Land plant-specific H3K27 methyltransferases ATXR5 and ATXR6 control plant development and stress responses

Xiaoyi Li; Jie Pan; Qian Liu; Huairen Zhang; Hui Li; Danhua Jiang

doi:10.1186/s13059-025-03801-5

. 2025 Sep 25;26:303. doi: 10.1186/s13059-025-03801-5

Land plant-specific H3K27 methyltransferases ATXR5 and ATXR6 control plant development and stress responses

Xiaoyi Li ^1,^2,^3,^#, Jie Pan ^2,^3,^#, Qian Liu ¹, Huairen Zhang ², Hui Li ^2,³, Danhua Jiang ^1,^2,^3,^✉

PMCID: PMC12461997 PMID: 40999518

Abstract

Background

Histone modifications are critical for transcriptional regulation. A notable genetic innovation in land plants is the emergence of histone lysine methyltransferases ATXR5/6, which specifically catalyze the repressive histone H3 lysine 27 monomethylation (H3K27me1). Current knowledge of ATXR5/6 function is largely based on Arabidopsis studies using a weak atxr5;atxr6 hypomorphic mutant, in which ATXR6 is still partially expressed and defects are primarily observed in heterochromatin. However, the significance for land plants to evolve these enzymes remains unclear.

Results

In this study, we generate strong atxr5;atxr6 mutants with further reduced ATXR6 expression in Arabidopsis to explore the broader roles of ATXR5/6. Our results show that ATXR5/6 are essential for plant reproductive development and play a critical role in supporting normal plant growth by repressing the transcription of stress responsive genes. In addition, ATXR5/6 are necessary for maintaining H3K27 trimethylation (H3K27me3), likely by providing H3K27me1 as a substrate for further methylation. We also demonstrate that the function of ATXR5/6 in regulating development and responsive genes is conserved in the monocot rice.

Conclusions

Our findings suggest that land plants evolved ATXR5/6 not only to maintain heterochromatin, but also to regulate development and environmental responses, providing new insights into the functional significance of ATXR5/6 in land plants.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13059-025-03801-5.

Background

In eukaryotes, epigenetic regulation, including post-translational modifications on histones, plays a pivotal role in determining the transcriptional activity [1]. Depending on their impact on transcription, histone modifications can either be “active” or “repressive.” A well-studied repressive histone modification is histone H3 lysine 9 (H3K9) methylation, which serves as a conserved mark of heterochromatin across yeast, animals, and plants [2, 3]. In Arabidopsis thaliana, H3K9 methylation is primarily catalyzed by histone methyltransferases Su(var)3–9 homolog 4/Kryptonite (SUVH4/KYP), SUVH5, and SUVH6 [4–7], and it predominantly silences the transcription of transposable elements (TEs) enriched at heterochromatin regions [5, 6, 8, 9].

Another repressive histone modification, the histone H3 lysine 27 trimethylation (H3K27me3), is primarily enriched at euchromatin regions and is associated with inactive protein-coding genes [10]. In animals, the enzymes responsible for catalyzing H3K27me3, namely the PRC2 complex, also deposit H3K27 monomethylation (H3K27me1) and H3K27 dimethylation (H3K27me2) [11, 12]. However, in land plants, two plant-specific H3K27me1 methyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6, have evolved (Additional file 1: Fig. S1) [13, 14]. H3K27me1 is mainly found at heterochromatin regions in Arabidopsis [13, 15]. A weak Arabidopsis atxr5;atxr6 double mutant exhibits decondensed heterochromatin, TE activation, and heterochromatin over-replication [13, 16]. Therefore, ATXR5/6-dependent H3K27me1 is regarded as a repressive histone modification that functions at heterochromatin in plants. However, the significance of land plants acquiring ATXR5/6 to deposit H3K27me1 remains unclear, especially given the presence of the already well-conserved heterochromatin mark H3K9 methylation. Although some genes at euchromatin are found to carry H3K27me1 in Arabidopsis [17, 18], this genic H3K27me1 may arise either from the activity of PRC2 or from the action of H3K27 demethylases, such as REF6, ELF6, and JMJ13, which can demethylate H3K27me3 to produce H3K27me1 [14, 17, 19, 20]. This hampers the efforts to identify the potential ATXR5/6-regulated genes through profiling H3K27me1 [17, 18]. Thus, it is of great interest to directly determine the biological importance of ATXR5/6.

In this study, we further investigate the functions of ATXR5/6 by generating strong atxr5;atxr6 mutants in Arabidopsis. We show that ATXR5/6 are essential for both vegetative and reproductive development and are required to repress stress responsive genes. Moreover, we provide genome-wide evidence that ATXR5/6-mediated H3K27me1 is necessary for the maintenance of H3K27me3, thereby contributing to H3K27me3-mediated transcriptional repression. In addition, we demonstrate that ATXR5/6 perform conserved functions in rice. These findings suggest that ATXR5/6 help balance plant growth and stress responses beyond their role in heterochromatin maintenance, offering new insights into the significance of ATXR5/6 in land plants.

Results

Loss of ATXR5 and ATXR6 results in strong developmental defects

The so far commonly used atxr5;atxr6 double mutant in Arabidopsis is a hypomorphic allele (referred to here as atxr5;atxr6^hyp), in which ATXR6 is still expressed at moderate levels because a T-DNA was inserted at its promoter region. The atxr5;atxr6^hyp mutant displays only a slightly reduced growth phenotype compared with wild-type (WT) Columbia (Col) [13, 18]. To generate stronger mutants, we transformed CRISPR/Cas9 constructs targeting ATXR6 into the atxr5;atxr6^hyp mutant and created deletions within the genic region of ATXR6 (atxr6^c-1 and atxr6^c-2), which result in premature termination of translation (Fig. 1a; Additional file 1: Fig. S2a). We failed to recover any atxr6^c-1 or atxr6^c-2 homozygotes from the progeny of atxr5;atxr5;atxr6^hyp;atxr6^c-1 or atxr5;atxr5;atxr6^hyp;atxr6^c-2 (Additional file 2: Table S1), indicating that null atxr5;atxr6 mutants are lethal. Examination of seeds in the siliques of atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 revealed seed abortion during seed development (Fig. 1b). Moreover, small ovule-like structures were observed, which appeared to either not be fertilized or ceased development shortly after fertilization (Fig. 1b). Reciprocal crosses indicated that the loss of ATXR5 and ATXR6 severely impaired female gametogenesis, although transmission was still observed at low rates: less than 10% compared to the expected 50% (Additional file 2: Table S2). Hence, we conclude that atxr5;atxr6^c-1 and atxr5;atxr6^c-2 mutants are not viable due to female gametophytic and embryonic lethality. This finding is consistent with recent reports showing that the atxr5;atxr6 null mutants are lethal [18, 21].

