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Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy

  1. Hikaru Sato
  2. Juan Santos-González
  3. Claudia Köhler  Is a corresponding author
  1. Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Sweden
  2. Max Planck Institute of Molecular Plant Physiology, Germany
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Cite this article as: eLife 2021;10:e64593 doi: 10.7554/eLife.64593

Abstract

Polycomb Repressive Complex 2 (PRC2)-mediated trimethylation of histone H3 on lysine 27 (H3K27me3) and methylation of histone 3 on lysine 9 (H3K9me) are two repressive epigenetic modifications that are typically localized in distinct regions of the genome. For reasons unknown, however, they co-occur in some organisms and special tissue types. In this study, we show that maternal alleles marked by H3K27me3 in the Arabidopsis endosperm were targeted by the H3K27me3 demethylase REF6 and became activated during germination. In contrast, maternal alleles marked by H3K27me3, H3K9me2, and CHG methylation (CHGm) are likely to be protected from REF6 targeting and remained silenced. Our study unveils that combinations of different repressive epigenetic modifications time a key adaptive trait by modulating access of REF6.

Introduction

Animals, fungi, and plants make use of two major epigenetic repressive modifications on histones that are destined to silence genes and transposable elements (TEs). The Polycomb repressive system applies the repressive trimethylation on lysine 27 of histone H3 (H3K27me3), which mainly serves to silence genes during defined stages of development (Mozgova and Hennig, 2015; Schuettengruber et al., 2017). In contrast, the repressive di- or trimethylation on lysine 9 of histone H3 (H3K9me2/3) is mainly associated with constitutively silenced heterochromatin, present in repeat-rich regions and TEs (Saksouk et al., 2015; Wendte and Schmitz, 2018). Repression mediated by the Polycomb system is transient and dynamically regulated, while repression mediated by H3K9me2/3 is generally stable during development (Mozgova and Hennig, 2015; Saksouk et al., 2015; Schuettengruber et al., 2017; Wendte and Schmitz, 2018). The establishment of H3K27me3 is mediated by the evolutionary conserved Polycomb repressive complex 2 (PRC2). In plants, there are distinct PRC2 complexes that are active during different stages of plant development (Mozgova and Hennig, 2015). In Arabidopsis, the EMBRYONIC FLOWER (EMF) and VERNALIZATION (VRN) PRC2 regulate sporophyte development, while the FERTILIZATION-INDEPENDENT SEED (FIS) PRC2 is active in the central cell of the female gametophyte and the descendent endosperm (Mozgova and Hennig, 2015). The establishment of H3K9me2 in Arabidopsis depends on the SU(VAR)3–9 homologous proteins (SUVHs), KRYPTONITE (or SUVH4), SUVH5 and SUVH6 that act in a feedback loop with CHG methylation (CHGm) established by the CHROMOMETHYLTRANSFERASE 3 (CMT3) (Kenchanmane Raju et al., 2019; Zhang et al., 2018). In animals and plants, H3K27me3 and H3K9me2 are generally exclusive repressive marks (Wiles and Selker, 2017); however, they co-occur in certain tissue types like the plant endosperm (Klosinska et al., 2016; Moreno-Romero et al., 2016; Weinhofer et al., 2010), as well as in specific organisms as filamentous fungi and bryophytes (Carlier et al., 2020; Montgomery et al., 2020). Furthermore, redistribution of H3K27me3 to heterochromatic regions occurs when constitutive heterochromatin is disrupted, indicating that specific features of heterochromatin prevent PRC2 recruitment (Deleris et al., 2012; Jamieson et al., 2016; Peters et al., 2003; Wiles and Selker, 2017).

Erasure of H3K27me3 and H3K9me2 is mediated by different types of JUMONJI lysine demethylases (KDMs) that belong to the KDM4 and KDM3 type, respectively (Cheng et al., 2020). RELATIVE OF EARLY FLOWERING 6 (REF6) and EARLY FLOWERING 6 (ELF6) function redundantly as major erasers of H3K27me3 during Arabidopsis development (Lu et al., 2011; Yan et al., 2018). The zinc-finger (ZnF) domains of REF6 recognize the sequence motif CTCTGYTY (Y = T or C) (Cui et al., 2016; Li et al., 2016). Binding of REF6 to this motif is negatively affected by non-CG methylation (Qiu et al., 2019), revealing cross-talk between different epigenetic modifications.

Seed development in flowering plants is initiated by a double fertilization event, where one of the sperm cells fertilizes the haploid egg, giving rise to the diploid embryo and the other sperm cell fertilizes the diploid central cell, initiating development of the triploid endosperm (Zhou and Dresselhaus, 2019). The endosperm regulates the transfer of nutrients to the developing embryo, similar as the placenta in mammals. (Costa et al., 2012; Lee et al., 2010; Linkies et al., 2009). The endosperm furthermore regulates seed dormancy, a process preventing germination of seeds under favourable conditions (Nonogaki, 2019). Both processes have been linked to genomic imprinting, an epigenetic phenomenon causing parent-of-origin specific gene expression (Batista and Kohler, 2020; Gehring and Satyaki, 2017; Piskurewicz et al., 2016). Imprinted genes are epigenetically modified during gametogenesis, establishing parental-specific expression patterns that are maintained after fertilization. Genomic imprinting leading to maternally biased expression is predominantly connected to DNA demethylation of maternal alleles mediated by the DNA glycosylase DEMETER (Gehring et al., 2009; Hsieh et al., 2011; Park et al., 2016). In contrast, paternally biased expression is predominantly connected to the presence of H3K27me3, H3K9me2, and CHGm on maternal alleles (Klosinska et al., 2016; Moreno-Romero et al., 2019; Moreno-Romero et al., 2016; Moreno-Romero et al., 2017). The FIS-PRC2 establishes H3K27me3 in the central cell, which is maintained on the maternal alleles after fertilization. Loss of FIS-PRC2 function impairs CHGm establishment, indicating that H3K27me3 is the primary imprinting mark and CHGm and H3K9me2 are recruited after H3K27me3 establishment (Moreno-Romero et al., 2019). The biological meaning of the co-occurance of H3K27me3, H3K9me2, and CHGm on the maternal alleles of paternally expressed genes (PEGs) is unclear. In this study, we reveal that genes marked by H3K27me3 on their maternal alleles are targeted by REF6 and become reactivated during germination. In contrast, REF6 fails to activate genes marked by H3K27me3, H3K9me2, and CHGm, consistent with the finding that DNA methylation prevents REF6 binding (Qiu et al., 2019). Among those genes with triple repressive marks was the ABA INSENSITIVE 3 (ABI3) gene, a known positive regulator of seed dormancy (Liu et al., 2013; Shu et al., 2016; Tian et al., 2020). Genes activated by REF6 are enriched for ethylene signaling, a process activated during germination. Consistently, loss of REF6 causes increased dormancy, as recently reported (Chen et al., 2020). Together, our study unveils a mechanism that employs the combination of different repressive epigenetic modifications to time a crucial developmental process.

Results

Genes marked by single H3K27me3 are expressed during germination

To understand the functional relevance of different combinations of repressive epigenetic modifications in the endosperm, we analyzed gene ontologies (GOs) of genes marked with single H3K27me3, single H3K9me2, and both modifications (referred to as H3K27me3/H3K9me2) using AgriGO v2.0 (http://systemsbiology.cau.edu.cn/agriGOv2/; Tian et al., 2017; Figure 1—figure supplement 1). Genes marked by single H3K9m2 were not enriched for any GO category and genes marked by H3K27me3/H3K9me2 only for one; in contrast, genes marked with single H3K27me3 were significantly enriched for many GOs (Figure 1—figure supplement 1). Interestingly, among those GOs was an enrichment of genes related to ethylene signaling (Figure 1—figure supplement 1; Dubois et al., 2018), a pathway which is activated in the endosperm tissue during germination to allow root protrusion through cell loosening (Linkies and Leubner-Metzger, 2012; Linkies et al., 2009) and known to release seed dormancy (Beaudoin et al., 2000; Corbineau et al., 2014). Supporting the GO enrichment, we found that genes with single H3K27me3 significantly overlapped with ethylene-inducible genes identified in a previous study (Das et al., 2016), while genes with single H3K9me2 and H3K27me3/H3K9me2 did not (Figure 1a). We analyzed the expression of ethylene-inducible genes marked by single H3K27me3 during germination in the endosperm using previously published data (Dekkers et al., 2013). We found that ethylene-inducible genes with single H3K27me3 were induced during germination, especially around the time point of endosperm rupture (p<0.05, Wilcoxon test) (Figure 1b), while there were no significant expression differences between different time points for genes with single H3K9me2 and genes with both H3K27me3/H3K9me2. Increased gene expression in the endosperm during germination was not restricted to ethylene inducible genes marked by H3K27me3, but also detectable for all H3K27me3 marked genes (Figure 1—figure supplement 2). This data reveal that genes with single H3K27me3 are initially suppressed during early endosperm development and become reactivated during germination, while genes with single H3K9me2 and genes with H3K27me3/H3K9me2 remain continuously suppressed throughout endosperm development and germination. This implies the presence of a molecular mechanism that allows to differentially regulate the expression of the three groups of epigenetically marked genes during germination.

Figure 1 with 2 supplements see all
Genes marked by H3K27me3 become activated in the endosperm during germination.

(a) Venn diagrams show overlaps between three groups of genes with different patterns of histone modifications and ethylene (ET)-inducible genes in seedlings. ET-inducible genes identified in a previous study (Das et al., 2016) were compared with genes marked by the indicated histone modifications in the developing endosperm (Moreno-Romero et al., 2016). Asterisks indicate significant overlap (p<0.0001, Fisher’s exact test). (b) Box plots show relative expression profiles of genes marked by the indicated histone modifications in the endosperm during germination. Time-course expression profiles of ethylene inducible genes with single H3K27me3 (top; 185 genes), single H3K9me2 (middle; 336 genes), and H3K27me3, H3K9me2 and CHG methylation (bottom; 227 genes). Expression data are endosperm-specific transcriptome data during germination (Dekkers et al., 2013). The time points of testa rupture and endosperm (Endo) rupture are shown. Asterisks indicate significant differences compared to the time point at 1 hr of imbibition (p<0.01, Wilcoxon test).

Genes with double H3K27me3/H3K9me2 modifications are suppressed through CHG methylation on REF6-binding sites during germination

Recent work revealed that the presence of CHG methylation (referred to as CHGm) prevents binding of the H3K27me3 histone demethylase RELATIVE OF EARLY FLOWERING 6 (REF6/JMJ12) to its target motif CTCTGYTY (Y = T or C) (Qiu et al., 2019). We recently showed that genes with dual H3K27me3/H3K9me2 modifications are marked by CHGm in the endosperm and that CHGm is established in the central cell (Moreno-Romero et al., 2019). We thus hypothesized that the presence of CHGm may prevent REF6 from targeting and activating those genes. To test this hypothesis, we identified genes with REF6 binding sites in their gene bodies and associated the presence of the REF6 motif, CHGm status, and expression during germination. We found that REF6 binding sites within genes that were marked either by single H3K9me2 or by H3K27me3/H3K9me2 were more frequently CHG methylated in the central cell (Figure 2a) and endosperm (Figure 2—figure supplement 1) compared to single H3K27me3 marked genes (Moreno-Romero et al., 2016; Park et al., 2016). Importantly, genes marked by H3K27me3/H3K9me2 were frequently marked by CHGm at position five within the REF6-binding site (Figure 2b). Methylation at position five was found to be sufficient to abolish the affinity of REF6 for this target site, while methylation at position three was less effective (Qiu et al., 2019). Consistent with the hypothesis that CHGm on REF6 binding sites prevents REF6 targeting and thus gene activation, we found that genes with non-methylated REF6 binding sites were significantly more frequently activated during germination than genes with methylated REF6 binding sites (Figure 2c). Together, this data supports the idea that the methylation status of REF6-binding sites affects the reactivation of genes in the endosperm during germination. While H3K27me3 marked genes can be activated during germination, genes with H3K27me3, H3K9me2, and CHGm fail to be activated since REF6 (or homologous proteins) recruitment is blocked by DNA methylation.

