H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity

H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity | 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 Article H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity Leor Eshed Williams, Tali Mandel, udi Landau, Tommy Kaplan, Yotam Cohen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5582331/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Plant callus cells possess a remarkable ability to regenerate organs that often differ from their respective origins or even entire new individuals. Yet, the mechanisms underlying their pluripotent state remain elusive. We propose a strategy that involves two independent mechanisms to endow callus cells with pluripotency: (1) maintaining a unique transcriptional profile, characterised by the expression of genes from diverse developmental pathways that allows rapid response to developmental cues; (2) preventing premature differentiation through H3K27 methylation-mediated silencing of key transcription factors such as WUCHEL and SPEECHLESS . This strategy relies on a mechanism to silence the pluripotency network upon regenerative stimuli, enabling a single developmental pathway to dominate. Our study reveals that the EMF2 complex, a key regulator of H3K27 tri-methylation, plays a crucial role in this process. Callus derived from the emf2 mutant, deficient in H3K27me3, exhibits severely impaired regeneration. Comparative analyses of chromatin states and transcription profiles between wild-type and emf2 calli revealed that the loss of EMF2 leads to upregulation of key transcription factors in callus, and identified the genes regulated solely by EMF2. Our findings suggest that suppressing pluripotency networks through H3K27me3 is essential for executing specific developmental programs to ensure effective regeneration. Biological sciences/Developmental biology/Pluripotency Biological sciences/Plant sciences/Plant development/Cell fate Biological sciences/Developmental biology/Reprogramming Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Certain plant cells retain plasticity and are capable, under appropriate stimuli such as wounding or hormone stimulation, to switch their developmental program, re-enter the cell cycle, and form a mass of less-differentiated pluripotent cells, termed callus 1 , 2 . These cells acquire the competence to rapidly respond to diverse stimuli and adopt new fates accordingly 3 – 5 , to regenerate organs that often differ from their respective origins. For example, auxin induces root regeneration, while a low auxin/cytokinin ratio promotes shoot induction 6 . Although numerous studies have investigated competency acquisition during callus formation, highlighting the significant role of epigenetic regulation in this process 7 , the mechanisms governing callus cell identity as proliferative, pluripotent cells with a high regenerative capacity remain elusive. The remarkable process of acquiring new cell fates and forming new lineages during organ regeneration requires the establishment of specific gene expression patterns and a cellular memory system to transmit these patterns through cell divisions to maintain cellular identity 5 . Central to controlling developmental gene expression during cell fate transitions is the tri-methylation of histone H3 at lysine 27 (H3K27me3), a repressive chromatin modification catalysed by the Polycomb Repressive Complex 2 (PRC2) 8 . The PRC2 was first elucidated in Drosophila melanogaster by identifying mutants exhibiting homeotic conversions due to ectopic expression of the Hox genes 9 . PRC2 is a four-core subunit complex, highly conserved across higher organisms 10 . It is the only known complex with H3K27me3 methyltransferase activity, and its importance in numerous organisms is highlighted by the severe developmental defects and even lethality caused by mutation in PRC2 subunits 11 . The subunits of the Drosophila PRC2, each encoded by a single-copy gene, include the catalytic component Enhancer of zeste (E(z)); the non-catalytic partners, Suppressor of zeste 12 (Su(z)12) and Extra sex combs (Esc), which directly contact E(Z) and are indispensable for the methyltransferase activity; and the Nucleosome remodelling factor 55 kDa (Nurf55) 12 . In the Arabidopsis thaliana model plant, the PRC2 core subunits, with the exception of the Esc homolog FERTILISATION INDEPENDENT ENDOSPERM (FIE), are encoded by multigene families and assemble in various combinations to form three distinct PRC2 complexes, named after their respective Su(z)12 homolog components: the EMBRYONIC FLOWER2 ( EMF2 ), the FERTILISATION INDEPENDENT SEED ( FIS2 ), and the VERNALISATION 2 ( VRN2 ) 13 . Each complex controls various