Fig. 1 — ATXR5 and ATXR6 regulate plant development. a Schematic view of the full-length genomic structure of *ATXR5* and *ATXR6*. Filled boxes indicate exons, the red dashed lines represent deleted regions in *atxr6*^c-1 and *atxr6*^c-2, and SALK_130637 and SAIL_240H01 are T-DNA insertions. b Seed developmental phenotypes of the indicated lines. Arrows indicate aborted seeds, while asterisks denote ovule-like structures. Scale bars, 1 mm. c Plant developmental phenotypes of the indicated lines. Scale bar, 1 cm. d Flowering phenotypes of the indicated lines. e Total number of primary rosette and cauline leaves at flowering for the indicated lines. Fifteen plants were scored for each line. Values are means ± SD. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences. f H3K27me1 levels in the indicated lines determined by Western blotting. H3 was employed as a loading control. The bar chart represents the quantification of Western blot signals from three biological replicates (Additional file 1: Fig. S2c). Values are means ± SD. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences. g Protein structure of ATXR5, the domains, and point mutations generated to deactivate ATXR5 are presented. h Plant developmental phenotypes of the complementation lines. Scale bar, 1 cm. i Total number of primary rosette and cauline leaves at flowering for the complementation lines. Fifteen plants were scored for each line. Values are means ± SD. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences

Although the atxr5;atxr6 null mutants are not viable, we observed that atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 displayed stronger developmental defects, such as significantly smaller plants, compared to atxr5;atxr6^hyp at the vegetative stage (Fig. 1c). Moreover, they exhibited early flowering phenotypes (Fig. 1d, e). Examination of ATXR6 expression revealed that its transcript levels were further reduced in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 compared to atxr5;atxr6^hyp (Additional file 1: Fig. S2b), consistent with the more severe developmental defects observed in these mutants.

To determine whether ATXR5/6 regulate development via its enzymatic activity, we first analyzed global H3K27me1 levels in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2. H3K27me1 levels were further diminished compared to atxr5;atxr6^hyp (Fig. 1f; Additional file 1: Fig. S2c), consistent with the more pronounced developmental defects. Next, we performed a complementation test with the full-length genomic ATXR5 and the SET domain activity disrupted ATXR5 (gATXR5 E217A&M221A and gATXR5 Y274N) (Fig. 1g) [16, 22]. Only WT ATXR5 fully rescued the developmental defects (Fig. 1h, I; Additional file 1: Fig. S2d), suggesting that the enzymatic activity of ATXR5/6 is crucial for plant development.

The function of ATXR5/6 in plant developmental control seems different from that of H3K9 methyltransferases, as loss of SUVH4, SUVH5, and SUVH6 does not induce significant developmental abnormalities (Additional file 1: Fig. S3a) [23]. The atxr5;atxr6^hyp mutant bears excess DNA due to the over-replication of heterochromatin, particularly in endoreduplicated cells [16]. However, a comparable increase in DNA content was observed in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 compared to atxr5;atxr6^hyp (Additional file 1: Fig. S3b, c), suggesting that the heterochromatin over-replication phenotype was not strongly enhanced in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2. Moreover, similar percentages of nuclei in atxr5;atxr6^hyp, atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 showed decondensation of chromocenters enriched with heterochromatin (Additional file 1: Fig. S3d). These results suggest that the additional developmental defects observed in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 compared to atxr5;atxr6^hyp are likely not due to heterochromatin misregulation. Since atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 exhibited similar phenotypes, we mainly used atxr5;atxr5;atxr6^hyp;atxr6^c-1 for subsequent studies.

Loss of ATXR5 and ATXR6 enhances plant resistance to bacterial infection

To explore potential reasons for the additional developmental abnormalities in atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared to atxr5;atxr6^hyp, we compared their transcriptome changes. Hundreds of TEs and genes were significantly misexpressed in atxr5;atxr6^hyp and atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared to WT Col (fold change > 2, P-adjust < 0.05), with the majority showing increased transcript levels (Fig. 2a; Additional file 2: Table S3, 4). This aligns with the notion that H3K27me1 is a repressive modification that inhibits transcription. atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr6^hyp had similar numbers of transcript level increased TEs, and nearly all of them were overlapped (Fig. 2a, b; Additional file 1: Fig. S4a). RT-qPCR validation of several overlapping TEs showed comparable activation levels in both genotypes (Additional file 1: Fig. S4b). These results confirm our earlier observations that the atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 mutants did not show stronger defects at heterochromatin compared to atxr5;atxr6^hyp (Additional file 1: Fig. S3b–d). On the other hand, a substantial number of genes (254) were significantly activated only in atxr5;atxr5;atxr6^hyp;atxr6^c-1, while a smaller set (96) was significantly activated only in atxr5;atxr6^hyp (Fig. 2b). Most of the genes and TEs that are identified to be significantly activated only in atxr5;atxr6^hyp also showed activation in atxr5;atxr5;atxr6^hyp;atxr6^c-1, albeit at relatively lower levels (Additional file 1: Fig. S4c–e). It is possible that the further activation of these genes and TEs in atxr5;atxr5;atxr6^hyp;atxr6^c-1 might trigger an unknown feedback mechanism that diminishes their expression. Together, these results suggest that the further loss of H3K27me1 in atxr5;atxr5;atxr6^hyp;atxr6^c-1 mainly affects the repression of genes rather than TEs.

Fig. 2 — Loss of ATXR5 and ATXR6 induces stress responses. a The number of significantly misexpressed TEs and genes in *atxr5;atxr6*^hyp and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. b Venn diagrams of transcript level significantly increased TEs and genes in *atxr5;atxr6*^hyp and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. c Gene ontology (GO) analysis on the transcript level significantly increased genes in *atxr5;atxr6*^hyp and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. Top 10 representative terms are listed and ranked by P value. GO terms specifically enriched in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 are highlighted in orange font. d and e Heatmap showing transcript levels of oxidative stress response (d) and abiotic stimulus response (e) genes determined by RNA-seq. Results from three biological replicates are shown. f Seedling growth phenotypes on 1/2MS plate supplemented with either no H₂O₂ or with 6 mM or 20 mM H₂O₂. Pictures were taken 10 days after germination. g The true leaf formation rate of seedlings germinated on 1/2MS plate supplemented with either no H₂O₂ or with 6 mM or 20 mM H₂O₂. For the 0 mM and 6 mM H₂O₂ treatments, rates were measured 10 days after germination, while for the 20 mM H₂O₂ treatment, rate was measured 15 days after germination. Values are means ± SD of three biological replicates. At least 79 seeds were sown for each replicate. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences. h Relative transcript levels of peroxidase-coding genes determined by RT-qPCR in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 with or without H₂O₂ treatment. Values are means ± SD of three biological replicates. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences. i Peroxidase activity in the indicated lines. Values are means ± SD of three biological replicates. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences. j H3K27me1 levels at peroxidase-coding genes determined by ChIP-qPCR in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 with or without H₂O₂ treatment. The amounts of immunoprecipitated DNA fragments were normalized to input DNA (mononucleosome). Values are means ± SD of three biological replicates. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences

We further performed gene ontology (GO) analysis with transcript level significantly increased genes in atxr5;atxr6^hyp or atxr5;atxr5;atxr6^hyp;atxr6^c-1 to assess the functions of ATXR5/6-repressed genes. Both groups were found to be enriched in pathways related to response to bacteria and defense response (Fig. 2c; Additional file 1: Fig. S4f). In addition, the DNA damage response pathway was also enriched (Fig. 2c; Additional file 1: Fig. S4g), consistent with previous studies showing that DNA damage repair genes are activated in atxr5;atxr6^hyp, likely due to genome instability [18, 24]. We next tested whether the loss of ATXR5/6-induced activation of defense responsive genes affects plant resistance to the virulent bacterium P. syringae pathovar tomato (Pst) DC3000. Compared to WT, less severe disease symptoms were observed on the leaves of atxr5;atxr6^hyp, atxr5;atxr5;atxr6^hyp;atxr6^c-1, and atxr5;atxr5;atxr6^hyp;atxr6^c-2 3 days after inoculation of Pst DC3000 (Additional file 1: Fig. S5a). Consistently, bacterial growth was lower in these mutants compared to WT (Additional file 1: Fig. S5b). However, it is important to note that the activation of DNA damage responses could enhance plant immunity [25, 26]. Moreover, both atxr5;atxr6^hyp and atxr5;atxr5;atxr6^hyp;atxr6^c-1 showed similar increase in DNA damage and defense responses, suggesting that these activations do not account for the additional developmental defects observed in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2.