Figure 2 with 1 supplement see all
Gene activation during germination depends on CHG methylation (CHGm) on REF6 binding sites (REF6-BS).

(a) Plot shows the percentage of REF6-BS with CHGm in the three groups of genes with different combinations of histone modifications. CHGm on REF6-BS in gene bodies was determined based on published DNA methylome data (Park et al., 2016). p values were calculated using pairwise Fisher’s exact test and Benjamini–Hochberg correction. (b) Plot shows the percentage of CHGm on different cytosine positions of REF6-BS in the three groups of genes with different combinations of histone modifications. CHGm on REF6-BS in gene bodies was determined based on published DNA methylome data (Park et al., 2016). Numbers above the REF6-BS indicate possible positions of DNA methylation, of which positions 3, 5, and 8 can be targeted by CHGm. p values were calculated using pairwise Fisher’s exact test and Benjamini–Hochberg correction and indicate significance for CHGm on position five in REF6-BS. (c) Plot shows the percentage of upregulated genes with double H3K27m3/H3K9me2 and single H3K27me3 in the endosperm during germination depending on CHGm on REF6-BS. Gene expression is based on previously published endosperm-specific transcriptome data during germination (Dekkers et al., 2013). Compared were expression levels between 1 and 38 hours of imbibition (1≦Log2FC, p<0.05). Asterisks indicate significant differences between two categories (*p<0.05; **p<0.01; pairwise Fisher’s exact test with Benjamini–Hochberg correction).

REF6 is required to promote germination

Loss of REF6 was recently reported to cause increased dormancy (Chen et al., 2020). There are two REF6 orthologs in Arabidopsis thaliana, EARLY FLOWERING 6 (ELF6), and JUMONJI 13 (JMJ13). The DNA-binding domains of REF6 and ELF6 share amino acids that are required for binding to REF6-sequence motifs (Qiu et al., 2019) contrasting with lack of conservation of those sites in JMJ13 (Figure 3—figure supplement 1). This suggests that ELF6 acts redundantly with REF6 to promote germination. We tested two independent mutant alleles of ref6 and elf6 that all showed late germination phenotypes, but the ref6 mutants were more strongly delayed than elf6 (Figure 3—figure supplement 2). To test whether there are genetic redundancies between REF6, ELF6, and JMJ13 in controlling germination, we analyzed double elf6 ref6 and triple elf6 ref6 jmj13 mutants (Yan et al., 2018) for their ability to germinate. The elf6 ref6 double was slightly, but significantly, more delayed compared to the ref6 single mutant, while the elf6 ref6 jmj13 triple mutant behaved like the efl6 ref6 double mutant (Figure 3a). This data reveal that REF6 is the major factor releasing seed dormancy and promoting germination and that ELF6 is partially redundant with REF6, consistent with the conserved DNA binding domain (Figure 3—figure supplement 1). Prolonged after-ripening or stratification of ref6 seeds decreased primary dormancy and promoted germination (Figure 3—figure supplement 3), similar to other dormancy-related mutants (MacGregor et al., 2019; Piskurewicz et al., 2016; Zheng et al., 2012). Loss of maternal, but not paternal REF6 caused a significant delay in germination (Figure 3b), revealing that REF6 acts in maternal tissues. Nevertheless, combined loss of maternal and paternal REF6 had a stronger effect than loss of the maternal REF6 (Figure 3b), indicating that REF6 also acts after fertilization. To test whether the maternal effect of ref6 is caused by a functional role of REF6 in maternal sporophytic tissues or in the female gametophyte, we analyzed the phenotypes of F1 seeds generated by crossing maternal heterozygous ref6-1 mutant plants with paternal wild-type plants. The F1 seeds showed significantly delayed germination compared to wild-type seeds, with germination frequencies being in between that of wild-type seeds and F1 seeds of homozygous ref6 mutants crossed with wild-type pollen (Figure 3c). Since REF6 is not imprinted (Hsieh et al., 2011; Pignatta et al., 2014), this data strongly suggests that REF6 acts before fertilization in the female gametophyte.

Figure 3 with 3 supplements see all
REF6 is the major H3K27me3 demethylase controlling seed dormancy.

(a) Plots show the percentage of germinating seeds of wild type, elf6-3 single, ref6-1 single, elf6-3 ref6C double, and elf6-3 ref6C jmj13G triple mutants with and without stratification. Error bars indicate SD from three biological replicates (n = 120 total). Letters above bars indicate significant differences among genotypes (p<0.05, Tukey’s multiple range test). (b) Germination phenotypes of seeds generated by reciprocal crosses between wild type and ref6-1. Plots show the percentage of germinating seeds with and without stratification. Error bars indicate SD from three biological replicates (n = 120 total). Letters above bars indicate significant differences among genotypes (p<0.05, Tukey’s multiple range test). (c) Plots show the percentage of germinating seeds with and without stratification of wild type (WT), F1 seeds generated by crossing maternal heterozygous or homozygous ref6-1 and paternal wild type. Error bars indicate SD from three biological replicates (n = 90 total). Letters above bars indicate significant differences among genotypes (p<0.05, Tukey’s multiple range test).

Endosperm-specific expression of REF6 is important for germination

Our data suggests a major role of REF6 in the endosperm to promote the expression of genes required for releasing dormancy. To further challenge this model, we tested whether expression of REF6 in the micropylar endosperm was sufficient to suppress the germination delay of ref6 mutants. We expressed REF6 in the ref6 mutant under control of the EARLY-PHYTOCHROME-RESPONSIVE1 (EPR1) promoter that is specifically expressed in the micropylar endosperm during germination (Dubreucq et al., 2000; Figure 4—figure supplement 1). Two independent transgenic EPR1::REF6; ref6 lines showed a significant suppression of the ref6 germination delay (Figure 4a), indicating that expression of REF6 in the micropylar endosperm is functionally required to release dormancy and promote germination. We also expressed REF6 under control of the TREHALOSE-6-PHOSPHATE SYNTHASE 1 (TPS1) promoter that is specifically active in the embryo during germination (Bae et al., 2009; Figure 4—figure supplement 1). However, TPS1::REF6 did not suppress the germination delay of ref6 (Figure 4b), indicating that REF6 has no functional role in the embryo during germination. In support of this notion, dissected ref6 embryos did not show any delay in growth compared to dissected wild-type embryos (Figure 4c,d). Together, this data supports our model that REF6 is required in the endosperm to promote the expression of genes required for releasing dormancy.

Figure 4 with 1 supplement see all
REF6 expression in the endosperm promotes germination.

(a) Germination phenotypes of the ref6-1 mutant and transgenic lines in the ref6-1 background expressing REF6 under control of the micropylar endosperm-specific EPR1 promoter. Plots show the percentage of germinating seeds of wild-type, ref6-1, and complemented lines (EPR1::REF6;ref6-1-a and b) with and without stratification. Error bars indicate SD from three biological replicates (n = 120 total). The letters above the bars indicate significant differences among plant lines (p<0.05, Tukey’s multiple range test). (b) Germination phenotypes of the ref6-1 mutant and transgenic lines in the ref6-1 background expressing REF6 under control of the embryo-specific TPS1 promoter during germination. Plots show the percentage of germinating seeds of wild-type, ref6-1, and complemented lines (TPS1::REF6;ref6-1 a, b, and c) with and without stratification. Details are shown in the legend of (a). (c) and (d) Germination phenotypes of ref6-1 mutant embryos dissected from seeds without (c) and with (d) stratification. Embryos of wild-type, ref6-1, and ref6-3 plants were manually dissected from seeds. Plots show the percentage of seedlings with root hairs and green cotyledons. Error bars indicate SD from three biological replicates (n = 90 total). The data at each time point were evaluated using one-way ANOVA, no significant differences were detected (p>0.05).

REF6 activates genes marked by single H3K27me3 during germination

To identify the genes controlled by REF6 during germination, we profiled the transcriptome of ref6 mutant endosperm during germination. We found more than twice as many significantly downregulated than upregulated genes in ref6 compared to wild type (1471 downregulated [Log2FC≦–1, p<0.05] vs 655 upregulated [Log2FC ≥ 1, p<0.05];Figure 5—source data 1), consistent with the proposed function of REF6 to activate genes during germination. Genes with single H3K27me3 and unmethylated REF6-binding sites were significantly enriched among downregulated but not upregulated genes (Figure 5a,b), supporting the model that REF6 activates genes with single H3K27me3 through the unmethylated REF6-binding sites. Furthermore, there was no significant enrichment of genes with single H3K9me2 among deregulated genes in ref6 (Figure 5—figure supplement 1). Additionally, while genes with double H3K27me3/H3K9me2 and CHG methylated REF6-binding sites were not significantly enriched among deregulated genes, genes with double H3K27me3/H3K9me2 and unmethylated REF6-binding sites were slightly enriched among downregulated genes, although the enrichment was not significant (p=0.0568, pairwise Fisher’s exact test) (Figure 5—figure supplement 2). The 59 genes with single H3K27me3 and REF6-binding sites that were downregulated in ref6 (Figure 5—figure supplement 3) were significantly enriched for germination- and ethylene-inducible genes, but not for gibberellin-inducible genes according to previous transcriptome analyses (Cao et al., 2006; Das et al., 2016; Dekkers et al., 2013; Figure 5c). These findings are consistent with the idea that REF6 promotes germination by activating germination-inducible genes acting in the ethylene pathway. Among the candidate REF6 target genes (Figure 5—figure supplement 3) was ETHYLENE RESPONSE DNA BINDING FACTOR 4 (EDF4) that was previously reported to mediate ethylene responses (Alonso et al., 2003; Chen et al., 2015). We confirmed significant suppression of EDF4 in the ref6 mutant endosperm (Figure 5d), supporting the proposed endosperm-specific function of REF6 during germination (Figure 4a,b). We furthermore found three genes encoding for ethylene biosynthesis enzymes converting the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) to biologically active ethylene (ACC oxidases, ACOs) significantly downregulated in ref6 (Figure 5—figure supplement 3). ACO genes were not marked by H3K27me3 in the 4 days after pollination (DAP) endosperm, suggesting that REF6 activates these genes at an earlier timepoint. REF6 was shown to target the ABA catabolism genes CYP707A1 and CYP707A3, but not CYP707A2 during seed development (Chen et al., 2020). We also found CYP707A1 and CYP707A3 slightly, but significantly suppressed in ref6 endosperm (Figure 5—figure supplement 3), consistent with the presence of REF6-BS in CYP707A1 and CYP707A3, but not CYP707A2. Like for ACO genes, we did not detect H3K27me3 on all three genes in 4 DAP endosperm (Moreno-Romero et al., 2016), suggesting that REF6 acts on these genes before this timepoint. To further test the contribution of the ethylene and ABA pathways to the delayed germination phenotypes in ref6 mutants, we analyzed germination of ref6 in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the ABA biosynthesis inhibitor fluridone, which both were reported to induce seed germination (Linkies et al., 2009; Martinez-Andujar et al., 2011). Treatment with only ACC or fluridone partially suppressed the ref6 delayed germination phenotype, but the germination ratios were significantly lower than non-treated wild type after stratification (Figure 5—figure supplement 4). In contrast, treatment of cyp707a2 mutants with fluridone resulted in comparable germination frequencies as for wild-type seeds without treatment (Figure 5—figure supplement 4), suggesting that induction of the ABA pathway can be effectively suppressed by fluridone, in line with previous data (Martinez-Andujar et al., 2011). The failure of fluridone to suppress the ref6 mutant phenotype is also consistent with previous findings showing that a mutation in the ABA biosynthesis gene ABA2 does not completely rescue the delayed germination phenotype of ref6-1 after stratification (Chen et al., 2020). Importantly however, treatment with both ACC and fluridone could completely suppress the delayed germination of ref6-1 after stratification (Figure 5—figure supplement 4). This data support the idea that loss of REF6 causes failure to activate the ethylene pathway (Figure 5c) and at the same time causes induction of the ABA pathway as previously shown (Chen et al., 2020).

