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

ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development Yueheng Zhou, Jianrui Li, Yingshuang Li, Xiaojie Li, Chunlei Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4998315/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract 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 mn8 endosperms, 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. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction 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. Results 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 (χ 2 = 0.013–0.215 < χ 2 0.05 = 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. 1 a). Compared with WT (B73) siblings, mn8 kernels were significantly smaller (Fig. 1 b, c). During sectioning of the kernels, both embryo and endosperm were markedly reduced in mn8 kernels compared with B73 (Fig. 1 d). When measured, the single kernel weight was significantly reduced than that of the WT (Fig. 1 e), and the embryo length was also found to be significantly shorter than that of the WT (Fig. 1 j). 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. 1 f-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. 1 k, l). The mn8 mutant seedlings were slight shorter than WT at the 7th day after germination (Additional file 1: Fig. S1 a). However, there was no significant difference in plant height at sexual maturity stage between the mn8 mutant and WT (Additional file 1: Fig. S1 b, 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. S1 d, 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. 2 a, 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. 2 i, 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. 2 b, 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. 2 j, o). At 12 DAP, the degeneration of maternal tissue introduced a gap between the endosperm and the pericarp in mn8 (Fig. 2 a, c, g). This gap enlarged during the later development stage of the mutant seed until maturity (Fig. 2 d, 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. 2 k, 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. 2 l, 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 F 1 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. 3 a). 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. 3 b). 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. 3 c). 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. 3 d, 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. 3 e, 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. 4 a). 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. 4 b). 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. 4 c). 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. 4 d). Y2H experiments demonstrated that both RING1 and MSI1 interacted with the N-terminal domain of EMF1a, but not with the C-terminal domain (Fig. 4 e). 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. 5 a). 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. 5 b). 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. 5 c, d). A metagene plot of H2Aub coverage at H2Aub-marked genes showed a significant reduce of H2Aub in mn8 compared with WT (Fig. 5 e). 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. 5 e). We found reduced levels of H3K27me3 in mn8 at H3K27me3/H2AK121ub-marked and only-H3K27me3 genes, as previously reported in Arabidopsis (Fig. 5 f) [ 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. 5 g). In addition to this, we found that only 21% of the H2Aub-marked genes were overlapped with H3K27me3-marked genes (Fig. 5 h), and the H2Aub levels of H3K27me3/H2Aub-marked genes were higher than that of only-H2Aub-marked genes (Fig. 5 i, 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 1.0). In the mn8 mutant, significantly more genes were up-regulated than down-regulated (Fig. 6 a), 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. 6 b), 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. 6 c, 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. 6 e, 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. 6 g, 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. 6 i). In the case of H2Aub-marked genes, about 60% showed either no or low expression (Fig. 6 i). 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. 6 j). 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. 7 a). 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. 7 b). 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. 7 b). 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. 7 c). 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. 7 d). 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. 7 g, 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. 7 i). In addition this, we further confirmed the modification levels of the selected genes by ChIP-qPCR analysis (Fig. 7 e, 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. Discussion 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. 5 b). 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. 5 b). 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. Methods 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 F 2 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 F 2 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 ( Bam HI and Xho I) 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 F 2 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 F 2 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. Declarations 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. 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Plant Cell. 2019; 31(2):465-485. Feng F, Qi W, Lv Y, Yan S, Xu L, Yang W, et al. OPAQUE11 Is a Central Hub of the Regulatory Network for Maize Endosperm Development and Nutrient Metabolism. Plant Cell. 2018; 30(2):375-396. Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, et al. Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol. 2002; 129(1):13-22. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007; 23(21):2947-2948. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016; 33(7):1870-1874. Qi X, Li S, Zhu Y, Zhao Q, Zhu D, Yu J. ZmDof3, a maize endosperm-specific Dof protein gene, regulates starch accumulation and aleurone development in maize endosperm. Plant Mol Biol. 2017; 93(1-2):7-20. Chen YF, Li LQ, Xu Q, Kong YH, Wang H, Wu WH. The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis . Plant Cell. 2009; 21(11):3554-3566. Yi F, Gu W, Chen J, Song N, Gao X, Zhang X, et al. High Temporal-Resolution Transcriptome Landscape of Early Maize Seed Development. Plant Cell. 2019; 31(5):974-992. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12):550. Tian T, Liu Y, Yan H, You Q, Yi X, Du Z, et al. agriGO v2.0: a GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Res 2017; 45(W1):W122-W129. Jiao Y, Peluso P, Shi J, Liang T, Stitzer MC, Wang B, et al. Improved maize reference genome with single-molecule technologies. Nature. 2017; 546(7659):524-527. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-359. Ramirez F, Ryan DP, Gruning B, Bhardwaj V, Kilpert F, Richter AS, et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016; 44(W1):W160-W165. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008; 9(9):R137. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotecnol. 2011; 29(1):24-26. Datasets 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. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Dec, 2024 Reviews received at journal 30 Nov, 2024 Reviewers agreed at journal 15 Nov, 2024 Reviews received at journal 23 Sep, 2024 Reviewers agreed at journal 08 Sep, 2024 Reviewers invited by journal 05 Sep, 2024 Editor assigned by journal 05 Sep, 2024 Submission checks completed at journal 30 Aug, 2024 First submitted to journal 29 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4998315","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":359234526,"identity":"acdcd4b3-e575-47ba-ab7a-74d827af9dc2","order_by":0,"name":"Yueheng Zhou","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yueheng","middleName":"","lastName":"Zhou","suffix":""},{"id":359234527,"identity":"f29ac9db-8368-4263-b3f2-4f6c1fef644c","order_by":1,"name":"Jianrui Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jianrui","middleName":"","lastName":"Li","suffix":""},{"id":359234528,"identity":"84fc40d8-d733-41c0-b2a2-b01dc5857d6b","order_by":2,"name":"Yingshuang Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yingshuang","middleName":"","lastName":"Li","suffix":""},{"id":359234529,"identity":"6e2fbdef-8995-47b0-a8b6-f07df35d8a97","order_by":3,"name":"Xiaojie Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaojie","middleName":"","lastName":"Li","suffix":""},{"id":359234530,"identity":"d7688ca5-a8e9-4313-a3dc-f9c14baf60d8","order_by":4,"name":"Chunlei Wang","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chunlei","middleName":"","lastName":"Wang","suffix":""},{"id":359234532,"identity":"196c239b-00d5-4cf5-9479-06b0ed8b84e9","order_by":5,"name":"Tong Li","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Li","suffix":""},{"id":359234536,"identity":"da78b5a5-6fde-4b83-af60-947f5051e822","order_by":6,"name":"Jian Chen","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Chen","suffix":""},{"id":359234542,"identity":"b16f0660-366a-439e-ae55-bb81c580f440","order_by":7,"name":"Weibin Song","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Weibin","middleName":"","lastName":"Song","suffix":""},{"id":359234543,"identity":"21142e6d-51da-4223-bb0c-984421b4c419","order_by":8,"name":"Jinsheng Lai","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jinsheng","middleName":"","lastName":"Lai","suffix":""},{"id":359234544,"identity":"7107f26a-bae6-4cfd-ad0c-a0070b1876c4","order_by":9,"name":"Haiming Zhao","email":"","orcid":"","institution":"China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haiming","middleName":"","lastName":"Zhao","suffix":""},{"id":359234545,"identity":"a25942e5-1f3b-4864-8d45-d721f5c4fefd","order_by":10,"name":"Zhijia Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYLACxgYJZjYG5gMQ3gHitbAlkKQFRPIYEKfF4PjZwy9/7rBg52M/8/HDzzYGOb4bCYyfC/BpOZOXZiF5BugwntzNkr1tDMaSNxKYpWfg03Igx8zAsA3kl9xtDLxtDIkbbiSwMfPg03L+jZlBIkgL/5tnjH/bGOoJa7mRY/zgIEiLRA4bM9CWBANCWiRvvDFjbARreWYsLXNOwnDmmYfN0vi08J3PMf74s60uWb4/+eHHN2U28nzHkw9+xqdF4QADmwSQTobyQWxINOEE8g0MzB+AtB1eVaNgFIyCUTCyAQDPMUkizhagugAAAABJRU5ErkJggg==","orcid":"","institution":"China Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Zhijia","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-08-29 14:08:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4998315/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4998315/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":65668175,"identity":"48c38cdb-d875-4192-ac48-15d32aea920c","added_by":"auto","created_at":"2024-10-01 06:37:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1753010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic and cytological analyses of maize \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emn8\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e kernels. (a)\u003c/strong\u003e Mature heterozygous F\u003csub\u003e2\u003c/sub\u003e ear of the \u003cem\u003emn8\u003c/em\u003e mutant. The arrows indicate \u003cem\u003emn8 \u003c/em\u003ekernels. \u003cstrong\u003e(b)\u003c/strong\u003e Comparison of the width and length of the mature B73 and \u003cem\u003emn8 \u003c/em\u003ekernels from a segregated F\u003csub\u003e2\u003c/sub\u003e ear\u003cem\u003e. \u003c/em\u003eBar = 1 cm.\u003cstrong\u003e (c) \u003c/strong\u003eB73 and \u003cem\u003emn8\u003c/em\u003e mature kernels viewed on a light box. Bar = 1 cm. \u003cstrong\u003e(d)\u003c/strong\u003e Longitudinal sections of mature B73 and \u003cem\u003emn8 \u003c/em\u003ekernels. Bar = 1 cm. \u003cstrong\u003e(e) \u003c/strong\u003eComparison of the single kernel weight of randomly selected mature B73 and\u003cem\u003e mn8\u003c/em\u003e kernels from segregated F\u003csub\u003e2\u003c/sub\u003e ears. The values are the means ± SE; n = 3 (***, P \u0026lt; 0.001, Student’s\u003cem\u003e t\u003c/em\u003e test).\u003cstrong\u003e (f),\u003c/strong\u003e \u003cstrong\u003e(g) \u003c/strong\u003eScanning electron microscopy images of the central starchy endosperm of mature B73 and \u003cem\u003emn8\u003c/em\u003e. Bars = 20 μm. SG, starch granule. \u003cstrong\u003e(h), (i) \u003c/strong\u003eTransmission electron microscopy images of B73 and \u003cem\u003emn8\u003c/em\u003e endosperms\u003cem\u003e \u003c/em\u003eat 15 DAP. Bars = 5 μm. SG, starch granule; PB, protein body. \u003cstrong\u003e(j) \u003c/strong\u003eEmbryo length (mm) of the B73 and \u003cem\u003emn8\u003c/em\u003e mutants. \u003cstrong\u003e(k)\u003c/strong\u003e Starch content of mature B73 and\u0026nbsp;\u003cem\u003emn8\u003c/em\u003e\u0026nbsp;endosperm relative to the kernel weight.\u003cstrong\u003e \u003c/strong\u003eThe values are the means ± SE; n = 3 (***, P \u0026lt; 0.001, Student’s \u003cem\u003et\u003c/em\u003e test). \u003cstrong\u003e(l)\u003c/strong\u003e Comparison of total protein content per kernel between mature B73 and \u003cem\u003emn8\u003c/em\u003e endosperms. The values are the means ± SE; n = 3 (***, P \u0026lt; 0.001, Student’s \u003cem\u003et\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/50ef0080a6a48bc098e75821.png"},{"id":65668174,"identity":"8ad8e032-6bf9-4a65-a006-0baeb1a9515b","added_by":"auto","created_at":"2024-10-01 06:37:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1947141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLongitudinal sections of B73 and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003emn8\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e kernels in maize. (a-h) \u003c/strong\u003eComparison\u003cstrong\u003e \u003c/strong\u003eof B73 and \u003cem\u003emn8 \u003c/em\u003ekernels at 6, 8, 12 and 15 d after pollination (DAP). (a-d) B73 kernels; (e-h) \u003cem\u003emn8\u003c/em\u003e kernels; Bar = 1 mm. \u003cstrong\u003e(i-r) \u003c/strong\u003eComparison\u003cstrong\u003e \u003c/strong\u003eof B73 and \u003cem\u003emn8 \u003c/em\u003eembryos at 6, 8, 12, 15 and 24 DAP. (i-m) B73 embryos; (n-r) \u003cem\u003emn8\u003c/em\u003eembryos; Bars = 100 μm in (i, j, n, o), and 1 mm in (k, l, m, p, q, r).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/2a0f967a07bc80979aa9e6cf.png"},{"id":65667836,"identity":"27ea5215-e237-4f5f-95bb-bfa50630dee6","added_by":"auto","created_at":"2024-10-01 06:29:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1410185,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMap-based cloning and validation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMn8. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e(a) \u003c/strong\u003eDiagram of the region identified by map-based cloning of \u003cem\u003emn8\u003c/em\u003e mutations. The \u003cem\u003eMn8\u003c/em\u003e locus was mapped to a region between markers 88.68 Mb and 89.1 Mb on chromosome 10. \u003cstrong\u003e(b)\u003c/strong\u003e Gene structure of \u003cem\u003eMn8\u003c/em\u003e and the location of the mutations in \u003cem\u003emn8\u003c/em\u003e and \u003cem\u003emn8C1\u003c/em\u003e. Exons are black boxes and introns are lines. \u003cstrong\u003e(c)\u003c/strong\u003e The sequence in the\u0026nbsp;\u003cem\u003eZm00001d024813\u003c/em\u003e\u0026nbsp;locus targeted using CRISPR/Cas9 and comparison of the width and length of the mature LH244 and \u003cem\u003emn8C1 \u003c/em\u003ekernels from a segregated F\u003csub\u003e2\u003c/sub\u003e ear.\u0026nbsp;The gRNA target sequence and the protospacer-adjacent motif (PAM) are shown in blue and pink, respectively.\u0026nbsp;\u0026nbsp;Alignment of mutant sequence from a transgenic line is indicated.\u0026nbsp;Red letter represent insertion. Bars = 2 cm \u003cstrong\u003e(d)\u003c/strong\u003e Mature ear of a self-pollinated heterozygous plant (\u003cem\u003emn8C1\u003c/em\u003e) showing segregation of wild-type (WT) and miniature kernels. The red arrows indicate miniature kernels. \u003cstrong\u003e(e) \u003c/strong\u003eAn allelism test was performed using a cross between \u003cem\u003emn8\u003c/em\u003e/+ and \u003cem\u003emn8C1\u003c/em\u003e/+. The red arrows indicate miniature kernels.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/dd05430095d057963426c004.png"},{"id":65667842,"identity":"9ee4ab6f-ec26-44b1-83da-172e25ae8ad4","added_by":"auto","created_at":"2024-10-01 06:29:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1236095,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZmEMF1a interacts with PRC1 and PRC2 components. (a) \u003c/strong\u003eYest two-hybrid (Y2H) assays showing that ZmEMF1a interacts with RING1 and MSI1. \u003cstrong\u003e(b)\u003c/strong\u003e Split-LUC assays showing that ZmEMF1a interacts with RING1 and MSI1. LHP1 or FIE1 does not interact with ZmEMF1a. \u003cstrong\u003e(c)\u003c/strong\u003e BiFC assays showing that ZmEMF1a\u003cstrong\u003e \u003c/strong\u003einteracts with RING1 and MSI1 in \u003cem\u003eN. benthamiana\u003c/em\u003e cells. Bars =50 μm. \u003cstrong\u003e(d)\u003c/strong\u003e Nuclear localization assays showing that the C-terminal of EMF1a is responsible for the EMF1a subnuclear pattern. Bars =2 μm. \u003cstrong\u003e(e)\u003c/strong\u003e Y2H assays showing that N-terminus of ZmEMF1a interacts with RING1 and MSI1, while the C-terminus does not interact.