ATXR5/6 repress plant oxidative stress response

Besides bacterial and DNA damage responses, GO analysis also revealed enrichment of oxidative stress and abiotic stimulus response pathways with transcript level significantly increased genes in atxr5;atxr5;atxr6^hyp;atxr6^c-1 but not in atxr5;atxr6^hyp (Fig. 2c–e), suggesting that the further loss of ATXR5/6 releases the repression of genes in these two pathways. To test whether loss of ATXR5/6 affects plant resistance to oxidative stress, seeds collected from WT Col, atxr5;atxr6^hyp, atxr5;atxr5;atxr6^hyp;atxr6^c-1, and atxr5;atxr5;atxr6^hyp;atxr6^c-2 were germinated on 1/2MS plates containing hydrogen peroxide. Among the progeny of atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2, about half are expected to be atxr5;atxr5;atxr6^hyp;atxr6^c-1 or atxr5;atxr5;atxr6^hyp;atxr6^c-2, respectively (Additional file 2: Table S1). By measuring the formation of true leaves, we found that the progeny of atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 exhibited greater resistance to hydrogen peroxide treatment compared to WT and the weak atxr5;atxr6^hyp mutant (Fig. 2f, g). Genotyping analysis confirmed that most plants forming true leaves under hydrogen peroxide treatment were indeed atxr5;atxr5;atxr6^hyp;atxr6^c-1 or atxr5;atxr5;atxr6^hyp;atxr6^c-2. Similarly, atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 displayed increased resistance to salt stress (Additional file 1: Fig. S6a, b), in agreement with the activation of abiotic stimulus response genes. The acquired resistance to oxidative and salt stresses in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 is unlikely to result from activation of DNA damage responses, as DNA damage responses were also triggered in the non-resistant atxr5;atxr6^hyp mutant. Furthermore, treatment with genotoxic stress in WT Col did not strongly enhance resistance to oxidative and salt stresses (Additional file 1: Fig. S6c).

Notably, several peroxidase-coding genes were activated in atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared to WT (Fig. 2d; Additional file 2: Table S3). Overexpression of peroxidase-coding genes has been reported to cause reduced plant growth, a phenotype similar to that observed in both atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 [27, 28]. In WT, the transcript levels of these peroxidase genes were induced by hydrogen peroxide treatment, suggesting that they are normally repressed in the absence of oxidative stress. However, their transcription was already activated in atxr5;atxr5;atxr6^hyp;atxr6^c-1, and to a much lesser extent in atxr5;atxr6^hyp, even without stress (Fig. 2h; Additional file 1: Fig. S6d). Consistent with this, peroxidase activity was higher in atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 than in WT and atxr5;atxr6^hyp (Fig. 2i).

To examine whether ATXR5/6 repress the oxidative stress response through its enzymatic activity, we compared the oxidative stress response phenotypes of atxr5;atxr5;atxr6^hyp;atxr6^c-1 complemented with either WT or SET domain mutated ATXR5. Only the WT ATXR5, and not the SET domain mutated ones, restored susceptibility to oxidative stress (Additional file 1: Fig. S6e). Furthermore, enzymatic activity impaired ATXR5 was unable to repress the transcription of peroxidase-coding genes or reduce the peroxidase activity (Additional file 1: Fig. S6f, g). Thus, the repression of the oxidative stress response by ATXR5/6 is dependent on their enzymatic activity. We further analyzed H3K27me1 levels at these genes by ChIP-qPCR. H3K27me1 levels carried by these genes were reduced upon loss of ATXR5/6 (Fig. 2j; Additional file 1: Fig. S6h). Moreover, oxidative stress induced a decrease in H3K27me1 at these loci in WT (Fig. 2j). Together, these results suggest that the ATXR5/6-mediated H3K27me1 represses peroxidase-coding genes under normal growth conditions.

ATXR5/6 repress active histone modifications at peroxidase-coding genes

It was reported that ATXR5/6-mediated H3K27me1 antagonizes the deposition of active histone acetylation, such as H3K27 acetylation (H3K27ac) and H3K36ac, at the heterochromatin [29]. We speculate that H3K27me1 may also inhibit other active modifications. At this point, we selected H3K27ac and H3K36 trimethylation (H3K36me3) to examine their levels at peroxidase-coding genes in atxr5;atxr5;atxr6^hyp;atxr6^c-1. Our results showed that H3K27ac and H3K36me3 levels were increased in atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared to WT (Fig. 3a, b), suggesting that the ATXR5/6-mediated H3K27me1 represses their deposition at genes.

Fig. 3 — ATXR5 and ATXR6 antagonize the accumulation of H3K27ac and H3K36me3 at peroxidase-coding genes. a and b H3K27ac (a) and H3K36me3 (b) levels at peroxidase-coding genes in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 determined by ChIP-qPCR. The amounts of immunoprecipitated DNA fragments were normalized to input DNA (mononucleosome). Values are means ± SD of three biological replicates. Statistical significance was determined by two-tailed Student’s t-test (**, P < 0.01). c and d Plant developmental phenotypes following the loss of *GCN5* (c) or *EFS* (d) in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. Scale bars, 1 cm. e and f Plant diameters following the loss of *GCN5* (e) or *EFS* (f) in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. Fifteen 3-week-old plants were measured for each line. g and h Relative transcript levels of peroxidase-coding genes following the loss of GCN5 (g) or EFS (h) in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 determined by RT-qPCR. Values are means ± SD of three biological replicates. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences

We then crossed atxr5;atxr5;atxr6^hyp;atxr6^c-1 with a mutant of GENERAL CONTROL NON-REPRESSED PROTEIN 5 (GCN5) or EARLY FLOWERING IN SHORT DAYS (EFS, also known as SDG8), which are histone acetylase and methyltransferase responsible for H3K27ac and H3K36me3 deposition, respectively [30, 31]. Although gcn5 and efs mutants also exhibit developmental defects, they partially rescued the growth defects in atxr5;atxr5;atxr6^hyp;atxr6^c-1, as indicated by increased plant size (diameter) and improved leaf formation (Fig. 3c–f; Additional file 1: Fig. S7a, b). Furthermore, the absence of GCN5 or EFS in atxr5;atxr5;atxr6^hyp;atxr6^c-1 partially inhibited the activation of peroxidase-coding genes and reduced the peroxidase activity (Fig. 3g, h; Additional file 1: Fig. S7c, d). Therefore, the H3K27me1 deposited by ATXR5/6 likely prevents the accumulation of active histone modifications, thereby inhibiting gene activation.