Figure 5 with 4 supplements see all
Genes marked by single H3K27me3 are activated by REF6 during germination.

(a) Volcano plot shows differentially expressed genes with single H3K27me3 and REF6-binding sites (REF6-BS) in ref6-1. The color of dots corresponds to the following gene categories: red dots represent genes with single H3K27me3 and non-CHG methylation (CHGm) on REF6-BS, orange dots represent genes with single H3K27me3 and REF6-BS (excluding non-CHGm REF6-BS), yellow dots represent genes with single H3K27me3 (excluding REF6-BS), and gray dots represent Arabidopsis all genes (excluding genes with single H3K27me3). Vertical and horizontal lines mark thresholds of up- and downregulated genes (1≦Log2FC, Log2FC≦–1 and p<0.05). (b) Plots show the percentage of up- (1≦Log2FC, p<0.05) and downregulated (Log2FC≦–1, p<0.05) genes in the endosperm of ref6-1 during germination. Compared are genes with single H3K27me3 and presence or absence of (CHGm on REF6-BS. CHGm was determined based on published data Park et al., 2016). Asterisks indicate significant differences compared to Arabidopsis all genes (*p<0.05, **p<0.01, pairwise Fisher’s exact test with Benjamini–Hochberg correction). (c) Plot shows the expression of genes with single H3K27me3 and REF6-BS that were downregulated in endosperm of ref6-1. Gene expression was calculated based on tissue-specific transcriptome data in the endosperm during germination (Dekkers et al., 2013). Expression levels were compared between 1 and 38 hours after imbibition. Expression changes were defined as follows: upregulated genes (1≦Log2FC and p<0.05), downregulated genes (Log2FC≦–1 and p<0.05), stably expressed genes (–1≦Log2FC≦1 and p<0.05) and non-expressed genes. Percentage of ethylene and gibberellin inducible genes was calculated based on transcriptome data from seedlings (Cao et al., 2006; Das et al., 2016). Asterisks indicate significant differences compared to Arabidopsis all genes (*p<0.01, **p<0.001, Fisher’s exact test). (d) Plot shows expression of EDF4 in the endosperm of ref6-1 during germination as determined by RT-qPCR. Embryos of wild-type, ref6-1, and ref6-3 mutants were dissected after 72 hr of stratification and 24 hr of incubation under normal conditions. Error bars indicate SD from technical triplicates. Asterisks indicate significant differences compared to wild type (p<0.01, Bonferroni-corrected Student’s t test).

Figure 5—source data 1

RNA-seq data in the ref6 mutant endosperm during germination.

https://cdn.elifesciences.org/articles/64593/elife-64593-fig5-data1-v2.xlsx

Together, our data support a model where REF6 is required to activate genes with single H3K27me3 and unmethylated REF6-binding sites in the endosperm during germination, among them genes in the ethylene and ABA catabolism pathway.

SUVH4/5/6 and CMT3 are required to promote germination

Our data indicate that REF6 is able to activate genes marked by single H3K27me3, but fails to access genes marked by H3K27me3/H3K9me2/CHGm due to the block of REF6 targeting by CHGm. The H3K9me2 modification is established by the redundantly acting SUVH4/5/6 histone methyltransferases and recruits CMT3 to establish DNA methylation in CHG context (Du et al., 2012; Ebbs and Bender, 2006; Jackson et al., 2002). Since the suvh456 mutant is strongly depleted for CHGm (Stroud et al., 2014), we speculated that in suvh456 REF6 targets should become accessible and activated. Previous work revealed that the suvh45 double mutant has increased dormancy (Zheng et al., 2012). Similarly, we found that suvh456 required prolonged after-ripening time and had reduced germination rates at 10 days after harvesting or required stratification when seeds developed under low temperature (Figure 6—figure supplement 1). Interestingly, the mutations in SUVH4/5/6 only caused a delay in germination when maternally, but not when paternally inherited (Figure 6a), revealing a maternal effect. Since SUVH4/5/6 genes are not regulated by genomic imprinting (Moreno-Romero et al., 2019; Pignatta et al., 2014), one possible explanation is that activity of SUVH4/5/6 in the central cell of the female gametophyte has a lasting effect until germination. Based on published expression studies, SUVH4 and SUVH6 are expressed in the central cell (Belmonte et al., 2013; Hsieh et al., 2011; Wuest et al., 2010), supporting this notion. Furthermore, we found a similar enrichment of methylated REF6 binding sites in genes marked with H3K9me2 and H3K27me3/H3K9me2 in central cells and developing endosperm (Figure 2a and Figure 2—figure supplement 1), supporting the idea that H3K9me2 and CHGm are established in the central cell. We tested this hypothesis by analyzing germination of F1 seeds generated from heterozygous suvh4/5/6 (suvh4/+ suvh5/+ suvh6/+) mutants pollinated with wild-type plants. Germination of these F1 seeds was clearly delayed (Figure 6b), supporting the idea that SUVH4/5/6 act in the female gametophyte. We tested whether indeed CHGm has a functional role in controlling seed dormancy by testing the effect of mutations in CMT3. Consistently, loss of CMT3 caused a delay in germination (Figure 6c) and prolonged after-ripening of cmt3 seeds decreased primary dormancy (Figure 6—figure supplement 2). Interestingly, contrary to the maternal effect of suvh4/5/6, loss of maternal and paternal CMT3 alleles was required to cause a delay in germination (Figure 6d). Based on DNA methylome data CHGm is established in the central cell before fertilization (Figure 2a; Park et al., 2016); nevertheless, the paternal CMT3 allele was sufficient to suppress delay of germination in cmt3. Previous work revealed that loss of SUVH4/5/6 causes severe depletion of both H3K9me2 and CHGm (Stroud et al., 2014; Underwood et al., 2018), while loss of CMT3 causes depletion of CHGm but only intermediate reductions of H3K9me2 (Stroud et al., 2014; Inagaki et al., 2010). Together, this suggests that H3K9me2 is established first and recruits CHGm and that CHGm establishment after fertilization is sufficient to control seed dormancy.

Figure 6 with 2 supplements see all
SUVH4/5/6 and CMT3 are necessary to promoter germination.

(a) Germination phenotypes of seeds generated by reciprocal crosses between wild-type and suvh456 triple mutants. Plots show the percentage of germinating seeds with and without stratification. Error bars indicate SD from three biological replicates (n = 120 total). The letters above the bars indicate significant differences between genotypes (p<0.05, Tukey’s multiple range test). (b) Plots show the percentage of germinating seeds with and without stratification of wild-type (WT) and F1 seeds generated by crossing maternal heterozygous or homozygous suvh456 and paternal wild type. Error bars indicate SD from three biological replicates (n = 90 total). Letters above bars indicate significant differences among genotypes (p<0.05, Tukey’s multiple range test). (c) Plots show percentage of germinating seeds of wild-type and cmt3-11 mutant with and without stratification. Error bars indicate SD from three biological replicates (n = 120 total). Asterisks indicate significant differences compared to wild type (p<0.05, Student’s t test). (d) Germination phenotypes of seeds generated by reciprocal crosses between wild-type and cmt3-11 mutant. Details are shown in the legend of (a).

SUVH4/5/6 suppress genes with H3K27me3/H3K9me2 and CHGm on REF6-binding sites

To further test the hypothesis that H3K9me2/CHGm block the access of REF6, we analyzed transcriptome data of suvh456 endosperm during germination. There were more than twice as many significantly up- than downregulated genes in suvh456 compared to wild type (3,203 upregulated [Log2FC ≥ 1, p<0.05] vs 1,502 downregulated [Log2FC ≦ –1, p<0.05]; Figure 7—source data 1). Importantly, genes marked with H3K27me3/H3K9me2 and CHGm on REF6 binding sites were significantly enriched among upregulated, but not downregulated genes (Figure 7a,b), supporting the idea that REF6 targeting is blocked by H3K9me2/CHGm in the endosperm. Consistently, REF6-binding sites were significantly enriched in the gene bodies of upregulated genes marked with H3K27me3/H3K9me2/CHGm (Figure 7c). REF6-binding sites were not significantly enriched among the downregulated genes in suvh456 (Figure 7—figure supplement 1). We also did not find significant differences among the up- and downregulated genes with single H3K9me2 (Figure 7—figure supplement 2), revealing that the suppressive effect of SUVH4/5/6 on gene repression connects to the presence of H3K27me3. Together, our data show that SUVH4/5/6 suppress genes with H3K27me3/H3K9me2/CHGm and that loss of SUVH4/5/6 causes upregulation of genes through REF6-binding sites. The ABA INSENSITIVE 3 (ABI3) gene was previously shown to be upregulated in suvh45 mutants (Zheng et al., 2012) and was also present among the 26 upregulated genes in suvh456 marked with H3K27me3/H3K9me2/CHGm and having REF6-binding sites (Figure 7—figure supplement 3). ABI3 is a B3 type transcription factor and positively regulates seed dormancy (Liu et al., 2013; Shu et al., 2016; Tian et al., 2020). HEAT SHOCK TRANSCRIPTION FACTOR C1 (HSFC1) was also reported to be induced in developing seeds by heat stress and suggested to activate HEAT SHOCK PROTEIN (HSP) genes that are abundant in seed maturation (Chiu et al., 2012). The upregulation of the two genes in the endosperm of suvh456 were also confirmed by RT-qPCR (Figure 7d). We found that genes with single H3K27me3 were significantly enriched for downregulated genes in the suvh456 endosperm (Figure 7—figure supplement 4), suggesting an indirect effect. Interestingly, we found RY binding motifs for B3 type transcription factors to be significantly enriched among downregulated genes in suvh456 containing single H3K27me3 (Figure 7—figure supplement 4). This raised the hypothesis that upregulated ABI3 in suvh456 suppressed genes with single H3K27me3. In support of this idea, we found that genes with single H3K27me3 significantly overlapped with previously identified direct target genes of ABI3 (Tian et al., 2020), while genes with single H3K9me2 and double H3K27me3/H3K9me2 did not significantly overlap (Figure 7—figure supplement 4).

Figure 7 with 5 supplements see all
SUVH4/5/6 are required for the suppression of genes with H3K27me3/H3K9me2 and CHG methylation (CHGm) on REF6-binding sites (REF6-BS).

