ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development

doi:10.21203/rs.3.rs-4998315/v1

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ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development

https://doi.org/10.21203/rs.3.rs-4998315/v1

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Polycomb group (PcG) proteins can silence gene expression by modifying histones, such as H2Aub and H3K27me3, which is crucial for maintaining cell type and tissue-specific gene expression patterns. However, little is known about the impact of gene regulation by PcG proteins through H2Aub and H3K27me3 during maize kernel development.Here, we characterized a maize miniature seed mutant mn8, and map-based cloning revealed that Mn8 encodes a plant specific PcG protein, ZmEMF1a. Mutation in ZmEMF1a leads to significantly reduced kernel size and weight. Molecular analyses showed that ZmEMF1ainteracts with PRC1 component ZmRING1 and PRC2 subunit ZmMSI1, which is required for H2Aub and H3K27me3 establishment. ZmEMF1a deficiency causes significant reduced levels of H2Aub and H3K27me3 in the genome. The combined analysis of ChIP-seq and RNA-seq data revealed that H2Aub is negatively correlated with gene expression in maize, unlike the positive association with expression of H2Aub in Arabidopsis. Compared with WT endosperms, elevated expressions of homology genes of cell proliferation repressors, such as DA1, BB1, ES22, MADS8 and MADS14, accompanied with decreases in H3K27me3 or H2Aub levels at these loci in mn8endosperms, indicating that lack of ZmEMF1a function impedes the deposition of H3K27me3 or H2Aub mark at cell division repressor genes. Taken together, our results show that ZmEMF1a plays a crucial role in regulating the expression of genes associated with maize kernel development through maintaining the modification levels of H2Aub and H3K27me3.

PcG complex

H2Aub

H3K27me3

kernel development

maize.

Polycomb group (PcG) proteins maintain the transcriptionally repressed state of genes involved in the normal development of eukaryotes by incorporating histone modifications within chromatin [1–3]. PcG proteins normally assemble into two types of Polycomb repressive complexes (PRC) with different histone-modifying activities: PRC1 and PRC2. PRC1 has histone H2A E3 ubiquitin ligase activity toward lysine 119, 120, or 121 in Drosophila, mammals or Arabidopsis, respectively [4–7], and PRC2 has histone H3 lysine 27 (H3K27) tri-methyltransferase activity [1, 8, 9]. PRC1 and PRC2 ultimately lead to the transcriptional repression by chromatin compaction, and other mechanisms are still under investigation. In animals, PRC2 deposits H3K27me3 mark at a specific gene, which subsequently recruits PRC1 due to its ability to bind to H3K27me3, thereby facilitating the monoubiquitination of H2A [6]. However, recent results indicate that a specific group of loci targeted by PRC2 requires the prior establishment of H2Aub1 for H3K27me3 to be deposited, revealing a hierarchical sequence where PRC1 acts initially and PRC2 follows [10, 11]. Despite the fact that the enzymatic activities of PRC2 and PRC1 are conserved between animals and plants, there are distinct differences in the complex composition and distribution of H2A monoubiquitination and H3K27me3 across the genome [12].

PRC2 core subunits are well conserved in plants compared to their vertebrate counterparts [1, 3]. In Arabidopsis, CURLY LEAF (CLF), MEDEA (MEA), and SWINGER (SWN) are homologs of the animal SET-domain-containing methyltransferase Enhancer of zeste (E(z)) [13–15]. FERTILIZATION INDEPENDENT SEED 2 (FIS2), EMBRYONIC FLOWER 2 (EMF2), and VERNALIZATION 2 (VRN2) are homologs of the scaffold protein Suppressor of zeste 12 (Su(z)12) [16–18]. FERTILIZATION INDEPENDENT ENDOSPERM (FIE) and MULTIPLE SUPPRESSOR OF IRA 1 (MSI1) are the equivalents of the H3K27me3 binding protein Extra sex combs (Esc) and the nucleosome-remodeling factor Nurf55, respectively [19, 20]. PRC2 complex in Arabidopsis contain single copy gene FIE, however, FIE is encoded by FIE1 and FIE2 in rice and maize [21, 22]. OsFIE2 and ZmFIE2 are expressed universally and are likely to be functional orthologs of Arabidopsis FIE [21]. Conversely, OsFIE1 and ZmFIE1 are expressed materially exclusively in endosperm, and have evolved a distinct function [23, 24].

PRC1 composition is less conserved, the vertebrate H2A E3 ubiquitin ligase module containing RING1A or RING1B and one of the six Polycomb RING finger (PCGF) proteins, while the one in Drosophila consists of Sex Comb Extra (Sce, also known as dRing) and Posterior Sex Combs (Psc) or Su(z)2 [25–28]. The E3 monoubiquitin ligase module can associate with other nonenzymatic activities to form canonical PRC1s or with other subunits to form variant PRC1s [28, 29]. Although the module in Arabidopsis containing one AtBMI1s (AtBMI1A/B/C) and one RING1 (AtRING1A or AtRING1B) protein has been identified, several canonical PRC1 components conserved in animals are missing in Arabidopsis, and instead, several plant-specific proteins are involved as PcG components in chromatin compaction and H3K27me3 reading [1, 30]. One such example is LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which is proposed to be a functional analog of Drosophila Pc due to its ability to bind the H3K27me3 mark and interact with other PRC1 components, including BMI1 and RING1 [4, 31–33]; nevertheless, it also co-purifies with PRC2 [34, 35]. Furthermore, in plants, there is an absence of proteins that possess a chromatin compaction domain known as PSC-CTR, which is crucial for the chromatin compaction process mediated by PRC1 in animals [36]. In the case of the flowering plant Arabidopsis, the plant-specific protein EMF1 serves a similar role to PSC-CTR, facilitating chromatin condensation during the process of Polycomb-mediated gene silencing [36, 37].