ATXR5/6 contribute to the maintenance of H3K27me3

The deposition of H3K27me1 by ATXR5/6 is considered replication coupled and primarily occurs on the replicative H3 variant H3.1 [22, 32]. Similarly, the maintenance of another repressive histone mark, H3K27me3, is also closely linked with DNA replication and depends on H3.1 [33–37]. It has been proposed that H3K27me1 deposition might facilitate the restoration of H3K27me3 after DNA replication [35, 38]. To assess the requirement of ATXR5/6 in H3K27me3 maintenance, we performed ChIP-seq to examine H3K27me3 profiles in WT and atxr5;atxr5;atxr6^hyp;atxr6^c-1. Interestingly, we observed a moderate reduction in H3K27me3 levels at H3K27me3-enriched regions in atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared with WT (Fig. 4a, b), and this reduction was consistently observed in both biological replicates performed (Additional file 1: Fig. S8a). Similarly, H3K27me3 levels were generally lower at H3K27me3-enriched genes in atxr5;atxr5;atxr6^hyp;atxr6^c-1 (Additional file 1: Fig. S8b), with the majority of these genes showing a loss of H3K27me3 in atxr5;atxr5;atxr6^hyp;atxr6^c-1 (Fig. 4c, d). This loss was accompanied by a moderate increase in gene transcription, particularly for genes with a stronger reduction in H3K27me3 (Fig. 4e; Additional file 1: Fig. S8c). H3K27me3-enriched regions are predominantly marked by the replicative histone variant H3.1, rather than the non-replicative H3.3 (Additional file 1: Fig. S8d). Furthermore, genes with greater H3K27me3 loss in the atxr5;atxr5;atxr6^hyp;atxr6^c-1 mutant tend to exhibit higher levels of both H3K27me3 and H3.1 in the wild type (Additional file 1: Fig. S8e). These observations are consistent with the preferential monomethylation of H3.1 at lysine 27 by ATXR5/6 [22]. The transcript levels of Polycomb group (PcG) genes, which regulate H3K27me3, as well as H3K27 demethylase-coding genes, were comparable between atxr5;atxr5;atxr6^hyp;atxr6^c-1 and WT (Additional file 1: Fig. S8f). Hence, the reduction of H3K27me3 in atxr5;atxr5;atxr6^hyp;atxr6^c-1 is not due to the misexpression of these genes.

Fig. 4 — ATXR5 and ATXR6 are required for the maintenance of H3K27me3. a Metaplots and heatmaps of H3K27me3 ChIP-seq signals in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 over H3K27me3-enriched peaks in WT Col. b Changes in H3K27me3 ChIP-seq signals in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 compared to Col over H3K27me3-enriched peaks in WT Col. c Scatter plot showing H3K27m3 ChIP-seq reads in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 for all H3K27me3-enriched genes in WT Col. d Distribution of H3K27me3-enriched genes based on changes in H3K27me3 signals in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 compared to Col. e Overall gene expression profiles of H3K27me3-enriched genes grouped according to changes in H3K27me3 levels in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 compared to Col. The expression values represent the average of three biological replicates. P value is based on the Manne-Whitney U test. f Genome browser view of H3K27me3 accumulation levels at the locus encompassing *AGL19* in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. g Average TPM values of *AGL19* in Col and *atxr5;atxr5;atxr6*^hyp;atxr6^c-1 determined by RNA-seq. Values are means ± SD of three biological replicates. Statistical significance was determined by two-tailed Student’s t-test (**, P < 0.01). h Flowering phenotypes following loss of *AGL19* in *atxr5;atxr5;atxr6*^hyp;atxr6^c-1. i Total number of primary rosette and cauline leaves at flowering for the indicated lines. Twenty plants were scored for each line. Values are means ± SD. The significance of differences was tested using one-way ANOVA with Tukey’s test (P < 0.05), with different letters indicating statistically significant differences

Both the atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 displayed early flowering phenotypes (Fig. 1d, e). We noted that the floral activator AGL19 and its upstream gene AT4G22960, which are both marked by H3K27me3, showed reduced H3K27me3 levels and increased transcript levels in atxr5;atxr5;atxr6^hyp;atxr6^c-1 compared to WT (Fig. 4f, g; Additional file 1: Fig. S9a). In addition, the H3K27 monomethylation activity of ATXR5 is required for repressing both flowering and AGL19 expression (Fig. 1i; Additional file 1: Fig. S9b). AGL19 is strongly derepressed in PcG mutants, contributing to the early flowering phenotype in the loss of function mutant of CURLF LEAF (CLF), a key H3K27me3 methyltransferase in Arabidopsis [39]. To determine whether the early flowering phenotype of atxr5;atxr5;atxr6^hyp;atxr6^c-1 is due to upregulated AGL19 expression, we generated an agl19 mutant (agl19^c) with CRISPR/Cas9 gene editing (Additional file 1: Fig. S9c, d) and introduced it into the atxr5;atxr5;atxr6^hyp;atxr6^c-1 background. Loss of AGL19 partially suppressed the early flowering phenotype (Fig. 4h, i), suggesting that ATXR5/6 repress flowering by assisting in the silencing of AGL19.

OsATXR5/6 regulate reproductive development and repress responsive genes in rice

ATXR5 and ATXR6 are conserved proteins across land plants. We thus further investigated their function in the monocot model plant rice (Oryza sativa) by knocking out their coding genes with CRISPR/Cas9 (Additional file 1: Fig. S10a–d). Similar to what we found in Arabidopsis, we were unable to recover the osatxr5^c;osatxr6^c double homozygous plants from the progeny of osatxr5^c;osatxr6^c/ + (Additional file 2: Table S5). While osatxr5^c;osatxr6^c/ + produced normal flower organs and pollen (Additional file 1: Fig. S10e, f), many empty grains were observed on the panicles, and its seed setting rate was lower than that of WT Nipponbare (NP) (Fig. 5a, b). The ratio of osatxr5^c to osatxr5^c;osatxr6^c/ + in the progeny of osatxr5^c;osatxr6^c/ + was approximately 1:2 (Additional file 2: Table S5). In addition, osatxr5^c;osatxr6^c mutations were successfully transmitted through both female and male gametes (Additional file 2: Table S6), suggesting that the osatxr5^c;osatxr6^c double homozygous is embryonic lethal. Thus, we conclude that OsATXR5/6 are essential for seed development but dispensable for gamete formation in rice.