processes and developmental programs with some redundancy, including cell fate specification, embryogenesis, vegetative growth, and flowering time 14 . EMF2 silences genes associated with floral induction and reproductive development to ensure sufficient vegetative growth before flowering. Accordingly, EMF2 mutations in Arabidopsis trigger flowering immediately upon germination, bypassing the vegetative growth phase 15 . The genomic distribution of H3K27me3 in plants is dynamic and strongly correlates with transcriptional repression, impacting hundreds of genes, many of which play roles in development 16 – 18 . ChIP-Seq analyses across diverse plant species have revealed extensive H3K27me3 reprogramming during biotic stress responses 19 and abiotic stress responses 20 , 21 , cell differentiation 22 , 23 , and developmental processes. For instance, the temporal and sequential expression of genes controlling cell fate switch during flower morphogenesis requires the differential deposition or removal of H3K27 methylation on numerous genes 18 , 24 . Other prominent examples include H3K27me3 redistribution during the differentiation of stem cells at the shoot apical meristem 25 or, conversely, during the acquisition of a pluripotent state, both in the male gametophyte 26 and callus formation 5 , 27 . H3K27me3 deposition was also shown to be essential for callus formation from leaf tissue. This is evidenced by the impaired callus formation observed in Arabidopsis leaf explants of PRC2 mutants, including CURLY LEAF ( CLF ), SWINGER ( SWN ), and EMF2 5 . Further support comes from defective callus formation in the hypocotyls of the HTR15 mutant, which encodes a histone H3 variant lacking the lysine 27 residue 28 . Here, we reveal a mechanism underlying callus cell identity, characterized by the expression of numerous genes from diverse developmental pathways while simultaneously silencing dominant genes that drive differentiation or organogenesis, thereby maintaining pluripotency. We demonstrate that callus derived from the EMF2 mutant exhibits severely impaired regenerative capacity, suggesting that the EMF2 complex is crucial for silencing pluripotency networks to enable commitment to a single developmental pathway. Furthermore, we emphasise the advantages of using callus as a model system for studying transcriptional regulation, as it minimises the averaging effects typical of other systems Results Pluripotent callus cells express genes from multiple developmental pathways. To enhance our understanding of pluripotency and regenerative competence, we compared the transcriptomes of six-week-old Arabidopsis calli derived from cotyledons, which exhibit a high capacity for regeneration, with those of leaves from three-week-old plants, which in Arabidopsis demonstrate little to no capacity for direct regeneration 29 . A total of 16,703 genes were expressed in callus and 16,694 in leaves (RPKM > 1) (Supplementary Data 1 and 2), with 12,109 genes exhibiting significant differential expression (cutoff of: |log2FC| > 0.6 and FDR < 0.05) (Supplementary Data 3), demonstrating the substantial dissimilarity between cells comprising the callus and the leaf (Fig. 1 a). As expected, Gene Ontology (GO) analysis revealed that the 6223 genes downregulated in calli (i.e. upregulated in leaves) were significantly enriched for photosynthesis-related biological processes (p < E-76), and plastid and thylakoid-related cellular components (p < E-69), (Supplementary Table 1). This reflects the function of the leaf as a photosynthesis factory and indicates that the leaf cells are differentiated 30 . Selected GO-enriched terms for the 5,886 up-regulated callus genes are shown in Fig. 1 b (full terms list in Supplementary Table 2). Out of the 3425 genes in the nucleic acid metabolic term (GO:0090304), 2574 are expressed in callus (p < 8.07E-75), suggesting high production of nucleic acids required for DNA synthesis in the highly proliferative cells. This is consistent with the enrichment of cell cycle genes, with 316 out of 423 genes involved in the cell cycle process (GO:0007049). The enrichment in response to endogenous stimulus (GO:0009719) was also predictable as the callus is cultured on media supplemented with the phytohormones auxin and cytokinin. However, what caught our attention is the significant enrichment in multicellular organismal development genes (GO:0007275). This was unexpected since callus is considered an unorganized, less differentiated mass of cells and has been shown to be enriched for root developmental genes 31 , 32 . A detailed GO analysis on the 1950 callus-developmental genes (Supplementary Table 3) confirmed that 339 genes are related to root development (GO:0048364; 