The EMF1 gene encodes a transcriptional regulator protein that includes the LXXLH motif [38], and it is known to interact with RING-finger proteins in Arabidopsis [39]. EMF1 also forms a complex with plant-unique BAH-domain-containing proteins SHORT LIFE (SHL) and EARLY BOLTING IN SHORT DAYS (EBS), which can read the H3K27me3 mark, to play PRC1-like roles, thereby implementing Polycomb silencing in higher plants [40]. However, EMF1 also interacts with PRC2 component MSI1 in vitro [41]. Genome-wide analysis of H3K27me3 modification and EMF1 binding in WT and emf1-2 mutants revealed that 58% of the EMF1-bound genes exhibited H3K27me3, and 44% of the genomic genes marked by H3K27me3 showed reduced H3K27me3 levels in emf1-2 mutants, suggesting that PRC2 function partially required EMF1 [37]. PRC2 activity may be regulated by EMF1 function in chromatin compaction, a recent report shows that in mouse embryonic stem cells, local chromatin compaction precedes and may regulate the formation of H3K27me3 [42]. Further work is required to fully understand the relationship among H2Aub, H3K27me3, and EMF1 in gene repression.

PcG proteins complexes play crucial functions in regulation of plant developmental transition and in controlling gene expression [37, 43]. FIS-PRC2 genes are considered to be repressors of endosperm formation in the absence of fertilization [44, 45]. After pollination, FIS-PRC2 complex is involved in endosperm development through repression of the expression of MADS-box gene AGAMOUS LIKE62 (AGL62), which suppresses cellularization phase of endosperm development [46, 47]. Endosperm development is characterized by four stages, that are coenocytic, cellularization, differentiation, and maturation [48]. The rapid expansion of endosperm volume during the differentiation stage is attributable to both cell proliferation and cell expansion. In Arabidopsis, several genetic factors controlled cell division to regulate seed size, including some components of the ubiquitin pathway. Arabidopsis DA1, encoding a predicted ubiquitin receptor, inhibits seed growth by restricting the period of cell proliferation. The da1-1 mutant plants represented large organs and seeds [49]. In maize, the mutation in homology gene ZmDA1 cause the same biological phenotype as da1-1 in Arabidopsis.

BIG BROTHER (BB) is a novel RING finger protein, which acts as a repressor of plant organ growth, small changes in the expression of BB can significantly affect the size of the organs [50]. WIDE AND THICK GRAIN1 (WTG1) is a functional deubiquitinating enzyme, loss of function of WTG1 produces wide and heavy grains [51]. The plant-specific transcription factor ABI3/VP1 (RAV1) negatively regulates plant growth, and overexpression of RAV1 results in reduced seed size and weight [52]. Previous research find that the orthologs of Arabidopsis BIN2, GSK2, interacts with GRAIN SIZE 2 (GS2)/GROWTH-REGULATING FACTOR 4 (OsGRF4) and inhibits its transcriptional activity and negatively regulate the grain size in rice [53]. Another study indicates that MADS1 interacts with GS3 and DEP1, promoting the transcription of downstream genes, thereby inhibiting grain growth of rice [54]. Many studies have well revealed the role of negative regulators in the regulation of seed development, including their interacting proteins and the regulatory networks on downstream genes. However, how the expression of these negative regulators is directly regulated remains unknown.

In this work, we isolated a small kernel mutant and map-based cloning and allelic analysis have demonstrated that the loss-of-function mutation in ZmEMF1a is responsible for the mutant phenotype. Further molecular investigations have demonstrated that ZmEMF1a interacted with the PcG proteins ZmRING1 and ZmMSI1, and the interactions were in the N-terminal of ZmEMF1a, not with the C-terminus. Genome-wide analyses revealed that loss of ZmEMF1a results in a significant decrease in the deposition of H2Aub and H3K27me3. A remarkable great number of genes, which were grouped to response to hormone, transcription factor activity and seed development, were up-regulated in mn8 mutant endosperm. Interestingly, H2Aub was negatively correlated with gene expression in maize, about 60% H2Aub-marked genes showed either no or low expression, contrary to the studies in Arabidopsis, which suggests that H2Aub modifications play different regulatory roles in maize compared to other plant species. In mn8 mutants, elevated expression of repressors in cell proliferation, such as DA1, BB1, MADS8, and MADS14, accompanied a reduction in H3K27me3 or H2Aub levels. In conclusion, ZmEMF1a plays an important role in maintaining the H2Aub and H3K27me3 modifications in maize, which is necessary for the kernel development.

Phenotype and Genetic Characterization of mn8

The mn8 mutant was isolated in the course of an EMS-induced mutant screen aiming at the cloning of mutants with defects in kernel development. The mutant was backcrossed to B73 for over three generations to clean the inessential mutational loci, meanwhile it was outcrossed with the inbred line Mo17. The miniature kernel trait: normal kernel trait on the ears of F2 plants segregated in accordance with a ratio of 1:3 (χ² = 0.013–0.215 < χ²0.05 = 3.84; Table S1), indicating that MN8 was a single gene. At maturity, the smaller kernels of mn8 can be clearly distinguished macroscopically from wild-type kernels from the same ear (Fig. 1a). Compared with WT (B73) siblings, mn8 kernels were significantly smaller (Fig. 1b, c). During sectioning of the kernels, both embryo and endosperm were markedly reduced in mn8 kernels compared with B73 (Fig. 1d). When measured, the single kernel weight was significantly reduced than that of the WT (Fig. 1e), and the embryo length was also found to be significantly shorter than that of the WT (Fig. 1j).

Transmission electron microscopy and scanning electron microscopy were used to evaluate the protein bodies and starch grains. Notably, we found that the immature endosperm of the mn8 mutant contained smaller protein bodies and starch grains than those in the WT (Fig. 1f-i). The starch content as a percentage of kernel weight and the average total protein content per kernel in the mn8 endosperm were significantly lower than those in the WT (Fig. 1k, l). The mn8 mutant seedlings were slight shorter than WT at the 7th day after germination (Additional file 1: Fig. S1a). However, there was no significant difference in plant height at sexual maturity stage between the mn8 mutant and WT (Additional file 1: Fig. S1b, c). We also found that the germination frequency and the number of leaves of mn8 were similar to those of the WT (Additional file 1: Fig. S1d, e). In summary, MN8 is specifically affects maize kernel development.