The osatxr5^c;osatxr6^c/ + mutant did not show obvious developmental defects at the vegetative stage (Fig. 5a). To assess the importance of OsATXR5/6 at this stage, we employed RNA interference (RNAi) strategy to knockdown OsATXR6 expression (osatxr6kd) in the osatxr5^c mutant, and selected two independent lines for further studies (Additional file 1: Fig. S11a). Both lines exhibited decreased global H3K27me1 levels and displayed reduced growth phenotypes compared to NP (Fig. 5c–e; Additional file 1: Fig. S11b–f). We next performed RNA-seq with NP, osatxr5^c;osatxr6kd-1, and osatxr5^c;osatxr6kd-2. Hundreds of TEs and genes were misexpressed in osatxr5^c;osatxr6kd-1 and osatxr5^c;osatxr6kd-2, with the majority being upregulated (Fig. 5f; Additional file 1: Fig. S11g; Additional file 2: Table S7, 8), further supporting the role of OsATXR5/6 in transcriptional repression. GO analysis revealed that the derepressed genes were primarily enriched in responsive pathways (Fig. 5g). Interestingly, DNA content analysis showed that the rice leaf predominantly contains 2C cells, and no extra DNA content was detected in osatxr5^c;osatxr6kd-1 and osatxr5^c;osatxr6kd-2 (Additional file 1: Fig. S11h, i). Moreover, DNA damage response genes were not activated in osatxr5^c;osatxr6kd-1 and osatxr5^c;osatxr6kd-2, including those homologous to genes induced in the Arabidopsis atxr5;atxr6 mutants (i.e., RAD51, BRCA1, GR1) (Fig. 5G; Additional file 1: Fig. S11j, k; Additional file 2: Table S7, 8) [24]. Thus, unlike in Arabidopsis, the loss of OsATXR5/6 causes transcriptional activation without compromising DNA stability in rice. Together, these results suggest that ATXR5/6 have conserved roles in regulating development and responsive genes in both Arabidopsis and rice.

Discussion

By generating strong atxr5;atxr6 mutants in Arabidopsis, we have demonstrated that ATXR5/6 are crucial for regulating both plant development and abiotic stress responses. The function of ATXR5/6 in these processes relies on their enzymatic activity but is likely independent of their role in heterochromatin regulation, as the heterochromatin defects are comparable in the weak atxr5;atxr6^hyp and the strong atxr5;atxr5;atxr6^hyp;atxr6^c-1 and atxr5;atxr5;atxr6^hyp;atxr6^c-2 mutants, with only the latter two showing clear developmental abnormalities and enhanced resistance to abiotic stresses (Figs. 1 and 2; Additional file 1: Fig. S2–6). This aligns with the idea that heterochromatin defects in Arabidopsis per se do not typically cause strong developmental defects [40, 41]. On the other hand, the further loss of ATXR5/6 in the strong atxr5;atxr6 mutant induces the derepression of many genes, including those responsive to oxidative stress and abiotic stimulus (Fig. 2a–e). ATXR5/6-mediated H3K27me1 is mainly enriched at heterochromatin regions [13, 17, 18]. It is possible that a partial loss of ATXR5/6 in the weak atxr5;atxr6^hyp mutant primarily impairs the deposition of H3K27me1 at heterochromatin, while a further loss of ATXR5/6 starts to cause defects at genic regions. This is further supported by the observations that complete loss of ATXR5/6 results in lethality, suggesting that a minimal level of ATXR5/6-mediated H3K27me1 is essential for plant viability.

Among the derepressed genes in the strong atxr5;atxr6 mutant, there is a group of peroxidase-coding genes, including those encoding class III peroxidases (i.e., PRX32, PRX49, PRX53, PRX71). Class III peroxidases are plant-specific peroxidases that can reduce peroxide and oxidize a wide range of substrates, such as lignin precursor [42]. Only a few class III peroxidases are found in some streptopyhte algae, but they have been greatly expanded in land plants [42, 43]. Therefore, class III peroxidases may play a critical role by adapting the land plants to a more oxygenated environment and enabling the formation of rigid cell walls suitable for terrestrial life [42–44]. As antioxidants, peroxidases are also important for plant responses to environmental stresses, with many being induced by stress signals [45, 46]. However, overexpression of peroxidases leads to reduced plant growth [27, 28], suggesting that their expression must be tightly controlled. It is likely that ATXR5/6 repress peroxidase-coding genes under normal growth conditions to balance plant growth and stress response.

We found that a loss of H3K27me1 at peroxidase-coding genes is accompanied by an increase in H3K27ac and H3K36me3 (Fig. 2j; Fig. 3a, b), suggesting that H3K27me1 represses active histone modifications. Introducing GCN5 and EFS mutations into the atxr5;atxr5;atxr6^hyp;atxr6^c-1 mutant partially suppressed the activation of peroxidase-coding genes (Fig. 3g, h). These observations are consistent with a recent study that reported an increase in H3K27ac at heterochromatin leading to heterochromatin defects and TE activation in atxr5;atxr6^hyp [29]. Therefore, AXTR5/6-mediated H3K27me1 appears to play a similar role in maintaining transcriptional silencing at both heterochromatin and genic regions by preventing the accumulation of active histone modifications.

In addition to depositing H3K27me1, we show that ATXR5/6 are also required for the maintenance of H3K27me3, a crucial histone modification in determining cellular identity (Fig. 4; Additional file 1: Fig. S8) [47]. This observation supports the previous hypothesis that the ATXR5/6-deposited H3K27me1 during DNA replication may serve as a substrate for the further catalyzation and rapid recovery of H3K27me3 after DNA replication in plants [35, 38]. Notably, H3K27me3 is only mildly affected in the strong atxr5;atxr6 mutant. This may be due to the residual ATXR6 still providing some H3K27me1, or the H3K27me3 methyltransferases may be capable of synthesizing most H3K27me3 directly from unmethylated H3K27. Nevertheless, this mild loss of H3K27me3 leads to moderate depression of H3K27me3-enriched genes (Fig. 4e; Additional file 1: Fig. S8c), and, in particular, the flowering time regulatory gene AGL19 becomes strongly activated upon loss of ATXR5/6, which contributes to the accelerated floral transition phenotype of the strong atxr5;atxr6 mutant, suggesting the importance of the ATXR5/6-assisted H3K27me3 maintenance in controlling development and cellular identity. We propose that land plants may have evolved ATXR5/6 for the strict maintenance of H3K27me3, which is necessary to support their complex developmental programs and the great diversity of their cell types. Additional studies are needed to distinguish the effects of ATXR5/6-dependent H3K27me1 and H3K27me3 in plant development and stress responses.

To test the function of ATXR5/6 in other plants, we generated the null osatxr5;osatxr6 mutant in rice and found that OsATXR5/6 are also essential for plant reproduction (Fig. 5a, b). However, unlike in Arabidopsis, OsATXR5/6 are required only for seed development and are dispensable for female gametogenesis. Further studies are needed to dissect the detailed functions of ATXR5/6 in plant reproductive development. In addition, by knocking down the expression of OsATXR5/6, we observed that the loss of OsATXR5/6 also leads to reduced plant growth and the depression of TEs and responsive genes (Fig. 5c–g; Additional file 1: Fig. S11c–g), suggesting the conserved function of OsATXR5/6 in repressing transcription and balancing plant growth and stress responses in rice. In Arabidopsis, the loss of ATXR5/6 results in genome instability, characterized by excess accumulation of heterochromatic DNA in endoreduplicated cells [16]. Interestingly, rice leaf cells lack endoreduplication, and no extra DNA was detected in the leaf nuclei of the osatxr5;osatxr6 knockdown mutants (Additional file 1: Fig. S11h, i). This suggests that loss of ATXR5/6 induces transcriptional activation independent from affecting genome stability, at least in rice.