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/9fb024dce5853f3eb0ba1328.png"},{"id":65667838,"identity":"3b83e3f1-abef-4ed8-b9e8-ebca6c3f9e65","added_by":"auto","created_at":"2024-10-01 06:29:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":432116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZmEMF1a knockout leads to genome-wide reduction of H2Aub and H3K27me3 modification. (a) \u003c/strong\u003eAnalysis of H3K27me3 and H2Aub levels of \u003cem\u003emn8\u003c/em\u003e and B73 endosperms by immunoblotting using anti-H3K27me3 and anti-H2Aub antibodies. Endosperms (12 DAP) were used to extract histone proteins. Total histone extracts were used for the analysis. Anti-H3 antibody was used as loading controls. \u003cstrong\u003e(b)\u003c/strong\u003e Percentage of peaks showing H2Aub and H3K27me3 within genic, intergenic and TE regions in the maize genome. \u003cstrong\u003e(c)\u003c/strong\u003e and \u003cstrong\u003e(d) \u003c/strong\u003eBox plots showing the H2Aub (c) and H3K27me3 (d) enrichment levels (quantified by Reads Per Kilobase per Million mapped reads [RPKM] values of B73 and \u003cem\u003emn8\u003c/em\u003e. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(e)\u003c/strong\u003e and \u003cstrong\u003e(f)\u003c/strong\u003e Metagene plot showing the H2Aub (e) and H3K27me3 (f) enrichment levels of H2Aub-marked, H2Aub/H3K27me3-marked and only-H2Aub-marked genes in B73 and \u003cem\u003emn8\u003c/em\u003e. \u003cstrong\u003e(g)\u003c/strong\u003e Box plots showing the peak length of H2Aub and H3K27me3 in B73. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(h)\u003c/strong\u003e Overlap between H2Aub-marked and H3K27me3-marked genes in B73. \u003cstrong\u003e(i) \u003c/strong\u003eMetagene plot showing the H2Aub enrichment level of only-H2Aub-marked genes and H2Aub/H3K27me3-marked genes in B73. \u003cstrong\u003e(j) \u003c/strong\u003eBoxplots show average signal of H2Aub of only-H2Aub-marked genes and H2Aub/H3K27me3-marked genes in B73. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test).\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/d1b08e568e797931cb0c94a4.png"},{"id":65668173,"identity":"b6002c97-024b-4b90-a164-367c16682044","added_by":"auto","created_at":"2024-10-01 06:37:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":428994,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe levels of H3K27me3 and H2Aub are negatively correlated with gene expression. (a) \u003c/strong\u003eVolcano plot showing the log\u003csub\u003e2\u003c/sub\u003e fold change and statistical significance of the differences in quantitative mRNA expression between B73 and \u003cem\u003emn8\u003c/em\u003e mutant. Significantly up- and downregulated genes in \u003cem\u003emn8\u003c/em\u003e are highlighted in red and green respectively.\u003cstrong\u003e (b) \u003c/strong\u003eGenes in B73 were divided into three classes based on the expression (FPKM values) levels, low, medium, and high expression levels. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(c)-(d) \u003c/strong\u003eBox plots showing H3K27me3 (c) and H2Aub (d) enrichment of low, medium and high expression levels of genes in B73. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(e)-(f) \u003c/strong\u003eMetagene plot showing the H2Aub (e) and H3K27me3 (f) enrichment levels of low, medium and high expressed genes throughout B73 genome. \u003cstrong\u003e(g) \u003c/strong\u003eGenes were divided into five classes based on the enrichment levels of H2Aub, including H2Aub-marked genes, none-H2Aub marked genes, genes with low enrichment levels, genes with medium enrichment levels, and genes with high enrichment levels. \u003cstrong\u003e(h) \u003c/strong\u003eBox plots showing the expression levels of the five classes genes in (g). Expression levels are indicated in FPKM. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(i)\u003c/strong\u003e Percentage of genes belonging to different expression level categories for H2Aub-, H3K27me3-, H2Aub/H3K27me3-, only-H2Aub-, and only-H3K27me3-marked genes. Expression levels are indicated in FPKM. \u003cstrong\u003e(j)\u003c/strong\u003e Box plots showing the expression levels of expressed-, non-expressed-, H2Aub-, H3K27me3-, H2Aub/H3K27me3-, only-H2Aub-, and only-H3K27me3-marked genes. Expression levels are indicated in FPKM. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test).\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/6c67e9c4bfda996e7fe546a1.png"},{"id":65668176,"identity":"cdc10b5a-3856-4a19-bcfe-67bff1c6b2fb","added_by":"auto","created_at":"2024-10-01 06:37:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":338594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eZmEMF1a is required for the expression of genes related to kernel development. (a) \u003c/strong\u003eThe most significantly enriched GO terms in the upregulated genes and their associated\u0026nbsp;\u003cem\u003eP\u003c/em\u003e-values are shown. Lower x-axis, −log\u003csub\u003e10\u003c/sub\u003e\u0026nbsp;(\u003cem\u003eP\u003c/em\u003e-value); upper\u0026nbsp;x-axis, the number of genes with a given GO term. \u003cstrong\u003e(b)\u003c/strong\u003e Metagene plot showing the H2Aub and H3K27me3 enrichment levels of upregulated genes in B73 and \u003cem\u003emn8\u003c/em\u003e.\u003cstrong\u003e (c)\u003c/strong\u003e The expression of genes that negatively regulate kernel development are quantified by RNA-seq analysis. The log\u003csub\u003e2\u003c/sub\u003e fold change values between \u003cem\u003emn8\u003c/em\u003e and B73 are shown as a heat map.\u003cstrong\u003e (d)\u003c/strong\u003e RT-quantitative PCR confirmation of 5 selected upregulated genes associated with negatively regulated kernel development in 12-DAP B73 and \u003cem\u003emn8\u003c/em\u003e endosperms. Values are means ±SE; n = 3 (*, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(e)\u003c/strong\u003e and \u003cstrong\u003e(f)\u003c/strong\u003e The graphs show ChIP-qPCR using anti-H2Aub and anti-H3K27me3 antibodies (IP) or no antibody (mock control) as a percentage of input DNA. Values are means ±SE; n = 3 (ns, no significant; *, P \u0026lt; 0.05; **, P \u0026lt; 0.01, Student’s t test). \u003cstrong\u003e(g)\u003c/strong\u003e-\u003cstrong\u003e(i)\u003c/strong\u003e ChIP-seq genome browser views of H3K27me3 and H2Aub marks on upregulated genes involved in kernel development. The blue diagrams indicate the gene structure underneath each panel.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/1ddecf8404a718b770f12b23.png"},{"id":65668875,"identity":"ca8e6681-6d3d-4d2e-bc82-6806a83dfa14","added_by":"auto","created_at":"2024-10-01 06:45:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10300666,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/b93291b6-508e-48aa-a37b-df451e4dd794.pdf"},{"id":65668177,"identity":"07069f6c-1d42-464d-ba3c-954dcbfd2930","added_by":"auto","created_at":"2024-10-01 06:37:37","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":3959918,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-4998315/v1/b6caf1347da9e39bbed81cd4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePolycomb group (PcG) proteins maintain the transcriptionally repressed state of genes involved in the normal development of eukaryotes by incorporating histone modifications within chromatin [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. 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 \u003cem\u003eDrosophila\u003c/em\u003e, mammals or \u003cem\u003eArabidopsis\u003c/em\u003e, respectively [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and PRC2 has histone H3 lysine 27 (H3K27) tri-methyltransferase activity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePRC2 core subunits are well conserved in plants compared to their vertebrate counterparts [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, CURLY LEAF (CLF), MEDEA (MEA), and SWINGER (SWN) are homologs of the animal SET-domain-containing methyltransferase Enhancer of zeste (E(z)) [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. 