers within the pericentromeric regions.

In organisms ranging from vertebrates to plants, major components of centromeres are rapidly evolving repeat sequences, such as tandem repeats (TRs) and transposable elements (TEs), which harbour centromere-specific histone H3 (CENH3)1,2. Complete centromere structures recently determined in human and Arabidopsis suggest frequent integration and purging of retrotransposons within the TR regions of centromeres3–5. Despite the high impact of ‘centrophilic’ retrotransposons on the paradox of rapid centromere evolution, the mechanisms involved in centromere targeting remain poorly understood in any organism. Here we show that both Ty3 and Ty1 long terminal repeat retrotransposons rapidly turnover within the centromeric TRs of Arabidopsis species. We demonstrate that the Ty1/Copia element Tal1 (Transposon of Arabidopsis lyrata 1) integrates de novo into regions occupied by CENH3 in Arabidopsis thaliana, and that ectopic expansion of the CENH3 region results in spread of Tal1 integration regions. The integration spectra of chimeric TEs reveal the key structural variations responsible for contrasting chromatin-targeting specificities to centromeres versus gene-rich regions, which have recurrently converted during the evolution of these TEs. Our findings show the impact of centromeric chromatin on TE-mediated rapid centromere evolution, with relevance across eukaryotic genomes. An Arabidopsis long terminal repeat retrotransposon integrates de novo into regions occupied by centromere-specific histone variant, showing the impact of centromeric chromatin on transposable element-mediated rapid centromere evolution, with relevance across eukaryotic genomes.

Paralogous variants of canonical histones guide accessibility to DNA and function as additional layers of genome regulation. Across eukaryotes, the mechanism of action and functional significance of several variants of core histones are well known except those of histone H4. Here we show that a variant of H4 (H4.V) expressing tissue-specifically among Oryza members mediated specific epigenetic changes contributing to salt tolerance. H4.V was incorporated into specific heterochromatic sites, where it blocked the deposition of active histone marks. Stress-dependent redistribution of H4.V enabled the incorporation of acetylated H4 lysine 5 (H4K5ac) in the gene bodies. The misexpression of H4.V led to defects in reproductive development and in mounting salt stress responses. H4.V formed homotypic nucleosomes and mediated these alterations by conferring distinct molecular properties to the nucleosomes, as seen with cryo electron microscopy structures and biochemical assays. These results reveal not only an H4 variant among plants but also a chromatin regulation that might have contributed to the adaptation of semi-aquatic Oryza members.

DNA methylation is a key epigenetic mark that impacts gene expression and represses transposable elements in eukaryotes. Numerous examples of cis elements targeted by DNA methylation, particularly at CG sites (mCG), have been reported to be under selective pressure in animals and plants. By contrast, there is limited knowledge of trans regulators of mCG leading to adaptation. Here, a genome-wide association study identifies CELL DIVISION CYCLE-ASSOCIATED PROTEIN 7 (CDCA7) as a major trans determinant of mCG in natural populations of Arabidopsis thaliana. CDCA7 or its paralogue physically binds the chromatin remodeller DECREASE IN DNA METHYLATION 1 (DDM1), which facilitates access of methyltransferases to DNA. Epigenomic analysis shows that while CDCA7 proteins control all DDM1-dependent processes, their predominant function is the maintenance of mCG. We identify a 26-bp promoter indel modulating CDCA7 expression in natural populations and determining the degree of mCG and transposable element silencing. The geographic distribution of CDCA7 alleles suggests that new alleles have repeatedly expanded to novel ecological niches, indicating a potential role in local adaptation. Our findings establish CDCA7 proteins as dedicated regulators of mCG and imply that DDM1 relies on alternative partners to regulate other chromatin features. Broadly, they illustrate how changes in global DNA methylation levels through transcriptional regulation of the epigenetic machinery have the capacity to facilitate local adaptation.

Autophagy sustains cellular health by recycling damaged or excess components through autophagosomes. Autophagy is mediated by conserved ATG proteins, among which the ubiquitin-like ATG8 proteins play a central role by linking cargo to the growing autophagosomes. Unlike most ATG proteins, the ATG8 gene family is significantly expanded in vascular plants, but its functional specialization remains poorly understood. Using transcriptional and translational reporters in Arabidopsis thaliana, we revealed that ATG8 isoforms are differentially expressed across tissues and form distinct autophagosomes. To explore ATG8 specialization, we generated the nonuple Δatg8 mutant, lacking all nine ATG8 isoforms. The mutant displayed hypersensitivity to carbon and nitrogen starvation, coupled with defects in bulk and selective autophagy, as shown by biochemical and ultrastructural analyses. Complementation experiments demonstrated that ATG8A could rescue both carbon and nitrogen starvation phenotypes, whereas ATG8H could only complement carbon starvation. Proximity labeling proteomics further identified isoform-specific interactors under nitrogen starvation, underscoring their functional divergence. These findings provide genetic evidence for functional specialization of ATG8 isoforms in plants and lay the foundation for investigating their roles in diverse cell types and stress conditions.