The mn8 Mutant kernels showed a developmental delay

To examine the developmental aberrations of the mn8 kernels, a detailed characterization of the development of both mn8 and WT kernels was achieved through analysis of cytological sections. At 6 DAP, the size of mn8 endosperm was about one-second of that of the WT (Fig. 2a, e). Both mn8 and WT embryos reached the transition stage, characterized by the formation of a distinct external cell layer, the protoderm, which marks the shift from radial to bilateral symmetry (Fig. 2i, n). At 8 DAP, the difference in endosperm volume between the mn8 and WT became more pronounced than at the early development stage, with the size of the mn8 endosperm being only one-third of that of its WT sibling (Fig. 2b, f). At this stage, WT embryo had reached the coleoptilar stage, characterized by the clear establishment of bilateral symmetry, the formation of the shoot apical meristem (SAM), the root apical meristem (RAM), and a separated scutellum. By contrast, the mn8 embryo still stayed at the transition stage, although they showed a remarkable increase in both the width and length of the embryo proper and suspensor (Fig. 2j, o). At 12 DAP, the degeneration of maternal tissue introduced a gap between the endosperm and the pericarp in mn8 (Fig. 2a, c, g). This gap enlarged during the later development stage of the mutant seed until maturity (Fig. 2d, h). At this period, the mn8 embryo had reached the coleoptilar stage, whereas the WT embryo had reached the late embryogenesis stage and had developed leaf primordia and a vascular system (Fig. 2k, p). The development of the mn8 embryo was delayed but not arrested. At 15 DAP, the mn8 embryo differentiated leaf primordia and a RAM. By 24 DAP, it had developed four to five leaf primordial, a well-formed scutellum, and an embryo axis (Fig. 2l, m, q, r).

Map-based cloning of mn8

The Mn8 mutation was induced by EMS, prompting us to performed map-based cloning to identify the mutation locus responsible for the mutant phenotypes. In the first step, mn8 in the B73 genetic background was outcrossed with the Mo17 inbred line. The heterozygous F₁ progeny was self-pollinated to create a mapping population. The mn8 locus was mapped to a 10-Mb interval on chromosome 10. Additional markers were used to narrow down the interval to 10 Mb, and markers CHR_10_87 Mb and CHR_10_97 Mb were defined as flanking markers for subsequent fine mapping. A population of 1,500 mutants was genotyped using the flanking markers, and a total of 62 genetic recombination events were identified. To increase marker density within this interval, 14 additional markers were developed, and the 62 recombinants were subsequently genotyped with these markers. The number of recombinants dropped considerably closer to 10-88.74 Mb and 10-89.01 Mb: there was 1 recombinant for 10-88.68 Mb, 1 for 10-89.1 Mb, and none for both 10-88.74 Mb and 10-89.01 Mb (Fig. 3a). These results revealed that the mutation was located between 10-88.68 Mb and 10-89.1 Mb, an interval that contained six predicted genes. A comparison of the nucleotide sequences of these candidate genes between the WT and mn8 plants revealed that only Zm00001d024813 contained a mutation expected to cause a loss of gene function. In mn8, a C to T conversion occurred at 2,972 bp downstream of the start codon, resulting in the amino acid Glu transitioning to a stop codon (Fig. 3b).

To confirm that the mutation in Zm00001d024813 accounted for the mn8 phenotype, we employed the CRISPR/Cas9 system to generate additional independent alleles. We constructed a pCAMBIA-derived CRISP-Cas9 binary vector containing gRNA expression cassettes targeting the 2nd exon of Zm00001d024813. Among 6 independent transformation events, several types of mutation were detected, and we select a T insertion mutant (mn8C1) for further genetic analysis. In mn8C1, the protein encoded by the mutated gene, Mn8C1, was predicted to be truncated because the T-insertion caused a frameshift, which introduced a premature stop codon. The phenotypes of the mn8C1 kernels exhibited similar defects to those of the mn8 (Fig. 3c). The mutants mn8C1, used in further work, were backcrossed to B73 to eliminate the CRISPR-Cas9 transgene. Ears of self-pollinated heterozygous mn8C1 segregated mutant kernels at the radio of 1:3 (mutant: WT) (Fig. 3d, Table S2). The kernels exhibiting the mn8-like phenotype were confirmed to be homozygous mutants of Zm00001d024813, as determined by genotyping with gene-specific primers. Allelic crosses between heterozygous mn8/+ and mn8C1/+ heterozygous produced ears that segregated kernels with normal and small kernels in the expected 3:1 ratio (Fig. 3e, Table S3), indicating that mn8 and mn8C1 are allelic. Taken together, these data provide evidence that the mutation in Zm00001d024813 is indeed responsible for the mutant phenotype observed in mn8.

Mn8 encodes an EMF1-like protein

Sequence analysis revealed that Mn8 contains 4 exons and 3 introns. The mature transcript of Mn8 features a 3,261-bp coding sequence that encodes an unknown protein, Zm00001d024813, comprising 1,086 amino acids. Homology analysis indicated that Mn8 exhibits the highest similarity with the Arabidopsis protein EMF1 (AT5G11530), with 36% identity and 50.48% similarity. In addition to MN8, there are three other homologous genes in maize that have been identified with predicted translation similarity to AtEMF1 through a BLASTp search of the NCBI non-redundant protein database. Here, we name MN8 as EMF1a, and the other three as EMF1b, EMF1c, and EMF1d, respectively. Protein sequence similarity analysis revealed that EMF1b shares 72% similarity with EMF1a, while EMF1c and EMF1d share 44% and 38%, respectively (Additional file 1: Fig. S2a). Protein sequence alignment showed that EMF1c and EMF1d were significantly shorter than EMF1a and appeared to be more like fragments of EMF1a (Additional file 1: Fig. S2b). EMF1a possessed a nuclear localization signal peptide (NLS) and an LXXLL motif, whereas EMF1b lacked the NLS motif, and both EMF1c and EMF1d lacked the LXXLL motif (Additional file 1: Fig. S2b). Despite their high similarity, the absence of important regions may lead to differences in their regulatory functions in maize.