(a) Volcano plot shows differentially expressed genes with double H3K27me3/H3K9me2 and CHGm on REF6-BS in suvh456. The color of dots corresponds to the following gene categories: red dots represent genes with double H3K27me3/H3K9me2 and CHGm on REF6-BS, orange dots represent genes with double H3K27me3/H3K9me2 and REF6-BS (excluding CHGm REF6-BS), yellow dots represent genes with double H3K27me3/H3K9me2 (excluding REF6-BS), and gray dots represent Arabidopsis all genes (excluding genes with H3K27me3/H3K9me2). Vertical and horizontal lines mark thresholds of up- and downregulated genes are shown (1≦Log2FC, Log2FC≦–1, p<0.05). (b) Plots show the percentage of up-(1≦Log2FC, p<0.05) and downregulated (Log2FC≦–1, p<0.05) genes in the endosperm of suvh456 during germination. Compared are genes with double H3K27me3/H3K9me2 and presence or absence of CHGm on REF6-BS. CHGm was determined based on published data (Park et al., 2016). Asterisks indicate significant differences compared to Arabidopsis all genes (p<0.05, pairwise Fisher’s exact test with Benjamini–Hochberg correction). (c) Plot shows the enrichment of REF6-BS among upregulated (1≦Log2FC, p<0.05) genes in the endosperm of suvh456 during germination. Compared are genes with the indicated combinations of histone modifications and presence (>0.04) or absence (≤0.04) of CHGm. CHGm levels were determined based on published data (Park et al., 2016). Asterisks indicate significant differences between two categories (p<0.05, Fisher’s exact test). (d) Plots show the expression of ABI3 and HSFC1 in the endosperm of suvh456 during germination as determined by RT-qPCR. Embryos of wild type and the suvh456 triple mutant were dissected after 72 hr of stratification and 24 hr of incubation under normal conditions. Error bars indicate SD from technical triplicates. Asterisks indicate significant differences between wild type and suvh456 (p<0.01, Student’s t test).

Figure 7—source data 1

RNA-seq data in the suvh456 mutant endosperm during germination.

https://cdn.elifesciences.org/articles/64593/elife-64593-fig7-data1-v2.xlsx

Parentally biased expression during germination is regulated by REF6 and SUVH456

Our data obtained in this (Figure 2) and previous studies (Moreno-Romero et al., 2019; Moreno-Romero et al., 2016) indicate that H3K27me3, H3K9me2, and CHGm are established in the central cell and control imprinted expression in the endosperm after fertilization. We next addressed the question whether maternal-specific epigenetic marks also control parental-specific gene expression in the endosperm during germination. Using previously published parental-specific transcriptome data of the endosperm during germination (F1 seeds of maternal Col and paternal Cvi accessions) (Piskurewicz et al., 2016), we found that genes with triple repressive marks H3K27me3/H3K9m2/CHGm on REF6-binding sites were significantly paternally biased compared to Arabidopsis all genes in both dormant and non-dormant seeds (Figure 8a, upper blue boxes), consistent with our data showing the relationship between triple repressive marks and expression during germination (Figure 2c). This data suggests that triple repressive marks are associated with paternally-biased gene expression in germinating endosperm, similar as in the developing endosperm (Moreno-Romero et al., 2019; Moreno-Romero et al., 2016). In contrast, genes with single H3K27me3 and unmethylated REF6-binding sites were significantly maternally biased in dormant seeds (Figure 8a, lower orange boxes, left panel), but not in non-dormant seeds (Figure 8a, lower gray boxes, right panel). The maternally-biased expression in dormant seeds supports the idea that genes with single H3K27me3 are reactivated during germination. We observed a weak maternal bias of genes marked with single H3K27me3 already in the developing endosperm at 4 DAP (Figure 8—figure supplement 1), suggesting that gene activation by removing single H3K27me3 starts before germination, consistent with the observed maternal effect of ref6 (Figure 3b,c). In non-dormant seeds, however, unbiased expression of genes with single H3K27me3 suggests that the paternal alleles of these genes are also activated. Indeed, this expression pattern has been previously reported for the germination-related CP1 gene, which is maternally-biased in dormant seeds, while biallelically expressed in non-dormant seeds (Piskurewicz et al., 2016). We did not observe a significant parental bias of genes with single H3K9me2 (Figure 8—figure supplement 1).

Figure 8 with 5 supplements see all
Maternal- and paternal-specific epigenetic marks regulate parental-specific gene expression in the endosperm during germination.

(a) Box plots show the mean values of maternal to total reads of genes expressed in the endosperm in dormant and non-dormant seeds. Compared are Arabidopsis all genes, all genes with double H3K27me3/H3K9me2 and single H3K27me3 depending on the presence or absence of CHG methylation (CHGm) on REF6-binding sites (REF6-BS) based on published data (Park et al., 2016). Parental-specific transcriptome data in the endosperm during germination from F1 seeds (maternal Col × paternal Cvi) were previously published (Piskurewicz et al., 2016). Boxes show medians and the interquartile range, and error bars show full-range excluding outliers. Numbers of genes in each category are shown beside the boxes. Asterisks indicates significant differences compared to Arabidopsis all genes (*p<0.05, **p<0.01, pairwise Wilcoxon test with Benjamini–Hochberg correction). Significantly paternally and maternally biased groups are shown in blue and orange, respectively. (b) Box plots show mean values of maternal to total reads in the endosperm of dormant and non-dormant seeds of Arabidopsis all genes, upregulated genes with double H3K27me3/H3K9me2 in suvh456, and downregulated genes with single H3K27me3 in ref6-1. Details are shown in the legend of (a).

Based on our data showing that ref6 mainly affected genes with single H3K27me3 while suvh4/5/6 mainly affected genes with triple repressive marks on REF6-binding sites (Figure 5a, b and Figure 7a,b), we selected genes that were likely to be regulated by REF6 and SUVH4/5/6 and analyzed the maternal expression ratios. Using the same published parental-specific transcriptome data (Piskurewicz et al., 2016), we found that genes with single H3K27me3 that were downregulated in ref6 were significantly maternally biased in both dormant and non-dormant seeds (Figure 8b, orange boxes). Conversely, upregulated genes in suvh456 that were marked with triple repressive marks were significantly paternally biased (Figure 8b, blue boxes). Nevertheless, the number of genes in each category was relatively small and further experiments should validate these conclusions. Together, this data suggests that REF6 and SUVH4/5/6 regulate genomic imprinting during germination.

The maternally biased expression of genes with single H3K27me3 suggests suppression of the paternal alleles by an epigenetic mechanism. Maternally biased gene expression during germination was previously reported to be mediated by the non-canonical RNA-directed DNA methylation (RdDM) pathway targeting the paternal alleles (Iwasaki et al., 2019). We therefore analyzed DNA methylation levels in genic as well as 2 kb up- and downstream regions on the paternal alleles of genes with single H3K27me3 on the maternal alleles using previously published data (Ibarra et al., 2012; Moreno-Romero et al., 2016). We found that paternal CHHm levels in downstream regions of genes with maternal H3K27me3 and in gene bodies of genes with maternal H3K27me3 and unmethylated REF6-binding sites were significantly higher compared to Arabidopsis all genes in 7–8DAP endosperm (Figure 8—figure supplement 2). CHGm levels in downstream regions were significantly higher in 4DAP endosperm, but this trend was not detected at 7–8DAP (Figure 8—figure supplement 3). Levels of CG methylation (CGm) were marginally increased in gene bodies of genes with maternal H3K27me3 and unmethylated REF6-binding sites in 4DAP and 7–8DAP endosperm (Figure 8—figure supplement 4). This data suggests that CHHm of genes with maternal H3K27me3 and unmethylated REF6-binding sites can be one possible factor to establish maternally biased expression of genes with single H3K27me3 in dormant seeds. However, detailed parental-specific DNA methylome data in dormant and non-dormant seeds are necessary to validate this conclusion.

Discussion

In this study, we uncovered the molecular function and physiological role of the endosperm-specific triple modification of the maternal genome with H3K27me3/H3K9me2 and CHGm. We found that genes marked by only H3K27me3 became reactivated during seed germination, a process that depends on the H3K27me3 demethylase REF6. In contrast, genes marked by triple repressive modifications remained silenced during germination because REF6 binding is repelled by H3K9me2/CHGm, consistent with previous work (Qiu et al., 2019). Supporting the functional requirement of REF6 to initiate germination, loss of REF6 caused higher seed dormancy (Figure 3, Figure 3—figure supplement 2; Chen et al., 2020), connected with decreased expression of many germination-inducible genes. Loss of SUVH4/5/6 also increased dormancy (Figure 6—figure supplement 1; Zheng et al., 2012), which was associated with increased expression of potential REF6 target genes. Since H3K9me2 acts in a feedback loop with CHGm (Du et al., 2012; Jackson et al., 2002; Lindroth et al., 2001; Malagnac et al., 2002), loss of SUVH4/5/6 is expected to expose unmethylated binding sites for REF6 that can then be activated by REF6. Consistent with this model, loss of CMT3 also caused increased dormancy (Figure 6b, Figure 6—figure supplement 2). Interestingly, among those upregulated SUVH4/5/6 targets was the positive regulator of seed dormancy ABI3 (Liu et al., 2013), providing a possible explanation for the increased dormancy in suvh4/5/6. Consistent with this notion we found that downregulated genes with single H3K27me3 were enriched for ABI3 binding motifs. Nevertheless, the small fraction of germination-inducible genes with triple repressive marks suggests possible additional mechanisms removing triple epigenetic marks.

Our work furthermore revealed the connection between maternal-specific repressive marks, gene imprinting, and seed dormancy. Imprinted genes were previously shown to play a role in regulating seed germination and were connected to the maternal inheritance of seed dormancy levels (Iwasaki et al., 2019; Piskurewicz et al., 2016). The non-canonical RdDM pathway was found to target and silence the paternal alleles of the dormancy-related gene ALN1 (Iwasaki et al., 2019). Our data suggests that in addition to RdDM, maternally biased expression of dormancy-related genes also requires REF6, which activates maternal H3K27me3 marked genes. We propose that both mechanisms together ensure the maternal control of seed dormancy; while REF6 regulates the time of activation of certain genes, RdDM-mediated CHHm on the paternal alleles prevents their activation. Interestingly, the timing of REF6 action probably differs for distinct genes. While some genes, like ALN1, CYP707A1, and CYP707A3 (Chen et al., 2020) are likely activated in the central cell of the female gametophyte and therefore lack detactable H3K27me3 in the endosperm, other genes are activated during endoperm development. To the latter category belongs the previously reported gene CP1, which is maternally biased in dormant seeds and biallelically expressed in non-dormant seeds (Piskurewicz et al., 2016) and marked with single H3K27me3 on the maternal alleles (Moreno-Romero et al., 2016). In contrast to genes with single H3K27me3, genes with triple repressive marks on maternal alleles are not targeted by REF6, causing the maternal alleles to be silenced and the paternal alleles to be active. Genes with triple repressive marks on REF6-binding sites were upregulated in suvh4/5/6 and were paternally biased during germination. Although more detailed parental-specific DNA methylome analyses in germinating endosperm are required to precisely determine the molecular mechanism repressing the paternal alleles of genes with maternal H3K27me3 during germination, this data supports the model that the presence of single H3K27me3 and triple repressive marks determines whether genes are maternally or paternally biased during germination, respectively (Figure 8—figure supplement 5).