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) [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. PRC2 complex in \u003cem\u003eArabidopsis\u003c/em\u003e contain single copy gene \u003cem\u003eFIE\u003c/em\u003e, however, FIE is encoded by \u003cem\u003eFIE1\u003c/em\u003e and \u003cem\u003eFIE2\u003c/em\u003e in rice and maize [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eOsFIE2\u003c/em\u003e and \u003cem\u003eZmFIE2\u003c/em\u003e are expressed universally and are likely to be functional orthologs of \u003cem\u003eArabidopsis\u003c/em\u003e FIE [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Conversely, OsFIE1 and ZmFIE1 are expressed materially exclusively in endosperm, and have evolved a distinct function [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePRC1 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 \u003cem\u003eDrosophila\u003c/em\u003e consists of Sex Comb Extra (Sce, also known as dRing) and Posterior Sex Combs (Psc) or Su(z)2 [\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The E3 monoubiquitin ligase module can associate with other nonenzymatic activities to form canonical PRC1s or with other subunits to form variant PRC1s [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Although the module in \u003cem\u003eArabidopsis\u003c/em\u003e 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 \u003cem\u003eArabidopsis\u003c/em\u003e, and instead, several plant-specific proteins are involved as PcG components in chromatin compaction and H3K27me3 reading [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. One such example is LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), which is proposed to be a functional analog of \u003cem\u003eDrosophila\u003c/em\u003e Pc due to its ability to bind the H3K27me3 mark and interact with other PRC1 components, including BMI1 and RING1 [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]; nevertheless, it also co-purifies with PRC2 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In the case of the flowering plant \u003cem\u003eArabidopsis\u003c/em\u003e, the plant-specific protein EMF1 serves a similar role to PSC-CTR, facilitating chromatin condensation during the process of Polycomb-mediated gene silencing [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe EMF1 gene encodes a transcriptional regulator protein that includes the LXXLH motif [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and it is known to interact with RING-finger proteins in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, EMF1 also interacts with PRC2 component MSI1 \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Genome-wide analysis of H3K27me3 modification and EMF1 binding in WT and \u003cem\u003eemf1-2\u003c/em\u003e 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 \u003cem\u003eemf1-2\u003c/em\u003e mutants, suggesting that PRC2 function partially required EMF1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Further work is required to fully understand the relationship among H2Aub, H3K27me3, and EMF1 in gene repression.\u003c/p\u003e \u003cp\u003ePcG proteins complexes play crucial functions in regulation of plant developmental transition and in controlling gene expression [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. FIS-PRC2 genes are considered to be repressors of endosperm formation in the absence of fertilization [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. After pollination, FIS-PRC2 complex is involved in endosperm development through repression of the expression of MADS-box gene \u003cem\u003eAGAMOUS LIKE62\u003c/em\u003e (\u003cem\u003eAGL62\u003c/em\u003e), which suppresses cellularization phase of endosperm development [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Endosperm development is characterized by four stages, that are coenocytic, cellularization, differentiation, and maturation [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The rapid expansion of endosperm volume during the differentiation stage is attributable to both cell proliferation and cell expansion. In \u003cem\u003eArabidopsis\u003c/em\u003e, several genetic factors controlled cell division to regulate seed size, including some components of the ubiquitin pathway. \u003cem\u003eArabidopsis DA1\u003c/em\u003e, encoding a predicted ubiquitin receptor, inhibits seed growth by restricting the period of cell proliferation. The \u003cem\u003eda1-1\u003c/em\u003e mutant plants represented large organs and seeds [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In maize, the mutation in homology gene \u003cem\u003eZmDA1\u003c/em\u003e cause the same biological phenotype as \u003cem\u003eda1-1\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBIG 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 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. WIDE AND THICK GRAIN1 (WTG1) is a functional deubiquitinating enzyme, loss of function of WTG1 produces wide and heavy grains [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The plant-specific transcription factor ABI3/VP1 (RAV1) negatively regulates plant growth, and overexpression of RAV1 results in reduced seed size and weight [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Previous research find that the orthologs of \u003cem\u003eArabidopsis\u003c/em\u003e 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 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Another study indicates that MADS1 interacts with GS3 and DEP1, promoting the transcription of downstream genes, thereby inhibiting grain growth of rice [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eIn this work, we isolated a small kernel mutant and map-based cloning and allelic analysis have demonstrated that the loss-of-function mutation in \u003cem\u003eZmEMF1a\u003c/em\u003e 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 \u003cem\u003emn8\u003c/em\u003e 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 \u003cem\u003eArabidopsis\u003c/em\u003e, which suggests that H2Aub modifications play different regulatory roles in maize compared to other plant species. In \u003cem\u003emn8\u003c/em\u003e mutants, elevated expression of repressors in cell proliferation, such as \u003cem\u003eDA1\u003c/em\u003e, \u003cem\u003eBB1\u003c/em\u003e, \u003cem\u003eMADS8\u003c/em\u003e, and \u003cem\u003eMADS14\u003c/em\u003e, 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.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhenotype and Genetic Characterization of\u003c/b\u003e \u003cb\u003emn8\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003emn8\u003c/em\u003e 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 (χ\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.013\u0026ndash;0.215\u0026thinsp;\u0026lt;\u0026thinsp;χ\u003csup\u003e2\u003c/sup\u003e0.05\u0026thinsp;=\u0026thinsp;3.84; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), indicating that \u003cem\u003eMN8\u003c/em\u003e was a single gene. At maturity, the smaller kernels of \u003cem\u003emn8\u003c/em\u003e can be clearly distinguished macroscopically from wild-type kernels from the same ear (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Compared with WT (B73) siblings, \u003cem\u003emn8\u003c/em\u003e kernels were significantly smaller (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, c). During sectioning of the kernels, both embryo and endosperm were markedly reduced in \u003cem\u003emn8\u003c/em\u003e kernels compared with B73 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). When measured, the single kernel weight was significantly reduced than that of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ee), and the embryo length was also found to be significantly shorter than that of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eTransmission 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 \u003cem\u003emn8\u003c/em\u003e mutant contained smaller protein bodies and starch grains than those in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ef-i). The starch content as a percentage of kernel weight and the average total protein content per kernel in the \u003cem\u003emn8\u003c/em\u003e endosperm were significantly lower than those in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e1\u003c/span\u003ek, l). The \u003cem\u003emn8\u003c/em\u003e mutant seedlings were slight shorter than WT at the 7th day after germination (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ea). However, there was no significant difference in plant height at sexual maturity stage between the \u003cem\u003emn8\u003c/em\u003e mutant and WT (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eb, c). We also found that the germination frequency and the number of leaves of \u003cem\u003emn8\u003c/em\u003e were similar to those of the WT (Additional file 1: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003ed, e). In summary, MN8 is specifically affects maize kernel development.