To further explore the evolutionary relationships among ZmEMF1s, a phylogenetic tree was constructed based on the full-length protein sequences of ZmEMF1a and its homologous from other plant species (Additional file 1: Fig. S3a). The phylogenetic tree revealed that ZmEMF1a and EMF1/CCP1 (EMF1-like protein in rice) were highly conserved in monocots and evolutionarily related to Arabidopsis EMF1. To better understand the role of ZmEMF1a in endosperm development, we analyzed its tissue expression patterns using published RNA sequencing (RNA-seq) data [55]. We found that ZmEMF1a was constitutively expressed in different tissues, but with higher expression in endosperm and seeds (Additional file 1: Fig. S3b). Subsequently, we collected seeds at different days after pollination (DAP) and examined the expression of ZmEMF1a by reverse transcription-quantitative PCR (RT-qPCR), which showed that its expression peaked at 10 to 14 DAP (Additional file 1: Fig. S3b). These results suggest that ZmEMF1a may play an important role in maize kernel development.

ZmEMF1a interacts with PRC1 and PRC2 components

In Arabidopsis, AtEMF1 can interact with both PRC1 RING-finger proteins and the PRC2 component MSI in vitro [4, 41]. To explore the possibility of interactions between ZmEMF1a and putative PcG proteins in maize, we performed a yeast two-hybrid (Y2H) assay. Employing a candidate-gene approach, we discovered that ZmEMF1a directly interacted with ZmRING1 and ZmMSI1 in yeast cells (Fig. 4a). To verify the interaction between ZmEMF1a and PRC components, split-luciferase complementation (LUC) imaging assays were used and confirmed the interactions between ZmEMF1a and both ZmRING1 and ZmMSI1, but not with ZmLHP1 or ZmFIE1 (Fig. 4b). We also analyzed the subnuclear localizations of ZmEMF1a, ZmRING1 and ZmMSI1 in maize protoplasts and Nicotiana benthamiana (N. benthamiana) leaves. The results showed that all proteins localized to the nucleus (Additional file 1: Fig. S4a, b), using AHL22-RFP as a nuclear marker [56]. This indicated that the interactions occurred in the nucleus. In addition, we performed the bimolecular fluorescence complementation (BiFC) assay to test these interactions in N. benthamiana. We observed the green fluorescent signal in the nucleus when ZmEMF1a was transiently expressed with ZmRING1 or ZmMSI1, but not when ZmEMF1a was expressed with ZmFIE1 (Fig. 4c).

The C to T point mutation in ZmEMF1a led to premature termination of protein translation, which in turn affected the kernel development in maize. To further explore the important role played by the C-terminal domain of EMF1a, we constructed vectors for subcellular localization and Y2H assays of EMF1a-N and EMF1a-C, respectively. The nuclear localization of the different domains was observed in the nuclei of the infiltrated N. benthamiana leaves. The results showed that GFP-EMF1a signal was concentrated in one spot within the nucleus. The GFP-EMF1a-N fusion protein was targeted mainly to the nucleolus, while GFP-EMF1a-C signal was indistinguishable from that of the GFP-EMF1a, indicating that the C-terminal domain is responsible for the subnuclear pattern of EMF1a (Fig. 4d). Y2H experiments demonstrated that both RING1 and MSI1 interacted with the N-terminal domain of EMF1a, but not with the C-terminal domain (Fig. 4e).

ZmEMF1a knockout leads to a genome-wide reduction in H2Aub and H3K27me3 modification

In eukaryotes, PcG proteins play important roles in maintaining gene silencing, which is involved in cellular and developmental processes [2, 3]. The major protein complex, PRC2, possesses H3K27 tri-methyltransferase activity, while PRC1 has histone H2A E3 ubiquitin ligase activity [5, 9]. In Arabidopsis, EMF1 plays a crucial role in H3K27me3 deposition; loss of EMF1 function results in a genome-wide reduction of H3K27me3, but does not affect the H2Aub modification compared to the WT [57]. H2AK119ub in mammals and H2AK121ub in Arabidopsis are observed within the consensus sequence PKKT [4, 5]. One of the maize H2A isoforms shows high similarity with both human H2A and Arabidopsis H2A and conserves the monoubiquitination PKKT sequence (Fig. S5a). Immunoblotting analysis with a commercial antibody revealed that H2Aub and H3K27me3 antibodies recognize target-sized bands in maize endosperms (Fig. S5b).

To investigate whether the ZmEMF1a mutation affects H3K27me3 and H2Aub modifications, we performed Western blotting on nuclear proteins isolated from both WT and mn8 endosperms at 12 DAP using anti-H3K27me3 and anti-H2Aub antibodies. Unexpectedly, although we observed a significant decrease in H3K27me3 levels in mn8 compared to the WT, as previously described in emf1-2 mutant of Arabidopsis, we also found markedly reduced levels of H2Aub in mn8 (Fig. 5a). In contrast, no difference was detected in emf1-2 compared to the WT in previous studies [57]. To further characterize the modification levels of H3K27me3 and H2Aub in the genome, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) to map the genome-wide localization of H3K27me3 and H2Aub marks in both WT and mn8 endosperm at 12 DAP. We obtained 66 to 125 million raw reads from each library, over 97% of these reads aligned to the B73 genome, and the Pearson correlation coefficients were high (Additional file 1: Fig. S6a), indicating a high mapping quality. After peak-calling, we found that the peak-marked genes showed a high degree of overlap between the two repetitions (Additional file 1: Fig. S6b). We then assessed two biological replicates for further analysis. Widespread localization of H2Aub marks has been reported in Arabidopsis and animals, the impact of this modification in maize is not yet fully understood. Distribution analysis of H3K27me3 and H2Aub peaks showed that the preferred location of H3K27me3 was intergenic regions, followed by transposable element (TE) regions. However, about 44% H2Aub peaks were usually located in the first exon of genes, and 29% H2Aub peaks located in TE regions (Fig. 5b). When analyzed the average genomic modification levels of H2Aub and H3K27me3, we also found reduced levels of H2Aub and H3K27me3 in mn8, which is consistent with the result of western blot (Fig. 5c, d).