The SUVH4/5/6 genes are not imprinted (Hsieh et al., 2011; Moreno-Romero et al., 2019; Pignatta et al., 2014); however, the delay of germination in suvh4/5/6 was under maternal control (Figure 6a). This indicates that maternal-specific triple repressive marks are established before fertilization in the central cell and maintained after fertilization, consistent with previous data (Moreno-Romero et al., 2019). This suggests that maternal-specific triple epigenetic marks of H3K27me3/H3K9me2 and CHGm contribute to maternally controlled seed dormancy (Iwasaki et al., 2019; Piskurewicz et al., 2016). Likewise, also REF6 is not imprinted (Hsieh et al., 2011; Pignatta et al., 2014), but ref6 had a maternal effect (Figure 3b), supporting the notion that derepression of some dormancy-controlling genes starts in the central cell of the female gametophyte. Nevertheless, the fact that some REF6 targets have detectable levels of H3K27me3 in the endosperm and since we could partially complement the ref6 mutant by expressing REF6 in the micropylar domain of the endosperm (Figure 5a), strongly suggests that REF6 also acts after fertilization. In support of this view, homozygous ref6 mutants had a stronger delay in germination than maternal ref6 mutants (Figure 3b).

Our data furthermore uncovered a relationship among REF6, ethylene, and ABA. Consistent with previous work showing that loss of ABA2 does not completely suppress the delayed germination of ref6 after stratification (Chen et al., 2020), we found that the ABA biosynthesis inhibitor fluridone was not sufficient to suppress delayed germination phenotypes of ref6 (Figure 5—figure supplement 4). Similarly, the ethylene precursor ACC caused only a partial suppression of the germination delay. However, the combined treatment with fluridone and ACC completely suppressed the ref6 germination phenotype (Figure 5—figure supplement 4), supporting our and previous data revealing that REF6 is required to activate ABA catabolism and ethylene signaling through removal of H3K27me3. Since the activation of the ABA catabolism genes CYP707A1 and CYC707A3 likely occurs in the central cell while activation of ethylene signaling genes occurs during endosperm development, we propose that there is a two-step maternal control of germination by REF6. The first step occurs in the central cell and leads to the suppression of ABA signaling, while the second step occurs in the endosperm and leads to the activation of ethylene signaling. This two-step control of germination may ensure the best timing of germination. Understanding by which mechanism the timing of REF6 action is regulated will be subject of future investigations.

Previous data showed that loss of FIS-PRC2 function causes upregulation of genes with both H3K27me3 and H3K9me2 (Hsieh et al., 2011; Moreno-Romero et al., 2016), suggesting that establishment of H3K9me2 depends on H3K27me3. This idea was substantiated by data showing that deficiency of FIS-PRC2 in the central cell causes depletion of CHGm (Moreno-Romero et al., 2019), pointing that H3K9me2/CHGm depend on FIS-PRC2. Also data shown in this study are consistent with this scenario; we demonstrate that loss of H3K9me2/CHGm only caused an effect if H3K27me3 was removed by REF6, revealing that H3K27me3 is the primary repressive mark. Additionally, we found that maternal loss of SUVH4/5/6 was sufficient to cause delayed gemination (Figure 6a), while that of CMT3 was not (Figure 6c). One possible explanation is that the effects caused by loss of SUVH4/5/6 and CMT3 function differ. While H3K9me2 and CHGm are severely depleted in suvh4/5/6; loss of CMT3 mainly affects CHGm with only intermediate effects on H3K9me2 (Stroud et al., 2014; Underwood et al., 2018). Thus, genes with H3K27me3/H3K9me2 can likely be suppressed by CMT3 after fertilization.

A possible mechanism is that the FIS2-PRC2 complex or H3K27me3 recruits SUVH4/5/6 and that H3K9me2 recruits CMT3, suggesting the presence of a molecular mechanism that distinguishes target genes to be marked by single H3K27me3 or triple marks of H3K27me3/H3K9me2 and CHGm. To identify this distinguishing mechanism remains an important future endeavor.

Our data bear striking similarities to maternal H3K27me3-dependent non-canonical imprinting reported in mammals (Chen et al., 2019; Hanna et al., 2019; Inoue et al., 2017a; Inoue et al., 2017b). In non-canonical imprinting, maternal-specific H3K27me3 inherited from oocytes serves as the primary epigenetic mark for imprinted paternal-specific expression. Most of this non-canonical imprinting is transient and restricted to preimplantation embryos, with only some genes important for placental development remain imprinted in extraembryonic cells (Inoue et al., 2017a). Maintenance of non-canonical imprinting is mediated by DNA methylation, which is specifically applied on the maternal alleles of previously H3K27me3 marked regions (Chen et al., 2019; Hanna et al., 2019). Thus, similar to the findings reported in this study, genes regulated by non-canonical imprinting in mammals are transiently imprinted by inheriting maternal-specific H3K27me3 and only few genes remain imprinting by establishing DNA methylation as secondary imprint. Genomic imprinting has independently evolved in flowering plants and mammals; however, rather remarkably, similar molecular mechanisms have been coopted to regulate imprinted genes in the accessory, nutrient-transferring tissues.

The combined presence of H3K27me3 with the heterochromatic modification H3K9me3 was also reported to occur on TEs and repeat-rich regions in the protozoa Tetrahymena and Paramecium (Frapporti et al., 2019; Liu et al., 2007; Zhao et al., 2019) and the fungus Podospora anserine (Carlier et al., 2020). In P. anserine, the presence of double repressive modifications is restricted to TEs and repeat-rich regions, while H3K27me3 alone is located in genic regions, indicating a similar molecular distinction of constitutively silenced regions marked by double modifications from regions that require activation during specific developmental stages marked by H3K27me3 alone. It is tempting to speculate that the co-occurrence of H3K9me3 and H3K27me3 may prevent H3K27me3 demethylases to target and activate those regions, similar to the findings reported in this study.

In summary, in this study, we uncovered a new molecular mechanism leading to maternal-specific dormancy control that distinguishes genes marked by either H3K27me3 alone from genes marked by the generally antagonistic repressive epigenetic modifications, H3K27me3, H3K9me2, and CHGm. We uncovered how the H3K27me3 demethylase REF6 controls release of dormancy and revealed the underlying mechanism for the H3K9me2 methylases SUVH4/5/6 in controlling dormancy and propose that these regulators form a regulatory network through ABA and ethylene pathways. Our data provide new insights into how maternal-specific epigenetic modifications control dormancy and thus ensure timely germination.

Materials and methods

Plant material and growing conditions

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All mutants used in this study are in the Col background. The T-DNA insertion lines of ref6-1 (SALK_001018) (Cui et al., 2016), ref6-3 (SAIL_747_A07) (Cui et al., 2016), elf6-3 (SALK_074694) (Tao et al., 2017), elf6-4 (SAIL_371_D08) (Tao et al., 2017), and an ethylmethane sulfonate-mutated line of cmt3-11 (Lindroth et al., 2001) were previous described. The T-DNA insertions were confirmed by amplifying the left border-flanking regions of the genomic DNA using the primer set given in Supplementary file 1. The suvh456 mutant was kindly provided by Judith Bender. The elf6-3 ref6C double and elf6-3 ref6C jmj13G triple mutants were kindly provided by Kerstin Kaufmann (Yan et al., 2018). The cyp707a2-1 and cyp707a2-2 mutants were kindly provided by Eiji Nambara (Kushiro et al., 2004). All plants were grown on half-strength Murashige and Skoog agar medium containing 1% sucrose or on soil under long-day conditions (16 hr light/8 hr darkness) at 21°C and a light intensity of 150 µE. Seeds were surface sterilized (5% sodium hypochlorite) before sawing on medium and 14-day-old plants were transferred into soil. For germinating assays, seeds from dry siliques were harvested on the same day 3 weeks after pollination and after-ripened at room temperature for 10 days unless otherwise indicated. Transgenic plants were generated through the floral dip method by using Agrogacterium tumefaciens GM3101 as described previously (Clough and Bent, 1998).

Germinating assays

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For germinating assays, seeds from 20 siliques were sterilized and grown on half-strength Murashige and Skoog medium with or without stratification at 4°C for 3 days. To analyze the effects of chemicals on germination, 1-aminocyclopropanecarboxylic acid (ACC) (Sigma-Aldrich) and fluridone (Merck Chemicals and Life Science AB) were added to half-strength Murashige and Skoog medium. For seed dormancy assays, plants were grown under normal conditions or under lower temperature (10°C). The sterilized seeds were sown on minimal media (0.9% agar) and grown under normal conditions above with or without stratification. For growth analysis of dissected embryos, seeds were sterilized and manually dissected after treatment of stratification as previously described (Piskurewicz et al., 2016). Root protrusion, root hair formation, and cotyledon greening were observed under a microscope (Leica, EZ4) at different times during germination, and photographs were taken by a high-resolution digital camera (Leica, M205 FA). Experiments were done in biological replicates, as indicated.

RNA preparation and qRT-PCR

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For gene expression analysis, embryos and endosperm were manually dissected from seeds that were imbibed for 24 hr as previously described (Endo et al., 2012; Piskurewicz et al., 2016). Total RNA from embryos and endosperm was extracted using MagMAX Plant RNA Isolation Kit (Thermo Fisher Scientific) according to the supplier’s instructions for RNA extraction from plant seeds. For quantitative RT-PCR, cDNA was synthesized from total RNA using the Thermo Scientific RevertAid First-Strand cDNA Synthesis Kit according to the supplier’s instructions. Quantitative RT-PCR analyses were performed using a BIO RAD CFX Connect Real-Time PCR Detection System. HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (Solis BioDyne) was used for the reactions. The primers used for quantitative RT-PCR are listed in Supplementary file 1. Triplicate measurements were generated for each cDNA sample, and the obtained values were normalized to ACT2.

RNA sequencing and data analysis mRNA was isolated from total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Kit (NEB) and cDNA libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB). Libraries were sequenced on Illumina NovaSeq platforms as paired-end through Novogene mRNA sequencing services. For each replicate, the 150 bp long reads were trimmed by removing the 20 bp from the 5’ end and 30 bp from the 3’ and mapped in single-end mode to the Arabidopsis (TAIR10) genome, masked for rRNA genes, using TopHat v2.1.0 (Trapnell et al., 2009) (parameters adjusted as -g 1 -a 10 -i 40 -I 5000 F 0 r 130). Mapped reads were counted and normalized to reads per kilobase per million mapped reads for chromosomal protein coding genes using GFOLD (Feng et al., 2012). Differentially regulated genes across the three replicates of each condition were detected using DESeq (v. 1.12.0) (Anders and Huber, 2010) and in R (v. 3.3.2) (R Development Core Team, 2016). Only changes in expression with a FDR of <0.05 were considered statistically significant. Overrepresentation analysis of hexamers on the promoters of sets of genes by RNA-sequencing data were performed as previously described (Maruyama et al., 2012) using 1 kb upstream sequences from the translational start sites obtained through Phytozome (version 12; https://phytozome.jgi.doe.gov/pz/portal.html).

Data analysis

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Published data of maternal-specific histone modifications of H3K27me3 and H3K9me2 in the developing endosperm derived from crosses of maternal Col and paternal Ler accessions (Moreno-Romero et al., 2019) were used to identify three groups of genes with H3K27me3, H3K9me2, and H3K27me3/H3K9me2. We used CHG methylation scores on gene bodies in central cells (Park et al., 2016) as defined in a previous study (Moreno-Romero et al., 2019). The time-course of expression profiles in the endosperm during germination was determined using previously published data of tissues containing micropylar and chalazal endosperm (Dekkers et al., 2013). To analyze the genomic loci containing CHG, CHH, and CG methylation, published DNA methylation data of central cells (Park et al., 2016) and the developing endosperm at 4 days (Moreno-Romero et al., 2016) and 7–8 days (Ibarra et al., 2012) after pollination were reanalyzed. For the analyses of methylated sites on REF6-binding sites, methylated CHG sites were defined as those containing one or more methylated cytosines among at least five reads in both replicates. For the analyses of parental-specific methylation, numbers of methylated and unmethylated cytosines of two replicates were pooled and methylated sites were defined as those containing one or more methylated cytosines among at least five reads. To analyze parental-specific gene expression patterns in the endosperm during germination, published data of endosperm-specific transcriptomes in F1 seeds generated by reciprocal crosses of Col and Cvi (Piskurewicz et al., 2016) was used. Maternal expression ratios were calculated as the maternal read counts to total read counts (maternal-specific read counts/total read counts).