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe\u003c/b\u003e \u003cb\u003emn8\u003c/b\u003e \u003cb\u003eMutant kernels showed a developmental delay\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo examine the developmental aberrations of the \u003cem\u003emn8\u003c/em\u003e kernels, a detailed characterization of the development of both \u003cem\u003emn8\u003c/em\u003e and WT kernels was achieved through analysis of cytological sections. At 6 DAP, the size of \u003cem\u003emn8\u003c/em\u003e endosperm was about one-second of that of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, e). Both \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ei, n). At 8 DAP, the difference in endosperm volume between the \u003cem\u003emn8\u003c/em\u003e and WT became more pronounced than at the early development stage, with the size of the \u003cem\u003emn8\u003c/em\u003e endosperm being only one-third of that of its WT sibling (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, 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 \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ej, o). At 12 DAP, the degeneration of maternal tissue introduced a gap between the endosperm and the pericarp in \u003cem\u003emn8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, c, g). This gap enlarged during the later development stage of the mutant seed until maturity (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, h). At this period, the \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003ek, p). The development of the \u003cem\u003emn8\u003c/em\u003e embryo was delayed but not arrested. At 15 DAP, the \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e2\u003c/span\u003el, m, q, r).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMap-based cloning of\u003c/b\u003e \u003cb\u003emn8\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eMn8\u003c/em\u003e 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, \u003cem\u003emn8\u003c/em\u003e in the B73 genetic background was outcrossed with the Mo17 inbred line. The heterozygous F\u003csub\u003e1\u003c/sub\u003e progeny was self-pollinated to create a mapping population. The \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). 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 \u003cem\u003emn8\u003c/em\u003e plants revealed that only \u003cem\u003eZm00001d024813\u003c/em\u003e contained a mutation expected to cause a loss of gene function. In \u003cem\u003emn8\u003c/em\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eTo confirm that the mutation in \u003cem\u003eZm00001d024813\u003c/em\u003e accounted for the \u003cem\u003emn8\u003c/em\u003e 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 \u003cem\u003eZm00001d024813\u003c/em\u003e. Among 6 independent transformation events, several types of mutation were detected, and we select a T insertion mutant (\u003cem\u003emn8C1\u003c/em\u003e) for further genetic analysis. In \u003cem\u003emn8C1\u003c/em\u003e, 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 \u003cem\u003emn8C1\u003c/em\u003e kernels exhibited similar defects to those of the \u003cem\u003emn8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The mutants \u003cem\u003emn8C1\u003c/em\u003e, used in further work, were backcrossed to B73 to eliminate the CRISPR-Cas9 transgene. Ears of self-pollinated heterozygous \u003cem\u003emn8C1\u003c/em\u003e segregated mutant kernels at the radio of 1:3 (mutant: WT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, Table S2). The kernels exhibiting the \u003cem\u003emn8\u003c/em\u003e-like phenotype were confirmed to be homozygous mutants of \u003cem\u003eZm00001d024813\u003c/em\u003e, as determined by genotyping with gene-specific primers. Allelic crosses between heterozygous \u003cem\u003emn8\u003c/em\u003e/+ and \u003cem\u003emn8C1\u003c/em\u003e/+ heterozygous produced ears that segregated kernels with normal and small kernels in the expected 3:1 ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e3\u003c/span\u003ee, Table S3), indicating that \u003cem\u003emn8\u003c/em\u003e and \u003cem\u003emn8C1\u003c/em\u003e are allelic. Taken together, these data provide evidence that the mutation in \u003cem\u003eZm00001d024813\u003c/em\u003e is indeed responsible for the mutant phenotype observed in \u003cem\u003emn8\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMn8\u003c/b\u003e \u003cb\u003eencodes an EMF1-like protein\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSequence analysis revealed that \u003cem\u003eMn8\u003c/em\u003e contains 4 exons and 3 introns. The mature transcript of \u003cem\u003eMn8\u003c/em\u003e features a 3,261-bp coding sequence that encodes an unknown protein, \u003cem\u003eZm00001d024813\u003c/em\u003e, comprising 1,086 amino acids. Homology analysis indicated that Mn8 exhibits the highest similarity with the \u003cem\u003eArabidopsis\u003c/em\u003e protein EMF1 (AT5G11530), with 36% identity and 50.48% similarity. In addition to \u003cem\u003eMN8\u003c/em\u003e, 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 \u003cem\u003eMN8\u003c/em\u003e as \u003cem\u003eEMF1a\u003c/em\u003e, and the other three as \u003cem\u003eEMF1b\u003c/em\u003e, \u003cem\u003eEMF1c\u003c/em\u003e, and \u003cem\u003eEMF1d\u003c/em\u003e, 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.\u003c/p\u003e \u003cp\u003eTo 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 \u003cem\u003eArabidopsis\u003c/em\u003e EMF1. To better understand the role of ZmEMF1a in endosperm development, we analyzed its tissue expression patterns using published RNA sequencing (RNA-seq) data [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. 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.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eZmEMF1a interacts with PRC1 and PRC2 components\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, AtEMF1 can interact with both PRC1 RING-finger proteins and the PRC2 component MSI \u003cem\u003ein vitro\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). 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.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). We also analyzed the subnuclear localizations of ZmEMF1a, ZmRING1 and ZmMSI1 in maize protoplasts and \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (\u003cem\u003eN. benthamiana\u003c/em\u003e) leaves. The results showed that all proteins localized to the nucleus (Additional file 1: Fig. S4a, b), using AHL22-RFP as a nuclear marker [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. This indicated that the interactions occurred in the nucleus. In addition, we performed the bimolecular fluorescence complementation (BiFC) assay to test these interactions in \u003cem\u003eN. benthamiana\u003c/em\u003e. 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.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003cp\u003eThe C to T point mutation in \u003cem\u003eZmEMF1a\u003c/em\u003e 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 \u003cem\u003eN. benthamiana\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Y2H experiments demonstrated that both RING1 and MSI1 interacted with the N-terminal domain of EMF1a, but not with the C-terminal domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig16\" class=\"InternalRef\"\u003e4\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eZmEMF1a knockout leads to a genome-wide reduction in H2Aub and H3K27me3 modification\u003c/h2\u003e \u003cp\u003eIn eukaryotes, PcG proteins play important roles in maintaining gene silencing, which is involved in cellular and developmental processes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The major protein complex, PRC2, possesses H3K27 tri-methyltransferase activity, while PRC1 has histone H2A E3 ubiquitin ligase activity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, EMF1 plays a crucial role in H3K27me3 deposition; loss of \u003cem\u003eEMF1\u003c/em\u003e function results in a genome-wide reduction of H3K27me3, but does not affect the H2Aub modification compared to the WT [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. H2AK119ub in mammals and H2AK121ub in \u003cem\u003eArabidopsis\u003c/em\u003e are observed within the consensus sequence PKKT [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. One of the maize H2A isoforms shows high similarity with both human H2A and \u003cem\u003eArabidopsis\u003c/em\u003e 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).