In conclusion, this study demonstrates that ATXR5/6 play conserved roles in controlling plant development and stress responses, which might be essential for terrestrial adaptation. This provides a possible explanation for the emergence of ATXR5/6 in land plants. Furthermore, our work highlights complex functions of ATXR5/6 in regulating transcriptional repression at both heterochromatin and genic regions through mediating H3K27me1 and H3K27me3. Further studies are required to clarify these functions in more detail.

Conclusions

ATXR5 and ATXR6 are land plant-specific histone methyltransferases that catalyze H3K27me1, yet the significance of their acquisition in land plants remains unclear. In this study, we investigated the roles of ATXR5/6 in the dicot Arabidopsis and the monocot rice and found that they are essential for reproductive development. Specifically, ATXR5/6 are required for both female gametogenesis and embryogenesis in Arabidopsis, but only for embryogenesis in rice. In addition, our results demonstrate that ATXR5/6 are necessary for vegetative development in both species and function to repress the transcription of responsive genes, highlighting a conserved role in balancing growth and stress responses. Furthermore, we show that ATXR5/6 contribute to the global maintenance of H3K27me3 in Arabidopsis, likely by supplying H3K27me1 for further methylation, which is critical for the full repression of H3K27me3-marked genes. Together, these findings offer new insights into the functional significance of ATXR5/6 in land plants and lay the foundation for future studies on their land plant-specific evolution.

Methods

Plant materials and growth conditions

The Arabidopsis atxr5;atxr6^hyp [13], gcn5 (Salk_150784) [48], and efs-3 [49] have been previously described. Arabidopsis plants were grown under long day conditions (16-h light/8-h dark) at 22 °C. Rice plants were grown in paddy fields in Beijing, China.

Plasmid construction for plant transformation

CRISPR-Cas9 gene editing constructs containing two single-guide RNAs targeting Arabidopsis ATXR6 or AGL19 were generated using pHSE401 as previously described (Xing et al., 2014). Constructs for editing OsATXR5 and OsATXR6 were generated using pYLCRISPR/Cas9P_ubi-H, following the methods described by [50]. Guide RNA sequences are listed in Additional file 2: Table S9. All the mutations were confirmed via Sanger sequencing. For the complementation test, the genomic DNA of ATXR5, including its promoter, was inserted into the pHGW vector [51]. The ATXR5 sequence was further mutated to generate gATXR5 E217A&M221A or gATXR5 Y274N. To knock down the expression of OsATXR6 (OsATXR6 RNAi), a 446 bp cDNA fragment, mainly from the 3′UTR region of OsATXR6, was PCR-amplified and cloned into the LH-FAD1390RNAi vector [52].

Measurement of plant diameter

Rosette diameter was measured as the distance from tip to tip of the longest rosette leaves in 3-week-old plants using ImageJ software. A total of 15 plants were measured for each line.

Pst DC3000 infection and bacteria number counting

Pst DC3000 infection was performed as previously described [53]. Briefly, Pst DC3000 was cultivated with KB medium containing rifampicin at 30 °C. After centrifugation, the bacteria were resuspended in sterilized double distilled water (ddH2O) to an OD₆₀₀ of 0.005. The bacterial suspension was infiltrated into Arabidopsis leaves using a syringe. Leaf discs with a 6-mm diameter were collected 3 h post-inoculation (0 DAI) or 3 days post-inoculation (3 DAI). The discs were washed with sterilized ddH₂O and ground in 100 μl of sterilized ddH2O. After adding 900 μl of sterilized ddH2O, the samples were thoroughly mixed and serially diluted 1:10. Then, 50 µl of each dilution was plated on TSA agar medium containing rifampicin, and colony numbers were counted after incubating at 30 °C for 2 days.

H2O2 and NaCl treatment

After-ripened seeds (harvested and stored at room temperature for at least 3 months) were sown on 1/2 MS medium, with or without H₂O₂ or NaCl. For the zeocin treatment, WT Col seeds were sown on 1/2 MS medium under the following conditions: without zeocin, with zeocin, without zeocin but with H₂O₂/NaCl, and with both zeocin and H₂O₂/NaCl. After stratification at 4 °C for 3 days, seeds were germinated under long-day conditions at 22 °C. Plants with true leaves were counted at 10, 14, or 15 days after germination. Two or three biological replicates were performed for each condition. For the H₂O₂ treatment in RT-qPCR and ChIP-qPCR experiments, 3-week-old Arabidopsis plants were sprayed with 20 mM H₂O₂ or with H₂O (as a mock control). Their leaves were collected 5 h after treatment.

Measurement of peroxidase activity

Peroxidase activity was measured using the peroxidase activity detection kit (Solarbio, BC0095) following the manufacturer’s instruction. Briefly, 0.1 g of leaves from 3-week-old Arabidopsis plants was homogenized on ice using a mortar and pestle in 1-ml extraction buffer. After centrifugation, the supernatant was mixed with buffer 1, 2, and 3, and the absorbance was then measured with a spectrometer at OD₄₇₀.

Western blotting

Nuclei extracts from leaves of 3-week-old Arabidopsis plants or 40-day-old rice were separated by SDS-PAGE and then transferred to a 0.2-μm nitrocellulose membrane (GE Healthcare). Proteins were detected with anti-H3K27me1 (Millipore, 07–448) or anti-H3 (Abcam, ab1791) antibodies. The intensity of the protein bands were quantified using ImageJ and normalized to the H3 loading control.

DNA content analysis

For DNA content analysis, leaves from 3-week-old Arabidopsis plants and 40-day-old rice plants were chopped in Galbraith buffer (20 mM MOPS, 45 mM MgCl2, 30 mM sodium citrate, and 0.1% Triton X-100) for Arabidopsis or lysis buffer (25 mM Tris–HCl pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton X-100, 2 mM spermine, and 10 mM β-Mercaptoethanol) for rice. Then, samples were filtered through a 30-μm filter, and nuclei were stained with DAPI. Flow cytometry profiles were obtained using a BD FACSAria II cell sorter.

Pollen viability examination

Mature rice pollen grains were stained with a 1% I₂-KI solution and observed under a light microscope to assess viability.

Immunofluorescence

Immunostaining of isolated leaf nuclei was performed as previously described [36]. H3K27me1 signals were detected with anti-H3K27me1 antibody (Millipore, 07–448). Images were captured with a Zeiss confocal laser scanning microscope.

RNA-seq

For RNA-seq analysis, total RNA was extracted from leaves of 3-week-old Arabidopsis plants or 40-day-old rice plants using the Minibest plant RNA extraction kit (Takara, 9769), and three independent biological replicates were performed. Sequencing libraries were prepared with the NEBNext Ultra RNA library prep kit for Illumina (NEB, 7530L) according to the manufacturer’s instruction. Prepared libraries were sequenced on a NovaSeq 6000 platform and paired-end 150 bp reads were generated. Adapter trimming was performed and low-quality reads were filtered with fastp version 0.20.1 [54]. Reads were aligned to the Arabidopsis genome (TAIR10) or the rice genome (RGAP, version 7.0) using Hisat2 version 2.1.10 [55]. Reads per gene were counted by HTseq version 0.11.2 [56]. Transcripts per million (TPM) values were generated using R. Differential gene expression analysis was performed using DESeq2 version 1.26.0 [57]. Genes were considered as differentially expressed if they exhibited a fold change greater than two and a P adjust value < 0.05. Gene ontology analysis was conducted with DAVID (https://david.ncifcrf.gov/) [58] for Arabidopsis and PlantRegMap (https://plantregmap.gao-lab.org/) [59] for rice.