Protein sequence alignment and gene ontology analysis

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The peptide sequences of REF6, ELF6, and JMJ13 were obtained from Phytozome (Phytozome v12, https://phytozome.jgi.doe.gov/pz/portal.html). Alignment of the proteins was performed using SnapGene and ClustalW and the result of the alignment was manually adjusted to detect conserved amino acids. Gene Ontology (GO) analyses were performed using the public GO database agriGO (Version 2.0) (Tian et al., 2017) and processed through REVIGO using p values (Supek et al., 2011).

Plasmid constructions

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To generate the constructs for complementation assays, the pB7WG vector (https://gatewayvectors.vib.be/) was digested with Eco32I and ligated to remove the cassette of the LR reaction. The promoter regions of EPR1 or TPS1 were inserted into the KpnI and XbaI sites of the vector, and the REF6 coding sequence was inserted into the AatII and Eco32I sites using the In-Fusion HD Cloning Kit (TaKaRa).

Data availability

All data generated in this study were deposited to the Gene Expression Omnibus and are available under the accession number GSE158907.

The following data sets were generated
    1. Köhler C
    (2021) NCBI Gene Expression Omnibus
    ID GSE158907. Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy.
The following previously published data sets were used
    1. Moreno-Romero J
    2. Jiang H
    3. Santos-González J
    4. Köhler C
    (2016) NCBI Gene Expression Omnibus
    ID GSE66585. Parental epigenetic asymmetry of PRC2-mediated histone modifications in the Arabidopsis endosperm.
    1. Park K
    2. Kim MY
    3. Vickers M Park
    (2016) NCBI Gene Expression Omnibus
    ID GSE89789. DNA demethylation is initiated in the central cells of Arabidopsis and rice.

References

Decision letter

  1. Daniel Zilberman
    Reviewing Editor; Institute of Science and Technology Austria, Austria
  2. Jürgen Kleine-Vehn
    Senior Editor; University of Freiburg, Germany
  3. Steven Penfield
    Reviewer; John Innes Centre, United Kingdom

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

Sato et al. describe the role of histone modifications and DNA methylation in the control of seed dormancy in Arabidopsis. The authors show that DNA methylation associated with a specific histone modification (H3K9me2) prevents the REF6 enzyme from removing another histone modification (H3K27me3). Genes marked with both histone modifications are therefore resistant to activation, and this is important for gene regulation in the endosperm during seed

Decision letter after peer review:

Thank you for submitting your article "Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by Daniel Zilberman as the Reviewing Editor and Jürgen Kleine-Vehn as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Steven Penfield (Reviewer #2).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We are also appending Steven Penfield's original review because we feel it may be helpful in drafting a revised manuscript, but only the revisions indicated in this letter need to be directly addressed.

Summary:

Sato et al. describe the role of histone marks and DNA methylation in the control of seed dormancy in Arabidopsis. This paper will be of interest to scientists working on epigenetics, seed development and germination. Most of the claims are supported by data and the authors have generated many genomic datasets and knowledge that could be useful for the study of seed dormancy in other plant species. The authors show that the REF6/H3K27me3 suvh456/H3K9me2 interaction is important for gene regulation in the endosperm during germination. The authors also investigate the role of REF6 in germination responses in seeds. This aspect of the study is intriguing, but less strongly supported and is in apparent disagreement with recently published conclusions.

Essential revisions:

1. The reviewers and editors felt that the most important revisions concern the narrative that REF6 and SUVH4-6 control dormancy via ethylene responses. The mechanism by which REF6 regulates dormancy is an essential component of the manuscript. However, the presented mechanism is not convincingly supported by the supplied data and differs from the conclusions of a recently published paper (Chen et al., Plant Physiology 2020), which implicates ABA catabolism during seed development. The authors should establish the relationship between REF6, ethylene and ABA signalling, and ideally reconcile their findings with those of Chen et al., 2020.

2. Related to the above point, seed dormancy should be assessed in the absence of dormancy-breaking stimuli, especially nitrate in the MS medium. Sowing freshly harvested seed on minimal media would result in higher quality data and be the normal approach in the field, showing that seed lots indeed show dormancy (germination <50% is better). Then the authors would clearly be able to show that dormancy rather than germination is affected.

3. The authors are not always clear on the mutant allele used in different experiments. A major caveat is that the ref6-1 and ref6-3 mutants used in germination experiments (Figure 3, Figure 3¬—figure supplement 2 and Figure 3-—figure supplement 3) and most of the molecular analyses are not a full knockout (null) mutants (Yan et al., 2019 and Antunez et al., 2020). The use of partial-loss-of-function REF6 alleles could be problematic, in particular for germination assays with seeds from reciprocal crosses. To address this issue, authors should use a loss-of-function mutant line ref6C, which they have used to generate the triple elf6-3 ref6C jmj13G mutant.

Similarly, the interpretations arising from the endosperm transcriptome analysis with ref6-1 mutant plants should be carefully considered in their conclusions.

4. The interpretation that suvh4/5/6 cause a maternal effect in germination due to the activity of these histone methylases in the central cell is not fully supported. Genetic analyses would be required to distinguish sporophytic and gametophytic effects of these mutants on seed germination. Similarly, the parent-of-origin effects on germination arising in the cmt3 mutant are far from clear. The role of suvh4/5/6 and cmt3 in parent-of-origin effects on germination should be critically assessed and described in the manuscript accordingly.

5. The expression of REF6 in the micropylar endosperm and embryo using EPR1::REF6 and TPS1::REF6 should be confirmed experimentally (eg. RT-PCR with dissected tissues).

6. The strong claims about the effects of CHGm on REF6 should be moderated. The model communicated by the authors involves a binary distinction between genes that only have H3K27me3 (accessible to REF6) and genes that also have H3K9me2 and CHGm (inaccessible to REF6). However, the data do not support such a strong conclusion. For example, Figure 2c shows significant but modest differences between control genes (about 90% are not upregulated) and genes with REF6 target domains that lack CHGm (about 80% not upregulated). Furthermore, because CHGm is not absolute and rarely reaches 50%, the effects of CHGm on REF6 recruitment in endosperm are likely to be quantitative rather than qualitative. These limitations should be clearly explained.

7. Statistical approaches are not always deployed appropriately. Throughout the manuscript, statistical comparisons are performed between multiple test groups and a single control group. This is appropriate when differences with the control group are key, but not appropriate when differences between the test groups are of primary interest. In such cases, the authors should use ANOVA or a similar approach that can simultaneously evaluate multiple groups.

An example of this issue is in Figure 2c. The lower four bars indicate that genes without CHGm are significantly upregulated, but not genes with CHGm. However, there are many fewer CHGm genes. The key issue is whether genes with and without CHGm behave differently from each other. A similar example is in Figure 5b. Genes with and without CHGm are likely not different from each other, but genes with CHGm do not reach significance in comparison to the control group because they are fewer (116) than genes without CHGm (488). Figure 8a also suffers from this problem. The top three blue boxes are marked as significant, and the following three grey boxes are not, but the blue and grey boxes are probably not significantly different from each other. The authors should carefully reassess the statistical comparisons and adjust their conclusions accordingly.

8. The regulation of genomic imprinting during germination by REF6 and SUVH4/5/6 is supported by a small number of genes (Figure 8). Similarly, the silencing of paternal alleles of maternally-expressed genes is linked to a very small level of CHH methylation on genes. The authors should acknowledge that regulation of genomic imprinting during germination by REF6 and SUVH4/5/6 is not fully demonstrated by the data shown, and the authors should moderate their claims accordingly.

Reviewer #2:

The study by Sato et al. examines the role of chromatin remodelling in the endosperm during dormancy and germination control. The challenge with any study if this kind is that individual epigenetic processes are very general, required at multiple stages for the regulation of seed development, germination and associated metabolism. When a phenotype is observed in mutants lacking a chromatin remodelling process this may be a product of multiple effects, or specific effects on a limited number of loci at one developmental stage. In this study the authors show that the REF6/H3K27me3 suvh456/H3K9me2 interaction is important for gene regulation in the endosperm during germination. I find this interesting and the authors understandably would like to expand this to show that functionally REF6 is required for germination responses in seeds. In my view this aspect of the study is less successful, perhaps reflecting the fact that the authors are world leaders in the field of plant epigenetics but less experienced in the study of seed physiology.

1. Lets start with reviewing what I believe is shown to the highest degree of confidence. In my view the data strongly support the conclusion that the ref6 mutants have germination phenotype which is conferred through endosperm-specific REF6 activity. Although the phenotypes are very weak, the authors take care to generate consistent data across experiments. Cmt3 and suvh456 mutants seem to have a maternal effect on dormancy consistent with previous studies of the maternal roles of these genes in the endosperm. The interaction between H3K27me3, H3K9me2, REF6 and CHG methylation is clearly described on the context of chromatin remodelling during germination, and I agree looks similar in seeds to that shown in a recent study (Qi et al., 2019). It seems clear that REF6-mediated epigenetic remodelling plays a role in the induction of ethylene responsive genes but also for genes in cell wall remodelling, lipid metabolism and other processes known to occur in the endosperm during germination, perhaps in response to ethylene. I would contrast the function of REF6 with that of PICKLE (Ogas et al., 1998) which is required for silencing of the seed expressed genes upon the transition to vegetative growth.

2. There is one experimental issue that make some of the data hard to interpret. The authors would ideally take more care to distinguish clearly between dormancy and germination of non-dormant seeds. Firstly, the dormancy assays are confounded by the fact that a nitrate source was added to the germination medium, a strong dormancy-breaking signal. As such all seed lots behave as if non-dormant in all the experiments, although a difference in germination speed between mutant and wt is recorded. It is not clear from the data provided whether without nitrate the seeds would actually show dormancy, but my feeling is that they would, but only then can it be studied. Certainly, where seeds lots germinate 100% after a few days of imbibition the field would not ascribe any dormancy to them, and would not usually use such experiments to make judgements about dormancy or use the word dormancy to describe the phenotypes. However, the authors rightly intuit that differences in germination speed can result from residual activity of dormancy-inducing pathways even if they have insufficient activity to completely prevent germination (Bassel and Finch-Savage, 2016). Evidence that this is happening is that germination speed is further increased by cold stratification, another dormancy-breaking signal. However, sowing freshly harvested seed on minimal media would result in higher quality data and be the normal approach in the field, showing that seed lots indeed show dormancy (germination <50% is better). Then the authors would clearly be able to show that dormancy rather than germination is affected.

3. The main implied narrative in the abstract that these marks cooperate to regulate dormancy loss through reactivation of maternal copies of ethylene response genes is only partly supported by the data in my view. There is no evidence presented to show that only maternal copies are activated, or describing how imprinting affects the activation rate. So the wider significance was less clear, in contrast to Piskurewicz 2016 who showed that dormancy status affected the degree of imprinting, and that imprinting of these genes affected the rate of protein catabolism or dormancy loss.

4. In my view more supporting evidence is required to show that REF6 directly affects endosperm responses to ethylene. I would inhibit germination with paclobutrazol to remove secondary effects and apply ethylene or a precursor, comparing the transcriptional response of wt and ref6 seeds.