A metagene plot of H2Aub coverage at H2Aub-marked genes showed a significant reduce of H2Aub in mn8 compared with WT (Fig. 5e). We next analyzed the coverage at H2Aub/H3K27me3 and only-H2Aub genes separately, and the results showed that both H2Aub/H3K27me3 and only-H2Aub genes with reduced levels of H2Aub in mn8 compared to WT (Fig. 5e). We found reduced levels of H3K27me3 in mn8 at H3K27me3/H2AK121ub-marked and only-H3K27me3 genes, as previously reported in Arabidopsis (Fig. 5f) [57]. Peak length analysis showed that H2Aub peaks were significantly shorter than H3K27me3 peaks, covering on average 0.7 kb and 2.3 kb, respectively (Fig. 5g). In addition to this, we found that only 21% of the H2Aub-marked genes were overlapped with H3K27me3-marked genes (Fig. 5h), and the H2Aub levels of H3K27me3/H2Aub-marked genes were higher than that of only-H2Aub-marked genes (Fig. 5i, j). These results showed that both H2Aub and H3K27me3 levels were significantly decreased in mn8 compared to WT in maize, which suggests that EMF1 regulates the expression of maize kernel development-related genes by modulating H2Aub and H3K27me3 modifications..

The level of H2Aub is negatively correlated with gene expression

Previous research revealed that H3K27me3 is a repressive in Arabidopsis, while H2Aub is positively correlated with gene expression [11, 58]. To determine the relationship between H3K27me3 and H2Aub modifications and gene transcription, we performed RNA-seq on WT and mn8 endosperm at 12 DAP, using the same tissue as that used in the ChIP-seq experiments (Additional file 1: Fig. S7a, b). From the RNA-seq analysis, we identified 5,604 significantly differentially expressed genes (DEGs) based on a differential expression threshold (P-value < 0.05 and absolute fold change > 1.0). In the mn8 mutant, significantly more genes were up-regulated than down-regulated (Fig. 6a), which strongly suggests that EMF1a functions in gene repression. We then divided all the protein-coding genes into three classes based on their expression level: low, medium and high expression (Fig. 6b), and analyzed the deposition of H3K27me3 and H2Aub. The results showed that the levels of H3K27me3 and H2Aub modifications gradually decrease as gene expression levels increase (Fig. 6c, d). The plots of the H3K27me3 and H2Aub abundance across these three classes revealed that both modifications were highly enriched around genes with low expression and less enriched around those with high expression (Fig. 6e, f).

To further confirm the inhibitory effect of H2Aub on gene expression, We classified H2Aub-marked genes into five categories based on their modification levels. We then determined the mean expression levels of genes within each category, and the result showed that genes not marked by H2Aub had a higher expression level compared to those that were H2Aub-marked. Moreover, as the levels of H2Aub modification increased, the levels of gene expression correspondingly decreased (Fig. 6g, h). H3K27me3 is a repressive mark, and most of the genes marked only by H3K27me3 (only-H3K27me3-marked) were not expressed or showed very low expression levels (Fig. 6i). In the case of H2Aub-marked genes, about 60% showed either no or low expression (Fig. 6i). At the genome-wide level, genes marked by H2Aub or H3K27me3 (H2Aub-marked or H3K27me3-marked) showed significantly lower expression levels than all the expressed genes. Moreover, genes marked by both H2Aub and H3K27me3 (H2Aub/H3K27me3-marked) exhibited lower expression than those marked only by H2Aub (only-H2Aub-marked), but this difference was not observed in only-H3K27me3-marked genes. This suggests that the repressive effect of H2Aub is weaker than that of H3K27me3 (Fig. 6j).

ZmEMF1a is required for the expression of genes related to kernel development

The absence of the EMF1a gene results in the upregulation of a large number of genes. Through Gene ontology (GO) enrichment analysis, it was observed that these up-regulated genes are associated with various biological processes, including response to hormones like abscisic acid, auxin, and brassinosteroid, reproductive process, transcription factor activity, and seed development (Fig. 7a). Considering the established roles of H3K27me3 and H2Aub in the repression of transcription, we conducted an analysis to assess the enrichment of these modifications among genes that were up-regulated in mn8. Our findings showed that the levels of H3K27me3 were significantly reduced in the up-regulated genes of mn8 when compared to their WT counterparts (Fig. 7b). In contrast, the levels of H2Aub among these up-regulated genes exhibited minimal variance between mn8 and WT. These observations suggest that the mutation of EMF1a is primarily responsible for the up-regulation of genes, attributable to the diminished levels of H3K27me3 modification (Fig. 7b). Moreover, EMF1a mutation also increased the expression of cell division-related genes, such as DA1, BB1, BB2, MADS8, MADS14 and bZIP75. In addition, GSK2, a homologous gene of BIN2, down-regulation of GSK2 expression levels resulted in long and heavy grains, was also up-regulated in mn8. ES22, encoding a MADS-type transcription factor, negatively regulated starch accumulation, was significantly up-regulated in mn8 (Fig. 7c). Five of these negative regulators were analyzed using RT-qPCR, and the results were consistent with the RNA-seq analysis of the WT and mn8 transcriptomes (Fig. 7d). We utilized the ChIP-seq Genome Browser to view H2AK121ub and H3K27me3 occupancy of selected genes, and we found that the level of H3K27me3 modification of ZmDA1 was significantly higher in the WT than in the mn8, and similar reduction of H2K27me3 levels in MADS8, MADS14 and ES22 were found (Fig. 7g, h; Additional file 1: Fig. S9a, b). In mn8 mutants, H2Aub levels in the BB1 locus were significantly reduced, suggesting that EMF1a is required for H2Aub labeling in the BB1 region (Fig. 7i). In addition this, we further confirmed the modification levels of the selected genes by ChIP-qPCR analysis (Fig. 7e, f), consistent with their increased transcriptional levels. Taken together, EMF1a is important to maintain the enrichment of H2Aub and H3K27me3 during cell proliferation, which is essential for maize kernel development.