\u003c/p\u003e \u003cp\u003eTo investigate whether the ZmEMF1a mutation affects H3K27me3 and H2Aub modifications, we performed Western blotting on nuclear proteins isolated from both WT and \u003cem\u003emn8\u003c/em\u003e endosperms at 12 DAP using anti-H3K27me3 and anti-H2Aub antibodies. Unexpectedly, although we observed a significant decrease in H3K27me3 levels in \u003cem\u003emn8\u003c/em\u003e compared to the WT, as previously described in \u003cem\u003eemf1-2\u003c/em\u003e mutant of \u003cem\u003eArabidopsis\u003c/em\u003e, we also found markedly reduced levels of H2Aub in \u003cem\u003emn8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In contrast, no difference was detected in \u003cem\u003eemf1-2\u003c/em\u003e compared to the WT in previous studies [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. 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 \u003cem\u003emn8\u003c/em\u003e endosperm at 12 DAP. We obtained 66 to 125\u0026nbsp;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 \u003cem\u003eArabidopsis\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). When analyzed the average genomic modification levels of H2Aub and H3K27me3, we also found reduced levels of H2Aub and H3K27me3 in \u003cem\u003emn8\u003c/em\u003e, which is consistent with the result of western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, d).\u003c/p\u003e \u003cp\u003eA metagene plot of H2Aub coverage at H2Aub-marked genes showed a significant reduce of H2Aub in \u003cem\u003emn8\u003c/em\u003e compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). 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 \u003cem\u003emn8\u003c/em\u003e compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ee). We found reduced levels of H3K27me3 in \u003cem\u003emn8\u003c/em\u003e at H3K27me3/H2AK121ub-marked and only-H3K27me3 genes, as previously reported in \u003cem\u003eArabidopsis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ef) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. 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.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003eg). In addition to this, we found that only 21% of the H2Aub-marked genes were overlapped with H3K27me3-marked genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003eh), and the H2Aub levels of H3K27me3/H2Aub-marked genes were higher than that of only-H2Aub-marked genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003ei, j). These results showed that both H2Aub and H3K27me3 levels were significantly decreased in \u003cem\u003emn8\u003c/em\u003e compared to WT in maize, which suggests that EMF1 regulates the expression of maize kernel development-related genes by modulating H2Aub and H3K27me3 modifications..\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe level of H2Aub is negatively correlated with gene expression\u003c/h2\u003e \u003cp\u003ePrevious research revealed that H3K27me3 is a repressive in \u003cem\u003eArabidopsis\u003c/em\u003e, while H2Aub is positively correlated with gene expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. To determine the relationship between H3K27me3 and H2Aub modifications and gene transcription, we performed RNA-seq on WT and \u003cem\u003emn8\u003c/em\u003e 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and absolute fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.0). In the \u003cem\u003emn8\u003c/em\u003e mutant, significantly more genes were up-regulated than down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ea), 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eb), 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eg, 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). In the case of H2Aub-marked genes, about 60% showed either no or low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ei). 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.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003ej).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eZmEMF1a is required for the expression of genes related to kernel development\u003c/h2\u003e \u003cp\u003eThe absence of the \u003cem\u003eEMF1a\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). 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 \u003cem\u003emn8\u003c/em\u003e. Our findings showed that the levels of H3K27me3 were significantly reduced in the up-regulated genes of \u003cem\u003emn8\u003c/em\u003e when compared to their WT counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). In contrast, the levels of H2Aub among these up-regulated genes exhibited minimal variance between \u003cem\u003emn8\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Moreover, EMF1a mutation also increased the expression of cell division-related genes, such as \u003cem\u003eDA1\u003c/em\u003e, \u003cem\u003eBB1\u003c/em\u003e, \u003cem\u003eBB2\u003c/em\u003e, \u003cem\u003eMADS8\u003c/em\u003e, \u003cem\u003eMADS14\u003c/em\u003e and \u003cem\u003ebZIP75\u003c/em\u003e. In addition, \u003cem\u003eGSK2\u003c/em\u003e, a homologous gene of \u003cem\u003eBIN2\u003c/em\u003e, down-regulation of \u003cem\u003eGSK2\u003c/em\u003e expression levels resulted in long and heavy grains, was also up-regulated in \u003cem\u003emn8\u003c/em\u003e. ES22, encoding a MADS-type transcription factor, negatively regulated starch accumulation, was significantly up-regulated in \u003cem\u003emn8\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). Five of these negative regulators were analyzed using RT-qPCR, and the results were consistent with the RNA-seq analysis of the WT and \u003cem\u003emn8\u003c/em\u003e transcriptomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003ed). 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 \u003cem\u003eZmDA1\u003c/em\u003e was significantly higher in the WT than in the \u003cem\u003emn8\u003c/em\u003e, and similar reduction of H2K27me3 levels in \u003cem\u003eMADS8\u003c/em\u003e, \u003cem\u003eMADS14\u003c/em\u003e and \u003cem\u003eES22\u003c/em\u003e were found (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eg, h; Additional file 1: Fig. S9a, b). In \u003cem\u003emn8\u003c/em\u003e mutants, H2Aub levels in the \u003cem\u003eBB1\u003c/em\u003e locus were significantly reduced, suggesting that EMF1a is required for H2Aub labeling in the \u003cem\u003eBB1\u003c/em\u003e region (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003ei). In addition this, we further confirmed the modification levels of the selected genes by ChIP-qPCR analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, 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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePcG 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 [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Suppressor of zeste 12 (SUZ12), a subunit of PRC2, is necessary for the catalytic activity of PRC2 [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Mutation of Suz12 in mice, the modifications of H3K27me2 and H3K27me3 are reduced drastically in \u003cem\u003eSuz12\u003c/em\u003e null embryos, resulting in a dramatic decrease in cell proliferation and an increase in apoptosis [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. EMF2, a Suz12 homologs in \u003cem\u003eArabidopsis\u003c/em\u003e, mutations in EMF2 lead to a decrease in the levels of H3K27me3 mark present in the chromatin of \u003cem\u003eSOC1\u003c/em\u003e and \u003cem\u003eFT\u003c/em\u003e, which affects the vegetative and flower development [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEMF1, the plant specific protein, was proposed to be a member of PRC1 for the chromatin compaction in vitro [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In recent years, it has been found that EMF1 interacts with PRC2 members, and is necessary for H3K27me3 marking [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, the classic maize kernel mutant \u003cem\u003emn8\u003c/em\u003e was cloned and the maize \u003cem\u003eMn8\u003c/em\u003e gene encodes the PRC1 component ZmEMF1a. In plants, EMF1 is involved in repressing both the vegetative to reproductive transition and flower initiation. In \u003cem\u003eArabidopsis\u003c/em\u003e, the strong \u003cem\u003eemf1-2\u003c/em\u003e