ChIP-seq

ChIP-seq was performed using leaves from 3-week-old Arabidopsis plants, which were fixed with 1% formaldehyde. After nuclei extraction, mononucleosomes were generated by digesting with micrococcal nuclease (NEB, M0247S). Immunoprecipitation was conducted with anti-H3K27me3 antibody (Millipore, 07–449) as previously described [60]. Two independent biological replicates were performed. Immunoprecipitated DNA were subjected to library preparation with the VAHTS universal DNA library prep kit for Illumina (Vazyme, ND607-02) according to the manufacturer’s instruction. Prepared libraries were sequenced on a NovaSeq 6000 platform and paired-end 150 bp reads were generated. Adapter trimming was performed and low-quality reads were filtered with fastp version 0.20.1 [54]. Reads were mapped to the Arabidopsis (TAIR10) genome with Bowtie2 version 2.4.2 [61]. Duplicate reads were filtered using Picard version 2.24.0 MarkDuplicates (https://github.com/broadinstitute/picard). H3K27me3 peaks in WT Col were called using MACS2 version 2.1.2 with input control and default parameters [62]. Only peaks identified in both biological replicates were retained. For data visualization, data from two biological replicates were merged, and bigwig coverage files were generated using deepTools utility bamCoverage with a bin size of 10 bp [63]. Average ChIP-seq profiles were generated using deepTools utility plotProfile. For the analysis of H3.1 and H3.3 signals over H3K27me3-enriched peaks and genes, the H3.1 and H3.3 ChIP-seq data were obtained from a previous publication [64].

RT-qPCR

Total RNA was extracted from leaves of 3-week-old Arabidopsis plants or 1-week-old rice seedlings with Minibest plant RNA extraction kit (Takara, 9769), and three independent biological replicates were performed. Reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, R312-02). Real-time quantitative PCR was conducted on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02). For normalization, TUB2 was used as the endogenous control in Arabidopsis, and OsUBQ5 was used in rice. Primers used for amplification are listed in Additional file 2: Table S10.

ChIP-qPCR

Leaves from 3-week-old Arabidopsis plants were fixed with 1% formaldehyde. After nuclei extraction, mononucleosomes were generated by digesting with micrococcal nuclease (NEB, M0247S). Immunoprecipitation was conducted with anti-H3K27me1 (Millipore, 07–448), anti-H3K27ac (Abclonal, a7253), or anti-H3K36me3 (Abcam, ab9050) antibody as previously described [60]. The amount of immunoprecipitated DNA was quantified by real-time PCR. Three independent biological replicates were performed. Primers used for amplification are listed in Additional file 2: Table S10.

Supplementary Information

13059_2025_3801_MOESM1_ESM.pdf^{(32.2MB, pdf)}

Additional file 1: Supplementary figures. Figs. S1–S11.

13059_2025_3801_MOESM2_ESM.xlsx^{(118.8KB, xlsx)}

Additional file 2: Supplementary tables. Tables S1–S10.

13059_2025_3801_MOESM3_ESM.pdf^{(495KB, pdf)}

Additional file 3: Uncropped images for the blots in Fig. S2c.

13059_2025_3801_MOESM4_ESM.pdf^{(608.2KB, pdf)}

Additional file 4: Uncropped images for the blots in Fig. S11b.

Acknowledgements

We thank Dr. Lei Li for the valuable advice on the bacterial infection experiment.

Peer review information

Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. The peer-review history is available in the online version of this article.

Authors’ contributions

X.L., J.P., and D.J. designed experiments; X.L., J.P., H.L., and D.J. performed experiments; X.L., J.P., Q.L., and H.Z. analyzed data; D.J. wrote the paper with the help from X.L., J.P., and Q.L.; All authors read and approved the final manuscript.

Funding

This work was supported by the National Key R&D Program of China (2023YFD1200704) and the intramural research support from Temasek Life Sciences Laboratory.

Data availability

The RNA-seq datasets for Arabidopsis and rice, as well as the H3K27me3 ChIP-seq dataset for Arabidopsis generated in this study, are available in the NCBI Gene Expression Omnibus under accession numbers GSE278500 [65], GSE278501 [66], and GSE278502 [67], respectively. The H3.1 and H3.3 ChIP-seq datasets were obtained from a previous publication and are available under accession number GSE34840 [68].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiaoyi Li and Jie Pan contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13059_2025_3801_MOESM1_ESM.pdf^{(32.2MB, pdf)}

Additional file 1: Supplementary figures. Figs. S1–S11.

13059_2025_3801_MOESM2_ESM.xlsx^{(118.8KB, xlsx)}

Additional file 2: Supplementary tables. Tables S1–S10.

13059_2025_3801_MOESM3_ESM.pdf^{(495KB, pdf)}

Additional file 3: Uncropped images for the blots in Fig. S2c.

13059_2025_3801_MOESM4_ESM.pdf^{(608.2KB, pdf)}

Additional file 4: Uncropped images for the blots in Fig. S11b.

Data Availability Statement

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[CR11] 11.Ebert A, Schotta G, Lein S, Kubicek S, Krauss V, Jenuwein T, et al. Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev. 2004;18:2973–83. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR12] 12.Shen X, Liu Y, Hsu YJ, Fujiwara Y, Kim J, Mao X, et al. EZH1 mediates methylation on histone H3 lysine 27 and complements EZH2 in maintaining stem cell identity and executing pluripotency. Mol Cell. 2008;32:491–502. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Jacob Y, Feng S, LeBlanc CA, Bernatavichute YV, Stroud H, Cokus S, et al. ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing. Nat Struct Mol Biol. 2009;16:763–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR14] 14.Jacob Y, Michaels SD. H3K27me1 is E(z) in animals, but not in plants. Epigenetics. 2009;4:366–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Mathieu O, Probst AV, Paszkowski J. Distinct regulation of histone H3 methylation at lysines 27 and 9 by CpG methylation in Arabidopsis. EMBO J. 2005;24:2783–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Jacob Y, Stroud H, Leblanc C, Feng S, Zhuo L, Caro E, et al. Regulation of heterochromatic DNA replication by histone H3 lysine 27 methyltransferases. Nature. 2010;466:987–91. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Antunez-Sanchez J, Naish M, Ramirez-Prado JS, Ohno S, Huang Y, Dawson A, Opassathian K, Manza-Mianza D, Ariel F, Raynaud C, et al. A new role for histone demethylases in the maintenance of plant genome integrity. Elife. 2020;9:e58533. [DOI] [PMC free article] [PubMed]

[CR18] 18.Potok ME, Zhong Z, Picard CL, Liu Q, Do T, Jacobsen CE, Sakr O, Naranbaatar B, Thilakaratne R, Khnkoyan Z, et al. The role of ATXR6 expression in modulating genome stability and transposable element repression in Arabidopsis. Proc Natl Acad Sci U S A. 2022;119:e2115570119. [DOI] [PMC free article] [PubMed]