PcG complexes play important roles in the regulation of eukaryotic gene expression. Two major members of the PcG complex, PRC1 and PRC2, catalyze the formation of H2Aub as well as H3K27me3 modifications, respectively, are associated with transcription repression [59–61]. Suppressor of zeste 12 (SUZ12), a subunit of PRC2, is necessary for the catalytic activity of PRC2 [62]. Mutation of Suz12 in mice, the modifications of H3K27me2 and H3K27me3 are reduced drastically in Suz12 null embryos, resulting in a dramatic decrease in cell proliferation and an increase in apoptosis [63]. EMF2, a Suz12 homologs in Arabidopsis, mutations in EMF2 lead to a decrease in the levels of H3K27me3 mark present in the chromatin of SOC1 and FT, which affects the vegetative and flower development [16, 64].

EMF1, the plant specific protein, was proposed to be a member of PRC1 for the chromatin compaction in vitro [36, 41]. In recent years, it has been found that EMF1 interacts with PRC2 members, and is necessary for H3K27me3 marking [37, 40]. In this study, the classic maize kernel mutant mn8 was cloned and the maize Mn8 gene encodes the PRC1 component ZmEMF1a. In plants, EMF1 is involved in repressing both the vegetative to reproductive transition and flower initiation. In Arabidopsis, the strong emf1-2 mutant exhibits a more severe phenotype than weak emf1-1 mutant, all lateral organs differentiate in carpelloid structures. In rice, loss-of-function mutations in EMF1-like gene CURVED CHIMERIC PALEA (CCP1) do not exhibit severe phenotype as emf1 mutants in Arabidopsis [65]. Compared with the WT, ccp1 displays decreased plant height, panicle length and seed setting rate but increased tilled. Previously published RNA sequencing (RNA-seq) data showed that ZmEMF1a was constitutively expressed throughout maize development but highest in endosperm and seeds [55]. Mutation of the EMF1a gene in maize resulted in smaller kernels but did not affect the height of mature plants, indicating different regulatory roles of EMF1 in plant growth and development.

Previous studies have shown that EMF1 plays an important role for the deposition of H3K27me3, and the emf1-2 mutant showed a decreased levels of H3K27me3 at gene body region. However, the average H2Aub signal levels of emf1-2 has no significant difference at both H2Aub/H3K27me3 and onlyH2Aub marked genes, compared with WT [66]. Interestingly, despite we found decreased levels of H3K27me3, the levels of H2Aub modification were also significantly reduced in mn8 mutant at both H2Aub/H3K27me3 and only-H2Aub marked genes. Widespread localization of H2Aub marks in animals and Arabidopsis has been recently reported, but the genome distribution of H2Aub in maize is not yet studied. Distribution analysis of H2Aub peaks across the genome showed that the preferred location of H2Aub was the exon of genes, followed by TE regions (Fig. 5b). Loss of EMF1a in maize leaded to a decrease in the proportion of peak in gene body region, and an increase in the proportion of TE and intergenic region (Fig. 5b).

In Drosophila, histone H2A monoubiquitination occurs mainly in the promoter region and represses the expression of target genes [5]. Recent study demonstrated that deposition of H2AK119ub1 is essential for maintaining repression of PcG target genes in embryonic stem cells (ESCs) with a fully catalytic inactive RING1B mutant [61]. In Arabidopsis, 60% of only-H2AK121ub genes were transcriptionally active, suggested that H2AK121ub might play a role in transcriptional activation [11]. In PRC1 mutants, the proportion of up-regulated genes was consistently higher than that of down-regulated genes only for PRC1-dependent genes, which indicated that H2Aub unlikely repressed genes directly [58]. To better understand the regulation roles of H2Aub for the gene regulation in maize, we performed ChIP-seq and RNA-seq using the same tissue. Surprisingly, 60% of only-H2Aub marked genes were lowly or not expressed genes, nearly 83% of only-H3K27me3 marked genes were lowly or not expressed genes. Genes with or without H2Aub were subdivided based on their levels of H2Aub. Genes that H2Aub-marked showed a lower expression than that of none-H2Aub-marked genes, and with increasing levels of H2Aub modification, genes expression gradually decreased. And the expression level of H3K27me3-marked genes was significantly lower than those of H2Aub-marked genes, the H2Aub/H3K27me3-marked genes also showed a lower expression than those of H2Aub-marked genes. Our data suggest that H2Aub is a repressive marker, but its repressive effect on gene expression is weaker than that of H3K27me3.

Few components of the ubiquitin pathway have been detected to play important roles in seed and organ size determination on plants. The ubiquitin receptor DA1 inhibits seed growth by restricting cell proliferation [49]. Genetic analyses demonstrate that DA1, TCP14 and TCP15 function in a common pathway to regulate cell division. E3 ligases EOD1, also known as BB, function independently synergistically with DA1 to regulate seed and organ size [49, 67]. We identify ZmDA1, ZmBB1 and ZmBB2 were significantly up-regulated in mn8 endosperms. Besides, the expression of both genes MADS8 and MADS14, homologs of OsMADS1 in maize, was up-regulated in mn8. OsMADS1 is best known for its negative regulatory role in the regulation of grain growth, mutations in the last intron of OsMADS1 cause splicing defects and produce long grains. In addition to this, we also found some other negative regulators of seed grain development was increased in mn8, such as WTG1, GSK2, RAV1, bZIP75 and ES22. The up-regulation of these genes was accompanied by a decrease in the level of H2Aub or H3K27me3 modifications. Collectively, our results demonstrate a crucial role of EMF1a in the regulation of maize kernel development through maintaining the modifications of H2Aub and H3K27me3.

Plant materials

mn8-1 mutant was isolated in the course of an EMS-induced mutant screen aiming at the cloning of mutants with defects in kernel development. The mutants were backcrossed into the B73 genetic background for over three times to eliminate potential mutations other than the mn8 mutation induced by the EMS treatment, meanwhile it was outcrossed with the inbred line Mo17. All maize plants were grown under natural conditions in the experimental field at Shangzhuang, China Agricultural University, Beijing. Immature kernels were harvested at 6, 8, 12, 15 and 24 DAP. Nicotiana benthamiana seeds were grown in a growth chamber under a photoperiod of 16 h:8 h (light:dark) and at a temperature of 22–23°C for germination.