5. If REF6 does affect the ethylene response, more evidence is required to show that this is linked to dormancy loss rather than germination promotion. The latter will occur in both dormant and non dormant seeds, and the seeds examined here are either non-dormant or have unknown dormancy because of the problems mentioned previously. The data in figure 1 only show an increase in the expression of H3K27me3-marked ethylene-inducible genes after testa rupture, ie after the germination process has already begun. This also suggests that the process is linked to the mechanics of germination rather than dormancy. This ought to be manifest in germination speed phenotypes even in non-dormant seeds, eg in a ref6 aba2 double mutant. Conversely, if REF6 is involved in dormancy loss maybe there is no dormancy phenotype until a dormancy-breaking signal is applied.

6. Timing of REF6 activity. Primary dormancy is established during seed development and seed maturation. Thus if seeds have different levels of primary dormancy, with some exceptions the cause is usually found in the processes that occur prior to desiccation. It is unclear when the EPR1:REF6 construct is expressed, whether it is also active prior to desiccation when it may affect the establishment of dormancy, rather than dormancy loss during germination. The unfortunate recent publication (Chen et al., 2020) which shows that REF6 affects ABA metabolism during seed maturation also seems to me to be a plausible mechanism for the ref6 dormancy phenotype. However, disagreement over the cause of the phenotype does not necessarily detract from the fact that REF6 is clearly affecting gene expression in the endosperm during germination, it just means that the significance of the process is less clear for the observed phenotype.

7. Also influencing my conclusions is the observation that the suvh456 effect is not limited to the endosperm but also observable in the embryo, where ABI3 is also higher expressed than wt (Figure 7d). This suggests that suvh456 mutant seeds have higher ABA levels or some global effect on dormancy that affects the whole seed and is capable of up-regulating ABI3 expression. I found the authors' discussion of ABI3 hard to follow. It is surprising that ABI3 reaches maximal expression in the mature seed, but is marked with the repressive H3K27me3. If I understand correctly the authors suggest that in suvh456 ABI3 is further activated by REF6. Again this is surprising because their seeds are germinating and during germination ABI3 expression falls rapidly (if the seeds were dormant then this would be more plausible). So it is possible that ABI3 regulation is several steps removed from the activity of the epigenetic process, or that REF6 acts on ABI3 earlier in seed development. Recent paper Chen et al., 2020 seems to shed some light on this, showing that ref6 seeds have higher ABA content. This would lead to an increase in dormancy and higher ABI3 expression, even in the embryo, because endospermic ABA is transported to the embryo to maintain dormancy (Kang et al., 2015).

So in conclusion I think the authors have discovered an interesting process which could well be very important for gene regulation during germination in the endosperm, but have not convinced me that the observed phenotype is due to disruption of this process. However, there may be other consequences that are observable if endosperm responses to ethylene are investigated in more detail, rather than focussing on the effect on primary dormancy. Alternatively, if the authors wish to explain the germination phenotype it may be worth expanding the study to events during the establishment of primary dormancy, taking into account the recent published observations of Chen et al., 2020.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Combinations of maternal-specific repressive epigenetic marks in the endosperm control seed dormancy" for further consideration by eLife. Your revised article has been evaluated by Jürgen Kleine-Vehn (Senior Editor), Daniel Zilberman (Reviewing Editor) and the original reviewers.

The manuscript has been greatly improved and the reviewers and editors are generally satisfied that the revisions address the majors points raised after initial review. A single important remaining issue remains, as explained in the three related major points below. Please consider these points and address them in the manuscript as appropriate.

1. Although they germinate slower than wild type, ref6 mutant response to ACC is similar to wt in terms of the type of germination increase promoted. Certainly ref6 does not appear to have low sensitivity to ACC. Therefore the authors' data suggest that ethylene responses are normal in ref6 mutants. The authors conclude that because ref6 germination deficiency is reversed by ACC (with fluridone), ref6 must be acting through ethylene signalling. How the authors reached this conclusion is not clear.

2. The authors may not be correctly interpretating the aba2 rescue of ref6 in Chen 2020. Chen 2020 show that aba2 more-or-less fully rescues the ref6 phenotype, and indeed any other finding would be very surprising. There are small differences but these are well within normal biological variation. The issue with the fluridone experiment is that fluridone inhibits ABA synthesis in imbibed seeds, and cyp707a2 is important for ABA catabolism in imbibed seeds. But according to Chen et al. REF6 mainly affects CYP707A1 and A3, which are active primarily during seed maturation, and therefore affect germination through the amount of ABA left over in mature seeds (Okamoto et al., 2006). In that context the failure of fluridone to completely suppress the ref6 phenotype, if caused by up-regulation of CYP707A1/3, is unsurprising.

3. New genetic data is presented that shows that the activity of REF6 in the central cell is sufficient to lead to most of the phenotypic effects. The H3K27me3 data used for figure 1 is from developing endosperm, after REF6 could have acted in the gametophyte. But the authors argue that REF6 is affecting de-methylation during dormancy loss or germination, or at least after the stage tissue was collected for the 2016 paper. So is the activity of REF6 described in the rest of the paper the weak phenotype attributed to the paternal effect? Could the differences (which are small) be simply due to gene dosage effects in the endosperm, if REF6 is a rate limiting factor, even if REF6 is not imprinted? If dormancy is determined by central cell REF6 activity, then it can't relate to the effects on imprinted gene expression during germination.

If the above is true, the statement in the manuscript title is mis-leading, because dormancy is determined by central cell ref6 function – the events during germination are clearly affected by REF6, but lack an obvious phenotypic outcome (in Col-0 at least). This is also pertinent to the normal ACC response in ref6 mutants. A clear statement on this issue is hence necessary.

https://doi.org/10.7554/eLife.64593.sa1

Author response

Essential revisions:

1. The reviewers and editors felt that the most important revisions concern the narrative that REF6 and SUVH4-6 control dormancy via ethylene responses. The mechanism by which REF6 regulates dormancy is an essential component of the manuscript. However, the presented mechanism is not convincingly supported by the supplied data and differs from the conclusions of a recently published paper (Chen et al., Plant Physiology 2020), which implicates ABA catabolism during seed development. The authors should establish the relationship between REF6, ethylene and ABA signalling, and ideally reconcile their findings with those of Chen et al., 2020.

To address this concern, we performed several experiments and in silico analyses. First, to test the contribution of the ethylene and ABA pathways to the delayed germination phenotypes in ref6, we analyzed germination of ref6 upon treatment with the ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC), and the ABA biosynthesis inhibitor fluridone, which were reported to induce seed germination (Linkies et al., 2009; Martinez-Andujar et al., 2011). Treatment with only ACC or fluridone failed to completely suppress the delayed germination of ref6 (Figure 5—figure supplement 4), consistent with previous data showing that loss of the ABA biosynthetic gene ABA2 does not completely rescue the delayed germination phenotype of ref6 after stratification (Chen et al., 2020). In contrast, fluridone could completely suppress the cyp707a2 germination phenotype (Figure 5—figure supplement 4), revealing that fluridone can effectively suppress ABA biosynthesis as previously shown (Martinez-Andujar et al., 2011). Supporting our transcriptome data showing that ethylene pathways were suppressed in ref6 (Figure 5c), we found that treatment with both, ACC and fluridone, could completely rescue the delayed germination phenotype of ref6 after stratification(Figure 5— figure supplement 4). We thus conclude that suppression of the ethylene pathway together with the previously revealed induction of the ABA pathway (Chen et al., 2020) account for the delay of germination in ref6. We included and discussed this new data on P11, L243 to L261.

Second, we have further investigated the contribution of SUVH4/5/6 in regulating germination. As shown in our previous version of the manuscript, the ABI3 gene has triple repressive marks on the maternal alleles and was upregulated in suvh456 endosperm during germination (Figure 7). We furthermore found that genes with single H3K27me3 tend to be suppressed in suvh456 endosperm probably through indirect effects (Figure 7—figure supplement 2). In the revised manuscript we include data showing that B3 type transcription factors-binding motifs (RY motifs) are enriched among the downregulated genes with single H3K27me3 in suvh456 (Figure 7—figure supplement 5). This data suggests that upregulated ABI3 in suvh456 suppresses genes with single H3K27me3. Supporting this idea, we found that genes with single H3K27me3 are significantly enriched among direct ABI3 target genes (Tian et al., 2020), while genes with single H3K9me2 and double H3K27me3/H3K9me2 are not enriched (Figure 7—figure supplement 5). Together, this data reveals a complex network between ABA and ethylene pathways in the endosperm that are both epigenetically and transcriptionally regulated by multiple factors. We have included this new data on P14, L332 to L338 and discussed on P17, L417 to L418, P18, L431 to L437, P19, L461 to L476 and P21, L525 to L526.

2. Related to the above point, seed dormancy should be assessed in the absence of dormancy-breaking stimuli, especially nitrate in the MS medium. Sowing freshly harvested seed on minimal media would result in higher quality data and be the normal approach in the field, showing that seed lots indeed show dormancy (germination <50% is better). Then the authors would clearly be able to show that dormancy rather than germination is affected.

To address this comment, we performed analyses of seed dormancy on minimal media (Figure 3figure supplement 3a, Figure 6—figure supplement 1a and Figure 6—figure supplement 2).

In addition to that, we further confirmed seed dormancy phenotypes in ref6 single and suvh456 triple mutant seeds that were developed under low temperature (Figure 3—figure supplement 3b, Figure 6figure supplement 1c). We included this new data on P8, L177 and P12, L276.

3. The authors are not always clear on the mutant allele used in different experiments. A major caveat is that the ref6-1 and ref6-3 mutants used in germination experiments (Figure 3, Figure 3¬—figure supplement 2 and Figure 3—figure supplement 3) and most of the molecular analyses are not a full knockout (null) mutants (Yan et al., 2019 and Antunez et al., 2020). The use of partial-loss-of-function REF6 alleles could be problematic, in particular for germination assays with seeds from reciprocal crosses. To address this issue, authors should use a loss-of-function mutant line ref6C, which they have used to generate the triple elf6-3 ref6C jmj13G mutant.

Similarly, the interpretations arising from the endosperm transcriptome analysis with ref6-1 mutant plants should be carefully considered in their conclusions.

To address this concern, we performed several new experiments and in silico analyses that support our conclusions reached with ref6-1 and ref6-3 alleles (see figures below).

In ref6-1/ref6-3 alleles, the T-DNAs are inserted into the coding region between the JumonjiC domain and Zinc-finger domain, and weak expression of REF6 mRNA was detected (Yan et al., 2019). This raises the possibility that the Zinc-finger domain-independent functions of REF6 still remain active in ref6-1 or ref6-3. It was previously shown that REF6 can be recruited by MADS-box transcription factors independently of the REF6 binding domain (Yan et al., 2019).

1. We obtained the ref6c null allele and compared the phenotype with ref6-1 and ref6-3 alleles. (Additional figure 1a), and also analyzed the parental-specific effects of ref6c (Additional figure 1b). The results show that the phenotypes of the mutant alleles are highly comparable, justifying the use of the ref6-1 and ref6-3 alleles for this study.

2. We investigated the possibility that REF6 is recruited to MADS-box motifs by analyzing whether predicted REF6 targets have REF6 binding sites. As shown in the manuscript, the presence of REF6binding sites is associated with the activation of genes with single H3K27me3 and double H3K27me3/H3K9me2 (Figure 2c). Consistently, REF6-binding sites are significantly enriched within gene bodies of upregulated genes with single H3K27me3 and double H3K27me3/H3K9me2 (additional figure 1c, left panel), while MADS-box binding sites on promoter regions are not enriched among the upregulated genes (additional figure 1c, right panel). Furthermore, we found that there were substantially fewer MADS-box binding sites (around 10%) than REF6-binding sites (40-60%) among upregulated genes. This data suggests that the possible recruitment of REF6 by MADS-box transcription factors in ref6-1 and ref6-3 is unlikely to have a large contribution to total gene activation. This data are in support with our observations that the ref6-1/ref6-3 alleles behave very similar to the ref6c allele and justify their use in this study.