[CR19] 19.Lu F, Cui X, Zhang S, Jenuwein T, Cao X. Arabidopsis REF6 is a histone H3 lysine 27 demethylase. Nat Genet. 2011;43:715–9. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Zheng S, Hu H, Ren H, Yang Z, Qiu Q, Qi W, et al. The Arabidopsis H3K27me3 demethylase JUMONJI 13 is a temperature and photoperiod dependent flowering repressor. Nat Commun. 2019;10:1303. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Zhao X, Wang J, Jin D, Cheng J, Chen H, Li Z, et al. AtMCM10 promotes DNA replication-coupled nucleosome assembly in Arabidopsis. J Integr Plant Biol. 2023;65:203–22. [DOI] [PubMed] [Google Scholar]

[CR22] 22.Jacob Y, Bergamin E, Donoghue MT, Mongeon V, LeBlanc C, Voigt P, et al. Selective methylation of histone H3 variant H3.1 regulates heterochromatin replication. Science. 2014;343:1249–53. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Yu CW, Tai R, Wang SC, Yang P, Luo M, Yang S, et al. Histone deacetylase6 acts in concert with histone methyltransferases SUVH4, SUVH5, and SUVH6 to regulate transposon silencing. Plant Cell. 2017;29:1970–83. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Stroud H, Hale CJ, Feng S, Caro E, Jacob Y, Michaels SD, et al. DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis. PLoS Genet. 2012;8:e1002808. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Wang S, Durrant WE, Song J, Spivey NW, Dong X. Arabidopsis BRCA2 and RAD51 proteins are specifically involved in defense gene transcription during plant immune responses. Proc Natl Acad Sci USA. 2010;107:22716–21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Yan S, Wang W, Marqués J, Mohan R, Saleh A, Durrant Wendy E, et al. Salicylic acid activates DNA damage responses to potentiate plant immunity. Mol Cell. 2013;52:602–10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Pedreira J, Herrera MT, Zarra I, Revilla G. The overexpression of AtPrx37, an apoplastic peroxidase, reduces growth in Arabidopsis. Physiol Plant. 2011;141:177–87. [DOI] [PubMed] [Google Scholar]

[CR28] 28.Raggi S, Ferrarini A, Delledonne M, Dunand C, Ranocha P, De Lorenzo G, et al. The Arabidopsis class III peroxidase AtPRX71 negatively regulates growth under physiological conditions and in response to cell wall damage. Plant Physiol. 2015;169:2513–25. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Dong J, LeBlanc C, Poulet A, Mermaz B, Villarino G, Webb KM, et al. H3.1K27me1 maintains transcriptional silencing and genome stability by preventing GCN5-mediated histone acetylation. Plant Cell. 2021;33:961–79. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Chen C, Li C, Wang Y, Renaud J, Tian G, Kambhampati S, et al. Cytosolic acetyl-CoA promotes histone acetylation predominantly at H3K27 in Arabidopsis. Nat Plants. 2017;3:814–24. [DOI] [PubMed] [Google Scholar]

[CR31] 31.Xu L, Zhao Z, Dong A, Soubigou-Taconnat L, Renou JP, Steinmetz A, et al. Di- and tri- but not monomethylation on histone H3 lysine 36 marks active transcription of genes involved in flowering time regulation and other processes in Arabidopsis thaliana. Mol Cell Biol. 2008;28:1348–60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Raynaud C, Sozzani R, Glab N, Domenichini S, Perennes C, Cella R, et al. Two cell-cycle regulated SET-domain proteins interact with proliferating cell nuclear antigen (PCNA) in Arabidopsis. Plant J. 2006;47:395–407. [DOI] [PubMed] [Google Scholar]

[CR33] 33.Del Olmo I, Lopez JA, Vazquez J, Raynaud C, Pineiro M, Jarillo JA. Arabidopsis DNA polymerase recruits components of Polycomb repressor complex to mediate epigenetic gene silencing. Nucleic Acids Res. 2016;44:5597–614. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR34] 34.Hyun Y, Yun H, Park K, Ohr H, Lee O, Kim DH, et al. The catalytic subunit of Arabidopsis DNA polymerase alpha ensures stable maintenance of histone modification. Development. 2013;140:156–66. [DOI] [PubMed] [Google Scholar]

[CR35] 35.Jiang D, Berger F. DNA replication-coupled histone modification maintains polycomb gene silencing in plants. Science. 2017;357:1146–9. [DOI] [PubMed] [Google Scholar]

[CR36] 36.Wang L, Xue M, Zhang H, Ma L, Jiang D. TONSOKU is required for the maintenance of repressive chromatin modifications in Arabidopsis. Cell Rep. 2023;42:112738. [DOI] [PubMed] [Google Scholar]

[CR37] 37.Zhou Y, Tergemina E, Cui H, Forderer A, Hartwig B, Velikkakam James G, et al. Ctf4-related protein recruits LHP1-PRC2 to maintain H3K27me3 levels in dividing cells in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2017;114:4833–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Borg M, Jiang D, Berger F. Histone variants take center stage in shaping the epigenome. Curr Opin Plant Biol. 2021;61:101991. [DOI] [PubMed] [Google Scholar]

[CR39] 39.Schonrock N, Bouveret R, Leroy O, Borghi L, Kohler C, Gruissem W, et al. Polycomb-group proteins repress the floral activator AGL19 in the FLC-independent vernalization pathway. Genes Dev. 2006;20:1667–78. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR40] 40.Vongs A, Kakutani T, Martienssen RA, Richards EJ. Arabidopsis thaliana DNA methylation mutants. Science. 1993;260:1926–8. [DOI] [PubMed] [Google Scholar]

[CR41] 41.Bourguet P, Picard CL, Yelagandula R, Pélissier T, Lorković ZJ, Feng S, et al. The histone variant H2A.W and linker histone H1 co-regulate heterochromatin accessibility and DNA methylation. Nat Commun. 2021;12:2683. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Land plant-specific H3K27 methyltransferases ATXR5 and ATXR6 control plant development and stress responses

Xiaoyi Li

Jie Pan

Qian Liu

Huairen Zhang

Hui Li

Danhua Jiang

Abstract

Background

Results

Conclusions

Supplementary Information

Background

Results

Loss of ATXR5 and ATXR6 results in strong developmental defects

Fig. 1.

Loss of ATXR5 and ATXR6 enhances plant resistance to bacterial infection

Fig. 2.

ATXR5/6 repress plant oxidative stress response

ATXR5/6 repress active histone modifications at peroxidase-coding genes

Fig. 3.

ATXR5/6 contribute to the maintenance of H3K27me3

Fig. 4.

OsATXR5/6 regulate reproductive development and repress responsive genes in rice

Fig. 5.

Discussion

Conclusions

Methods

Plant materials and growth conditions

Plasmid construction for plant transformation

Measurement of plant diameter

Pst DC3000 infection and bacteria number counting

H2O2 and NaCl treatment

Measurement of peroxidase activity

Western blotting

DNA content analysis

Pollen viability examination

Immunofluorescence

RNA-seq

ChIP-seq

RT-qPCR

ChIP-qPCR

Supplementary Information

Acknowledgements

Peer review information

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

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