Light microscopy, scanning electron microscopy and transmission electron microscopy

For light microscopy analysis, immature mn8 and B73 kernels were freshly collected and fixed overnight in formalin–acetic acid–alcohol (FAA) (50% [v/v] ethanol, 5% [v/v] acetic acid, and 3.7% [v/v] formaldehyde) and then dehydrated with gradient ethanol (50%, 70%, 85%, 95%, and 100% ethanol in water [v/v]) and substituted with gradient xylene solution (50%, 75% and 100% xylene in ethanol [v/v]). After infiltration in paraffin three times at 60°C, the samples were embedded in paraffin (P3683, Sigma-Aldrich) and sectioned into 10-µm slices using a microtome (RM2265, Leica). The sections were then dewaxed in xylene, rehydrated in a graded series of ethanol, and stained with fuchsin. Images were taken using a Leica microscope equipped with a digital camera.

For transmission electron microscopy analysis, mn8 and B73 endosperms at 15 DAP were freshly collected and prepared as previously described [68]. The sections were observed using Hitachi H7600 transmission electron microscope (Japan). For scanning electron microscopy analysis of starch grains, mature mn8 and B73 seeds were cut with a razor blade, sputter-coated with gold and observed by scanning electron microscope (S-3400N, Hitachi, Japan).

Measurement of protein and starch contents

For protein content measurements, mature B73 and mn8 kernels were collected from the same segregating ears and the endosperm samples were ground to a fine powder in liquid nitrogen. The total protein content was measured following the established methods detailed in prior study [69]. The protein levels were quantified using the BCA protein assay kit (Beyotime, P0006C) according to the supplied protocols. Total starch was measured according to the manufacturer's instructions using an amyloglucosidase/a-amylase starch assay kit (Megazyme).

Genetic Mapping of mn8 Locus

For the initial mapping of mn8, DNA samples isolated from 30 miniature kernels from segregating F₂ population were used for preliminary pooling, and the genotypes were confirmed with 44 genetic markers that were spaced roughly every 25 Mb apart across the 10 chromosomes. Genetic linkage with four markers on chromosome 10 (10-4.05, 10–29, 10–59 and 10-104.2) was determined. The addition F₂ population of 600 mutants was genotyped with this four markers and the region between 10–59 Mb and 10-104.2 Mb that contained the gene of interest was identified. Furthermore, the markers 10–87 and 10–97 were used to genotype an additional 1500 miniature kernel DNA samples, and the mutant locus was narrowed down the interval to a 10-Mb. 14 markers in this interval were developed to increase marker density and 62 recombinants were genotyped with these 14 markers. mn8 was localized to a 420-kb interval, as determined by analysis with the markers 10-88.68 and 10-89.1.

Maize Transformation

Transgenic plants were generated by Agrobactium tumefaciens–mediated maize transformation [70]. For CRISPR/Cas9 editing plants, sequencing was used to identify editing sites near the target position. One transgenic line with Zm00001d024813 knockout was obtained via CRISPR/Cas9, and it was selected for allelism tests.

Phylogenetic analysis

By performing a BLASTp search of the the full-length EMF1a protein sequence, we identified relevant homologs sequences from the NCBI nonredundant protein sequences database. The amino acid sequences were aligned by employing ClustalX2 [71], followed by the creation of a phylogenetic tree with the application of MEGA 7 software, which was executed using the neighbor-joining method [72].

RNA extraction and RT-qPCR

Total RNA was extracted from 12 DAP endosperm from mn8 and B73 using TRIzol reagent (Ambion) and reverse transcribed into cDNA with HiScript II Q RT SuperMix along with DNA elimination (Vazyme, R223). Then quantitative real-time PCR assays were performed with 2 × Ultra SYBR Mixture (CWBIO) on an ABI 7500 Real-Time PCR System (Applied Biosystem). The PCR conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The gene expression levels were determined by the ΔCt (threshold cycle) method, utilizing ZmActin7 gene as the internal control for normalization. Primer sequences for RT-qPCR are listed in Dataset S3.

Subcellular localization of the EMF1a protein

The full-length ORF of EMF1a without the stop codon was cloned into the pUC-GFP vector through a process involving restriction enzyme cleavage (BamHI and XhoI) followed by ligation (the primers used are listed in Dataset S3). The AHL22 gene, integrated with the RFP tag, was constructed as a positive control. Maize protoplasts were isolated and transformed based on a previously described protocol [73]. After transformation, the protoplasts were incubated in the dark for 16–20 h at 28°C, after which the fluorescence signals were observed utilizing a confocal laser microscope (Zeiss 880, Germany).

Yeast two-hybrid assays

To analyze the interaction between the EMF1a and PRC1 or PRC2 subunits, the coding sequences of PRC1 or PRC2 subunits were cloned into a pGBKT7 vector, the coding sequence of the EMF1a was cloned into the pGADT7 vector, and the resulting vectors were transformed into the yeast strain AH109. Subsequently, SD/-Leu/-Trp medium and SD/-His/-Leu/-Trp medium were used to detect the interactions.

Luciferase complementation image assays

To analyze the interaction between the EMF1a and PRC1 or PRC2 subunits, the full-length ORF of the PRC1 or PRC2 subunits were cloned into a pCAMBIA-nLuc vector, and the coding sequence of EMF1a was cloned into the pCAMBIA-cLuc vector. These constructs were transfected into Agrobacterium strain GV3101, and the transformants were then infiltrated into 6-week-old N. benthamiana leaves following the established method [74]. After incubation for 48 h under the condition of 16 h:8 h (light:dark), the leaves were incubated with 1 mM luciferin (Promega), and the luciferase signals were collected using a low-light cooled charge-coupled device camera.

Bimolecular fluorescence complementation assays

To confirm the interaction between EMF1a and RING1 or MSI1, the CDS of EMF1a was cloned into pSPYNE(R)173 vector, and the coding sequences of RING1 and MSI1 were cloned into pSPYCE(M) vector. The resulting constructs were transformed into Agrobacterium strain EHA105 and then infiltrated into N. benthamiana leaves. The YFP signals were monitored by confocal microscopy (Zeiss880; Carl Zeiss) with an excitation wavelength of 488 nm.