The complexity of varied modifications of chromatin composition is integrated in archetypal combinations called chromatin states that predict the local potential for transcription. The degree of conservation of chromatin states has not been established amongst plants, and how they interact with transcription factors is unknown. Here we identify and characterize chromatin states in the flowering plant Arabidopsis thaliana and the bryophyte Marchantia polymorpha , showing a large degree of functional conservation over more than 450 million years of land plant evolution. We used this new resource of conserved plant chromatin states to understand the influence of chromatin states on gene regulation. We established the preferential association of chromatin states with binding sites and activity of transcription factors. These associations define three main groups of transcription factors that bind upstream of the transcription start site, at the +1 nucleosome or further downstream of the transcription start site and broadly associate with distinct biological functions. The association with the +1 nucleosome defines a list of candidate pioneer factors we know little about in plants, compared to their important roles in animal stem cells and early development. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, https://ror.org/013tf3c58, P32054, P36231, PAT1104523, PAT6138924 European Union’s Framework Programme for Research and Innovation Horizon 2020, Marie Curie Skłodowska Grant Agreement no. 847548 (VIP2) Leverhulme Trust, https://ror.org/012mzw131, Early Career Fellow (ECF-2023-534) Isaac Newton Trust, https://ror.org/02gn6ta77, 23.08(f)

Nucleosomes are thought to be structural barriers to transcription, establishing a restrictive ground state that must be destabilized for gene expression. However, structural insights have revealed that transcription can proceed in the presence of nucleosomes, suggesting that this model is incomplete. Here, we reconstituted H2A.Z sequences resulting from more than a billion years of eukaryotic evolution in a single synthetic host system, interrogating their impact on transcription. We identified single-residue substitutions within the ultra-conserved core domain loop 2 (L2) of H2A.Z as sufficient to confer emergent properties and drive neofunctionalization. Such L2 neomorphs acquired a direct interaction with transcription elongation factor Spt6, rewiring gene expression by tuning polymerase processivity. We conclude that even minimal changes in histone sequences can transform their function, underscoring the evolutionary potential of the histone core domain to drive regulatory innovation and highlighting a previously unappreciated role of the histone core domain in transcriptional regulation.

Sexual reproduction results in the development of haploid and diploid cell states during the life cycle. In bryophytes, the dominant multicellular haploid phase produces motile sperm that swim through water to the egg to effect fertilization from which a relatively small diploid phase develops. In angiosperms, the reduced multicellular haploid phase produces non-motile sperm that is delivered to the egg through a pollen tube to effect fertilization from which the dominant diploid phase develops. These different life cycle characteristics are likely to impact the distribution of genetic variation among populations. However, little is known about the distribution of genetic variation among wild populations of bryophytes. To investigate how genetic variation is distributed among populations of a bryophyte and to establish the foundation for population genetics research in bryophytes, we described the genetic diversity of collections of Marchantia polymorpha subsp. ruderalis, a cosmopolitan ruderal liverwort. We identified 78 genetically unique (non-clonal) from a total of 209 sequenced accessions collected from 37 sites in Europe and Japan. There was no detectable population structure among European populations but significant genetic differentiation between Japanese and European populations. By associating genetic variation across the genome with global climate data, we showed that temperature and precipitation influence the frequency of potentially adaptive alleles. This collection establishes the core of an experimental platform that exploits natural genetic variation to answer diverse questions in biology.

Induction of DNA damage triggers rapid phosphorylation of the histone H2A.X (γH2A.X). In animals, mediator of DNA damage checkpoint 1 (MDC1) binds γH2A.X through a tandem BRCA1 carboxyl-terminal (tBRCT) domain and mediates recruitment of downstream effectors of DNA damage response (DDR). However, readers of this modification in plants have remained elusive. We show that from the Arabidopsis BRCT domain proteome, BCP1-4 proteins with tBRCT domains are involved in DDR. Through its tBRCT domain BCP4 binds γH2A.X in vitro and localizes to DNA damage-induced foci in an H2A.X-dependent manner. BCP4 also contains a domain that interacts directly with NBS1 and thus acts as a functional counterpart of MDC1. We also show that BCP1, that contains two tBRCT domains, co-localizes with γH2A.X but it does not bind γH2A.X suggesting functional similarity with human PAXIP1. A phylogenetic analysis supports that PAXIP1 and MDC1 in metazoa and their plant counterparts evolved independently from common ancestors with tBRCT domains. Collectively, our study reveals missing components and provides mechanistic and evolutionary insights into plant DDR.