mutant exhibits a more severe phenotype than weak \u003cem\u003eemf1-1\u003c/em\u003e mutant, all lateral organs differentiate in carpelloid structures. In rice, loss-of-function mutations in \u003cem\u003eEMF1\u003c/em\u003e-like gene \u003cem\u003eCURVED CHIMERIC PALEA\u003c/em\u003e (\u003cem\u003eCCP1\u003c/em\u003e) do not exhibit severe phenotype as \u003cem\u003eemf1\u003c/em\u003e mutants in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Compared with the WT, \u003cem\u003eccp1\u003c/em\u003e 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 [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Mutation of the \u003cem\u003eEMF1a\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that EMF1 plays an important role for the deposition of H3K27me3, and the \u003cem\u003eemf1-2\u003c/em\u003e mutant showed a decreased levels of H3K27me3 at gene body region. However, the average H2Aub signal levels of \u003cem\u003eemf1-2\u003c/em\u003e has no significant difference at both H2Aub/H3K27me3 and onlyH2Aub marked genes, compared with WT [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. Interestingly, despite we found decreased levels of H3K27me3, the levels of H2Aub modification were also significantly reduced in \u003cem\u003emn8\u003c/em\u003e mutant at both H2Aub/H3K27me3 and only-H2Aub marked genes. Widespread localization of H2Aub marks in animals and \u003cem\u003eArabidopsis\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Loss of \u003cem\u003eEMF1a\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig17\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eDrosophila\u003c/em\u003e, histone H2A monoubiquitination occurs mainly in the promoter region and represses the expression of target genes [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, 60% of only-H2AK121ub genes were transcriptionally active, suggested that H2AK121ub might play a role in transcriptional activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. 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 [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eFew 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 [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Genetic analyses demonstrate that \u003cem\u003eDA1\u003c/em\u003e, \u003cem\u003eTCP14\u003c/em\u003e and \u003cem\u003eTCP15\u003c/em\u003e function in a common pathway to regulate cell division. E3 ligases EOD1, also known as \u003cem\u003eBB\u003c/em\u003e, function independently synergistically with DA1 to regulate seed and organ size [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. We identify \u003cem\u003eZmDA1\u003c/em\u003e, \u003cem\u003eZmBB1\u003c/em\u003e and \u003cem\u003eZmBB2\u003c/em\u003e were significantly up-regulated in \u003cem\u003emn8\u003c/em\u003e endosperms. Besides, the expression of both genes MADS8 and MADS14, homologs of OsMADS1 in maize, was up-regulated in \u003cem\u003emn8\u003c/em\u003e. \u003cem\u003eOsMADS1\u003c/em\u003e is best known for its negative regulatory role in the regulation of grain growth, mutations in the last intron of \u003cem\u003eOsMADS1\u003c/em\u003e 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 \u003cem\u003emn8\u003c/em\u003e, 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.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003e \u003cem\u003emn8-1\u003c/em\u003e 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 \u003cem\u003emn8\u003c/em\u003e 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. \u003cem\u003eNicotiana benthamiana\u003c/em\u003e seeds were grown in a growth chamber under a photoperiod of 16 h:8 h (light:dark) and at a temperature of 22\u0026ndash;23\u0026deg;C for germination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eLight microscopy, scanning electron microscopy and transmission electron microscopy\u003c/h2\u003e \u003cp\u003eFor light microscopy analysis, immature \u003cem\u003emn8\u003c/em\u003e and B73 kernels were freshly collected and fixed overnight in formalin\u0026ndash;acetic acid\u0026ndash;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\u0026deg;C, the samples were embedded in paraffin (P3683, Sigma-Aldrich) and sectioned into 10-\u0026micro;m 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.\u003c/p\u003e \u003cp\u003eFor transmission electron microscopy analysis, \u003cem\u003emn8\u003c/em\u003e and B73 endosperms at 15 DAP were freshly collected and prepared as previously described [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. The sections were observed using Hitachi H7600 transmission electron microscope (Japan). For scanning electron microscopy analysis of starch grains, mature \u003cem\u003emn8\u003c/em\u003e and B73 seeds were cut with a razor blade, sputter-coated with gold and observed by scanning electron microscope (S-3400N, Hitachi, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of protein and starch contents\u003c/h2\u003e \u003cp\u003eFor protein content measurements, mature B73 and \u003cem\u003emn8\u003c/em\u003e 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 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. 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).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic Mapping of\u003c/b\u003e \u003cb\u003emn8\u003c/b\u003e \u003cb\u003eLocus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the initial mapping of \u003cem\u003emn8\u003c/em\u003e, DNA samples isolated from 30 miniature kernels from segregating F\u003csub\u003e2\u003c/sub\u003e 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\u0026ndash;29, 10\u0026ndash;59 and 10-104.2) was determined. The addition F\u003csub\u003e2\u003c/sub\u003e population of 600 mutants was genotyped with this four markers and the region between 10\u0026ndash;59 Mb and 10-104.2 Mb that contained the gene of interest was identified. Furthermore, the markers 10\u0026ndash;87 and 10\u0026ndash;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. \u003cem\u003emn8\u003c/em\u003e was localized to a 420-kb interval, as determined by analysis with the markers 10-88.68 and 10-89.1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMaize Transformation\u003c/h2\u003e \u003cp\u003eTransgenic plants were generated by \u003cem\u003eAgrobactium tumefaciens\u003c/em\u003e\u0026ndash;mediated maize transformation [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. For CRISPR/Cas9 editing plants, sequencing was used to identify editing sites near the target position. One transgenic line with \u003cem\u003eZm00001d024813\u003c/em\u003e knockout was obtained via CRISPR/Cas9, and it was selected for allelism tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis\u003c/h2\u003e \u003cp\u003eBy 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 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e], followed by the creation of a phylogenetic tree with the application of MEGA 7 software, which was executed using the neighbor-joining method [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and RT-qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 12 DAP endosperm from \u003cem\u003emn8\u003c/em\u003e 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 \u0026times; Ultra SYBR Mixture (CWBIO) on an ABI 7500 Real-Time PCR System (Applied Biosystem). The PCR conditions were 95\u0026deg;C for 10 min, followed by 40 cycles of 95\u0026deg;C for 15 s, and 60\u0026deg;C for 1 min. The gene expression levels were determined by the ΔCt (threshold cycle) method, utilizing \u003cem\u003eZmActin7\u003c/em\u003e gene as the internal control for normalization. Primer sequences for RT-qPCR are listed in Dataset S3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization of the EMF1a protein\u003c/h2\u003e \u003cp\u003eThe full-length ORF of EMF1a without the stop codon was cloned into the pUC-GFP vector through a process involving restriction enzyme cleavage (\u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eXho\u003c/em\u003eI) followed by ligation (the primers used are listed in Dataset S3). The \u003cem\u003eAHL22\u003c/em\u003e gene, integrated with the RFP tag, was constructed as a positive control. Maize protoplasts were isolated and transformed based on a previously described protocol [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. After transformation, the protoplasts were incubated in the dark for 16\u0026ndash;20 h at 28\u0026deg;C, after which the fluorescence signals were observed utilizing a confocal laser microscope (Zeiss 880, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assays\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003eEMF1a\u003c/em\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase complementation image assays\u003c/h2\u003e \u003cp\u003eTo 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 \u003cem\u003eEMF1a\u003c/em\u003e was cloned into the pCAMBIA-cLuc vector. These constructs were transfected into \u003cem\u003eAgrobacterium\u003c/em\u003e strain GV3101, and the transformants were then infiltrated into 6-week-old \u003cem\u003eN. benthamiana\u003c/em\u003e leaves following the established method [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation assays\u003c/h2\u003e \u003cp\u003eTo confirm the interaction between EMF1a and RING1 or MSI1, the CDS of \u003cem\u003eEMF1a\u003c/em\u003e was cloned into pSPYNE(R)173 vector, and the coding sequences of \u003cem\u003eRING1\u003c/em\u003e and \u003cem\u003eMSI1\u003c/em\u003e were cloned into pSPYCE(M) vector. The resulting constructs were transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e strain EHA105 and then infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The YFP signals were monitored by confocal microscopy (Zeiss880; Carl Zeiss) with an excitation wavelength of 488 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq and data analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from \u003cem\u003emn8\u003c/em\u003e and B73 endosperms obtained from the same F\u003csub\u003e2\u003c/sub\u003e 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 [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. Significant DEGs were identified with an adjusted \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and log2 (|fold change|)\u0026thinsp;\u0026ge;\u0026thinsp;1 using DESeq2 software in R (v.1.30.0, R package) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. GO enrichment was performed via the AgriGO (version 2.0) online program [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eChIP-seq and ChIP-qPCR\u003c/h2\u003e \u003cp\u003eTwo biological replicates of \u003cem\u003emn8\u003c/em\u003e and B73 endosperm tissues were obtained from two independent F\u003csub\u003e2\u003c/sub\u003e ears at 12 DAP. ChIP-seq with Anti-H2Aub and anti-H3K27me3 were performed as previously described [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. 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\u0026ndash;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 \u0026micro;L ChIP-grade protein A/G beads (Millipore) for 2 h at 4\u0026deg;C. The mixture was then incubated overnight with 5 \u0026micro;g antibody at 4\u0026deg;C, followed by incubation with 20 \u0026micro;L ChIP-grade protein A/G beads for 2 h at 4\u0026deg;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 \u0026micro;g 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).\u003c/p\u003e \u003cp\u003eFor 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\u0026times;Ultra SYBR Mixture (CWBIO) on a 7500 Real-Time PCR System (Applied Biosystem).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eChIP-seq data analysis\u003c/h2\u003e \u003cp\u003eRaw fastq data were trimmed using Trim Galore (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\u003c/span\u003e\u003cspan address=\"https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to remove adapters and low-quality bases, and quality control was then performed using FastQC (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/\u003c/span\u003e\u003cspan address=\"http://www.bioinformatics.babraham.ac.uk/projects/fastqc/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The reads were mapped to the maize reference genome B73 (AGPv4) using Bowtie2 [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e], and duplicated reads were removed for subsequent analysis. A correlation analysis between biological replicates was performed using deepTools [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Peak calling was performed separately for each biological replicate using MACS2 [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e] 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) [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. 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 \u003cem\u003emn8\u003c/em\u003e were developed using the computeMatrix and plotProfile programs.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by National Natural Science Foundation of China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e(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).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll 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. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMozgova I, Hennig L. The polycomb group protein regulatory network. Annu Rev Plant Biol\u003cem\u003e.\u003c/em\u003e 2015; 66:269-296.\u003c/li\u003e\n\u003cli\u003eRingrose L, Paro R. Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. 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Nat Biotecnol. 2011; 29(1):24-26.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Datasets","content":"\u003cp\u003eDatasets 1 to 3 are not available with this version.\u003c/p\u003e\u003cp\u003eDataset1\u0026nbsp;Genes used in the yeast two-hybrid experiments.\u003c/p\u003e\n\u003cp\u003eDataset2 DEGs in \u003cem\u003emn8\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eDataset3 Primers used in this study.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"genome-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gbio","sideBox":"Learn more about [Genome Biology](https://genomebiology.biomedcentral.com/)","snPcode":"13059","submissionUrl":"https://submission.springernature.com/new-submission/13059/3","title":"Genome Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PcG complex, H2Aub, H3K27me3, kernel development, maize.","lastPublishedDoi":"10.21203/rs.3.rs-4998315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4998315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePolycomb 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 \u003cem\u003emn8\u003c/em\u003e, and map-based cloning revealed that \u003cem\u003eMn8\u003c/em\u003e encodes a plant specific PcG protein, ZmEMF1a. Mutation in \u003cem\u003eZmEMF1a\u003c/em\u003e 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 \u003cem\u003eArabidopsis\u003c/em\u003e. Compared with WT endosperms, elevated expressions of homology genes of cell proliferation repressors, such as \u003cem\u003eDA1\u003c/em\u003e, \u003cem\u003eBB1\u003c/em\u003e, \u003cem\u003eES22, MADS8\u003c/em\u003e and \u003cem\u003eMADS14\u003c/em\u003e, accompanied with decreases in H3K27me3 or H2Aub levels at these loci in \u003cem\u003emn8\u003c/em\u003eendosperms, 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.\u003c/p\u003e","manuscriptTitle":"ZmEMF1a is required for the maintainence of H2Aub and H3K27me3 modifications in maize kernel development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-01 06:29:32","doi":"10.21203/rs.3.rs-4998315/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-18T09:37:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-30T06:06:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23855654838937833044455251875522779790","date":"2024-11-15T07:03:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-23T17:03:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48567341685211790207303654286986952830","date":"2024-09-08T20:17:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-05T09:26:12+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-05T09:11:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-30T10:06:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Genome Biology","date":"2024-08-29T14:07:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"genome-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gbio","sideBox":"Learn more about [Genome Biology](https://genomebiology.biomedcentral.com/)","snPcode":"13059","submissionUrl":"https://submission.springernature.com/new-submission/13059/3","title":"Genome Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"96417ae5-7272-4f72-9b85-b0aad36b42f1","owner":[],"postedDate":"October 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T08:23:44+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-01 06:29:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4998315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4998315","identity":"rs-4998315","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}