RNA-seq and data analysis

Total RNA was extracted from mn8 and B73 endosperms obtained from the same F₂ ears at 12 DAP (30 endosperms per replicate). Three biological replicates were collected from the kernels of three independent ears. Total RNA was extracted with TRIzol reagent (Ambion), and DNA was digested with RNase free DNase I (NEB). cDNA libraries were constructed using a VAHTS mRNA-seq Library Prep Kit (Vazyme) and sequenced using an Illumina NovaSeq 6000 (BerryGenomics). Read mapping and analysis were performed in accordance with a previously described method [75]. Significant DEGs were identified with an adjusted P value < 0.05 and log2 (|fold change|) ≥ 1 using DESeq2 software in R (v.1.30.0, R package) [76]. GO enrichment was performed via the AgriGO (version 2.0) online program [77].

ChIP-seq and ChIP-qPCR

Two biological replicates of mn8 and B73 endosperm tissues were obtained from two independent F₂ ears at 12 DAP. ChIP-seq with Anti-H2Aub and anti-H3K27me3 were performed as previously described [56]. Briefly, nuclei (from 2.0 g of fresh tissue per sample) were lysed with nuclear lysis buffer (50 mM Tris-HCl [pH 7.5], 10 mM EDTA [pH 8.0], 1% SDS, protease inhibitor and phosphatase inhibitor) and subjected to high-speed sonication at 200–600 bp using a Bioruptor instrument. Anti-H2Aub (Abcam,) and anti-H3K27me3 (Abcam,) antibodies were used for chromatin immunoprecipitation. The chromatin solutions were pre-cleared with 50 µL ChIP-grade protein A/G beads (Millipore) for 2 h at 4°C. The mixture was then incubated overnight with 5 µg antibody at 4°C, followed by incubation with 20 µL ChIP-grade protein A/G beads for 2 h at 4°C on a rotating tube shaker. The beads were washed four times using wash buffer (20 mM Tris-HCl [pH 7.5], 2 mM EDTA [pH 8.0], 1% Triton X-100, 0.1% SDS and 500 mM NaCl) and eluted with elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA [pH 8.0], 1% SDS, 10 µg RNase A). The prepared DNA was then recovered using a QIAquick PCR purification kit (Qiagen), and quality was checked using the Qubit (Thermo Fisher). The DNA libraries were constructed using a VAHTS DNA Library Prep Kit (Vazyme) and sequenced on the NovaSeq 6000 platform with 150-bp paired-end reads (BerryGenomics).

For ChIP-qPCR analysis, the obtained DNA from ChIP was diluted and analyzed using specific DNA primers (the primers used are listed in Dataset S3). The qPCR was performed using 2×Ultra SYBR Mixture (CWBIO) on a 7500 Real-Time PCR System (Applied Biosystem).

ChIP-seq data analysis

Raw fastq data were trimmed using Trim Galore (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapters and low-quality bases, and quality control was then performed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were mapped to the maize reference genome B73 (AGPv4) using Bowtie2 [78, 79], and duplicated reads were removed for subsequent analysis. A correlation analysis between biological replicates was performed using deepTools [80]. Peak calling was performed separately for each biological replicate using MACS2 [81] with a q-value cutoff of 0.01. The peaks were transformed into bigWig files using the bamCoverage tool from the deepTools, a crucial step for visualization purposes in the Integrative Genomics Viewer (IGV) [82]. Peak annotation or peak-related genes were performed using bedtools intersect. Normalized fold enrichment profiles were created by employing the callpeak function with the -SPMR flag, and then feeding the resulting bedgraph outputs into the bdgcmp function, utilizing the setting -m FE. Metagene plots illustrating the coverage of H2Aub and H3K27me3 marks at specific genomic loci between the B73 and mn8 were developed using the computeMatrix and plotProfile programs.

Acknowledgements

This research was supported by National Natural Science Foundation of China

(31901496, 32272143, 31971959, 32271541 and 62031003), The Science and Techonology Innovation 2030-Major Project (2022ZD04020), China Postdoctoral Science Foundation (2020M670535), National Postdoctoral Program for Innovative Talents (BX20190376).

Conflict of interest

The authors declare no competing interests.

Author contributions

Z.Y., H.Z., Y.Z. and J.L. designed the experiments; Z.Y., J.L., X.L, C.L. and Y.L. performed the experiments; J.L., Y.Z., Y. L., X.L., and T.L. performed the data analysis; Z.Y. and J.L. wrote the paper; J.L., Z.Y., H.Z., W.S., J.C. and J.L. reviewed and discussed the results.

Data availability

All supporting data from this study are available in the article and Supplementary Information files, or from the corresponding author upon reasonable request. The RNA-seq and ChIP-seq data associated with this study have been deposited in the NCBI SRA under the Accession Number PRJNA1149556 and PRJNA1152525.

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Datasets 1 to 3 are not available with this version.

Dataset1 Genes used in the yeast two-hybrid experiments.

Dataset2 DEGs in mn8.

Dataset3 Primers used in this study.

No competing interests reported.

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ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development

Status:

Version 1

Abstract

Figures

Introduction

Results

ZmEMF1a interacts with PRC1 and PRC2 components

ZmEMF1a knockout leads to a genome-wide reduction in H2Aub and H3K27me3 modification

The level of H2Aub is negatively correlated with gene expression

ZmEMF1a is required for the expression of genes related to kernel development

Discussion

Methods

Plant materials

Light microscopy, scanning electron microscopy and transmission electron microscopy

Measurement of protein and starch contents

Maize Transformation

Phylogenetic analysis

RNA extraction and RT-qPCR

Subcellular localization of the EMF1a protein

Yeast two-hybrid assays

Luciferase complementation image assays

Bimolecular fluorescence complementation assays

RNA-seq and data analysis

ChIP-seq and ChIP-qPCR

ChIP-seq data analysis

Declarations

References

Datasets

Additional Declarations

Supplementary Files

Status:

Version 1