4. The interpretation that suvh4/5/6 cause a maternal effect in germination due to the activity of these histone methylases in the central cell is not fully supported. Genetic analyses would be required to distinguish sporophytic and gametophytic effects of these mutants on seed germination. Similarly, the parent-of-origin effects on germination arising in the cmt3 mutant are far from clear. The role of suvh4/5/6 and cmt3 in parent-of-origin effects on germination should be critically assessed and described in the manuscript accordingly.

To address this concern, we analyzed the gametophytic effect of suvh456 on germination using F1 seeds generated by pollinating heterozygous suvh456 mutants with wild-type pollen. The results support the conclusion that suvh456 caused delayed germination through gametophytic effects (Figure 6b). We described this new data on P12, L287 to L290.

Additionally, we analyzed the cause of the maternal effect of ref6 by analyzing the phenotypes of F1 seeds generated by pollinating heterozygous ref6-1 with wild-type pollen. The results support the conclusion that the maternal effect of ref6 is caused by a gametophytic effect (Figure 3c). We described this new data on P8, L183 to P9, L191.

The cmt3-11 mutant did not have a maternal effect on germination (Figure 6d), indicating that CMT3 acts after fertilization on H3K9me2 marks established in the female gametophyte. One possible explanation for the difference between suvh456 and cmt3 mutants is different effect on H3K9me2; while loss of SUVH4/5/6 causes severe depletion of both, H3K9me2 and CHGm (Stroud et al., 2014; Underwood et al., 2018), loss of CMT3 causes depletion of CHGm but has only intermediate effects on on H3K9me2 (Stroud et al., 2014; Inagaki et al., 2010). This suggests that genes with H3K27me3/H3K9me2 are suppressed by the paternal CMT3 after fertilization. We have discussed this data on P13, L298 to L301 and P20, L485 to L489.

5. The expression of REF6 in the micropylar endosperm and embryo using EPR1::REF6 and TPS1::REF6 should be confirmed experimentally (eg. RT-PCR with dissected tissues).

We confirmed the tissue-specific expression of REF6 in EPR1::REF6;ref6-1 and TPS1::REF6;ref6-1 (Figure 4—figure supplement 1).

6. The strong claims about the effects of CHGm on REF6 should be moderated. The model communicated by the authors involves a binary distinction between genes that only have H3K27me3 (accessible to REF6) and genes that also have H3K9me2 and CHGm (inaccessible to REF6). However, the data do not support such a strong conclusion. For example, Figure 2c shows significant but modest differences between control genes (about 90% are not upregulated) and genes with REF6 target domains that lack CHGm (about 80% not upregulated). Furthermore, because CHGm is not absolute and rarely reaches 50%, the effects of CHGm on REF6 recruitment in endosperm are likely to be quantitative rather than qualitative. These limitations should be clearly explained.

To address this concern, we moderated our claim about the relationship between CHGm directly on REF6 binding sites and REF6 accessibility. As the editor suggests about 10% of genes with H3K27me3/H3K9me2 and CHGm on REF6 binding sites are induced during germination, suggesting unknown mechanisms can remove the triple repressive marks or paternal alleles of some of those genes are activated during germination. It will be interesting to study whether and how the strong suppressing mechanism can be released. More detailed analyses of individual genes with triple repressive marks will be required to reveal the complete molecular mechanisms establishing and removing triple repressive marks. We have discussed these points on P17, L418 to L420.

7. Statistical approaches are not always deployed appropriately. Throughout the manuscript, statistical comparisons are performed between multiple test groups and a single control group. This is appropriate when differences with the control group are key, but not appropriate when differences between the test groups are of primary interest. In such cases, the authors should use ANOVA or a similar approach that can simultaneously evaluate multiple groups.

An example of this issue is in Figure 2c. The lower four bars indicate that genes without CHGm are significantly upregulated, but not genes with CHGm. However, there are many fewer CHGm genes. The key issue is whether genes with and without CHGm behave differently from each other. A similar example is in Figure 5b. Genes with and without CHGm are likely not different from each other, but genes with CHGm do not reach significance in comparison to the control group because they are fewer (116) than genes without CHGm (488). Figure 8a also suffers from this problem. The top three blue boxes are marked as significant, and the following three grey boxes are not, but the blue and grey boxes are probably not significantly different from each other. The authors should carefully reassess the statistical comparisons and adjust their conclusions accordingly.

To address this concern, we reanalyzed our data using pairwise statistical tests with multiple testing correction. The statistical methods are described in the legend of each figure.

8. The regulation of genomic imprinting during germination by REF6 and SUVH4/5/6 is supported by a small number of genes (Figure 8). Similarly, the silencing of paternal alleles of maternally-expressed genes is linked to a very small level of CHH methylation on genes. The authors should acknowledge that regulation of genomic imprinting during germination by REF6 and SUVH4/5/6 is not fully demonstrated by the data shown, and the authors should moderate their claims accordingly.

To address this concern, we moderated our claims and specifically noted that further studies are required to validate the conclusions. Modifications are on P16, L376 to L377, L395 to L397 and P18, L441 to L443.

We also reanalyzed the DNA methylation data using pairwise Wilcoxon test and included up-and downstream regions in the analyses (Figure 8—figure supplement 2, 3 and 4). Also for this analyses we toned down the conclusions and included the modifications on P16, L383 to L397.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Essential revisions:

1. Although they germinate slower than wild type, ref6 mutant response to ACC is similar to wt in terms of the type of germination increase promoted. Certainly ref6 does not appear to have low sensitivity to ACC. Therefore the authors' data suggest that ethylene responses are normal in ref6 mutants. The authors conclude that because ref6 germination deficiency is reversed by ACC (with fluridone), ref6 must be acting through ethylene signalling. How the authors reached this conclusion is not clear.

We agree with the reviewer that the ref6 mutant can respond to ethylene; however, also ethylene biosynthesis is negatively affected in ref6 mutants (Figure 5—figure supplement 3); thus by adding ACC, ethylene production likely increases and thus ethylene signalling. Furthermore, since ethylene biosynthesis and signalling are in a feedback loop and ethylene was shown to directly regulate its biosynthesis (doi.org/10.1111/nph.16873), it is well possible that by adding ACC and thus highly increasing ethylene levels, ethylene signalling is enhanced. Since the combined treatment of fluridone and ACC completely suppressed the ref6 mutant phenotype, we consider our interpretation that REF6 acts (as well) through ethylene signalling justified. We discussed the requirement of REF6 to induce ethylene biosynthesis on P11, L239 to P11, L244.

2. The authors may not be correctly interpretating the aba2 rescue of ref6 in Chen 2020. Chen 2020 show that aba2 more-or-less fully rescues the ref6 phenotype, and indeed any other finding would be very surprising. There are small differences but these are well within normal biological variation. The issue with the fluridone experiment is that fluridone inhibits ABA synthesis in imbibed seeds, and cyp707a2 is important for ABA catabolism in imbibed seeds. But according to Chen et al. REF6 mainly affects CYP707A1 and A3, which are active primarily during seed maturation, and therefore affect germination through the amount of ABA left over in mature seeds (Okamoto et al., 2006). In that context the failure of fluridone to completely suppress the ref6 phenotype, if caused by up-regulation of CYP707A1/3, is unsurprising.

The data from Chen et al., 2000 show that the ref6 aba2 double mutant has lower germination rates than the aba2 single mutant after stratification (Figure 2C). While the difference has not been statistically evaluated, a germination difference of 50% versus 90% at 1.5 DAP seems to be highly significant. Since the data are generated in biological triplicates; we consider it unlikely that the difference is solely due to biological variation.

3. New genetic data is presented that shows that the activity of REF6 in the central cell is sufficient to lead to most of the phenotypic effects. The H3K27me3 data used for figure 1 is from developing endosperm, after REF6 could have acted in the gametophyte. But the authors argue that REF6 is affecting de-methylation during dormancy loss or germination, or at least after the stage tissue was collected for the 2016 paper. So is the activity of REF6 described in the rest of the paper the weak phenotype attributed to the paternal effect? Could the differences (which are small) be simply due to gene dosage effects in the endosperm, if REF6 is a rate limiting factor, even if REF6 is not imprinted? If dormancy is determined by central cell REF6 activity, then it can't relate to the effects on imprinted gene expression during germination.

If the above is true, the statement in the manuscript title is mis-leading, because dormancy is determined by central cell ref6 function – the events during germination are clearly affected by REF6, but lack an obvious phenotypic outcome (in Col-0 at least). This is also pertinent to the normal ACC response in ref6 mutants. A clear statement on this issue is hence necessary.

While ref6 has a maternal effect, the fact that many REF6 targets have detectable levels of H3K27me3 in the endosperm and since we could partially complement the ref6 mutant by expressing REF6 in the micropylar domain of the endosperm (Figure 5a), strongly suggests that REF6 also acts after fertilization. In support of this, homozygous ref6 mutants have a stronger delay in germination than mutants inheriting a maternal ref6 and a wild-type paternal allele (Figure 3b). Nevertheless, independent of when REF6 acts; the consequence of REF6 activity occurs during germination. We had discussed the idea that there are two types of genes that are either activated in the central cell or later on in the endosperm in the manuscript lines 472 to 495. In summary, we do not think that our title is misleading, the combination of epigenetic modifications present in the endosperm (independent on when they are established) determines the activity status of the alleles during germination.

https://doi.org/10.7554/eLife.64593.sa2

Article and author information

Author details

  1. Hikaru Sato

    Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Uppsala, Sweden
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing - original draft, Writing - review and editing
    Competing interests
    none
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7628-0414
  2. Juan Santos-González

    Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Uppsala, Sweden
    Contribution
    Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Visualization
    Competing interests
    none
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8712-9776
  3. Claudia Köhler

    1. Department of Plant Biology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Centre for Plant Biology, Uppsala, Sweden
    2. Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
    Contribution
    Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing - review and editing
    For correspondence
    claudia.kohler@slu.se
    Competing interests
    none
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2619-4857

Funding

Vetenskapsrådet (2017-04119)

  • Claudia Köhler

Knut och Alice Wallenbergs Stiftelse (2018-0206)

  • Claudia Köhler

Goran Gustafsson Foundation for Research in Natural Sciences and Medicine

  • Claudia Köhler

Human Frontier Science Program (LT000162/2018-L)

  • Hikaru Sato

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Cecilia Wärdig for technical support and Katarzyna Dziasek for helpful comments on the manuscript. This research was supported by grants from the Swedish Research Council VR (2017–04119, to CK), a grant from the Knut and Alice Wallenberg Foundation (2018–0206, to CK), a grant from the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine (to CK), and a fellowship grant from the HFSP (to HS).

Senior Editor

  1. Jürgen Kleine-Vehn, University of Freiburg, Germany

Reviewing Editor

  1. Daniel Zilberman, Institute of Science and Technology Austria, Austria

Reviewer

  1. Steven Penfield, John Innes Centre, United Kingdom

Publication history

  1. Received: November 4, 2020
  2. Preprint posted: November 12, 2020 (view preprint)
  3. Accepted: August 23, 2021
  4. Accepted Manuscript published: August 24, 2021 (version 1)
  5. Version of Record published: August 31, 2021 (version 2)

Copyright

© 2021, Sato et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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