432 genes), including the root marker genes WUSCHEL RELATED HOMEOBOX 5 (WOX5) , SHORT ROOT ( SHR ) and SOMBRERO 33 – 35 . Most striking was the significant enrichment of genes participating in many other developmental pathways, including shoot, leaf, flower, embryo, and seed development (Fig. 1 c). For example, out of the 282 genes in the leaf development term (GO:0048366), 197 (70%) are expressed in callus, including key transcription factors (TFs) essential for leaf morphogenesis such as ASYMMETRIC LEAVES 1 ( AS1 ), CUP-SHAPED COTYLEDON 2 ( CUC2 ), AUXIN RESPONSE FACTOR 3/ and 4 ( ARF3/4 ) and the five members of the Class III HD-ZIP genes 36 . Another example is the activation of meristem development genes (135 out of 174 in the term - GO:0048507), from which many are participating in shoot and inflorescent meristem development, like KNOTTED1-LIKE HOMEOBOX GENE 6 ( KNAT6 ) and KNAT1 37 . Remarkably, the callus does not express genes specific to stem cells at the shoot apical meristem, such as CLAVATA3 ( CLV3 ), AINTEGUMENTA-LIKE 7 , and INHIBITOR OF GROWTH 1 38,39 . The expression of numerous genes from diverse developmental programs in the callus may enable a rapid response to stimuli and facilitate de novo organogenesis without requiring the release of transcriptional silencing or undergoing the complex, multistep transcription process. In this scenario, we hypothesized that genes encoding for TFs, that can independently induce differentiation or de novo organogenesis must be silenced to maintain callus cell pluripotency and identity. Indeed, genes that can induce cell differentiation into stomata, trichomes, xylem tracheary cells, root hair, and others are not expressed (RPKM < 1) in the callus. One example is the SPEECHLESS ( SPCH ), MUTE , and FAMA TFs regulating stomatal development and guard cell differentiation 40 , 41 . Key TFs that are sufficient to trigger de novo organogenesis, for instance, the WUSCHEL (WUS ) gene, that its expression alone in callus, leaves, or roots, induces shoot meristem formation 42 , 43 , the LEAFY ( LFY ) that can direct flower formation 44 or AGAMOUS ( AG ), that its miss-expression leads to carpel formation 45 , 46 are also not expressed in the callus. H3K4me3 enrichment in the callus correlates with gene expression. Acquiring new cell fates and maintaining cell identity rely on the establishment of lineage-specific transcriptional programs, which are primarily regulated by epigenetic mechanisms 47 . To characterize the H3K4me3 and H3K27me3 epigenetic marks, which are associated with transcriptional activation and repression, respectively 48 , 49 , we performed ChIP-seq analyses on nuclei isolated from callus using H3K4me3- and H3K27me3-specific antibodies. In total, 16,161 genes were found to be marked by H3K4me3 (Supplementary Data 4), with an enrichment typically covering ~ 800bp of the gene, starting at the transcription start site (TSS) and peaking within ~ 200bp downstream to the TSS (Fig. 2 a), consistent with the hallmark of H3K4me3 at gene TSSs 49 . The H3K4me3 coverage profile across the TSS for all expressed genes, categorized into four expression level quantiles, demonstrates a strong correlation between H3K4me3 and gene activation (Fig. 2 a), suggesting its consistent role in transcription. Overlapping the callus-expressed genes with the H3K4me3-marked genes reveals three groups (Fig. 2 b): 14,502 expressed genes marked by H3K4me3, accounting for 87% of all expressed genes, indicating that H3K4me3 functions as a general mechanism rather than a regulator of a specific gene set; 2,201 expressed genes-not marked by H3K4me3, indicating that the presence of the H3K4me3 mark is not imperative for gene expression. However, as this analysis was performed at a population level, some genes may show mixed signals, with H3K4me3 and transcription co-occurring in some cells but absent in others. This averaging dilutes the H3K4me3 signal below the cutoff, while expression levels remain above it; 1659 non-expressed-marked by H3K4me3 genes, including known bivalent genes like FLOWERING LOCUS C , NGATHA3 and BLADE ON PETIOLE 1 , which are marked with both H3K4me3 and H3K27me3 and silenced 50 , 51 , suggesting that some genes within this group are potential candidates for bivalency. Other genes in this group might be marked with H3K4me3 to be poised for later expression, as was suggested for zebrafish 52 . An example for genes in each group is given in Fig. 2 c. The H3K27me3 mark in the callus is associated with a specific set of silent genes Mapping the H3K27me3 mark in the callus reveals enrichment across the gene body of silenced genes (Fig. 3 a), consistent with other reports 53 . However, only 3413 genes out of the 12,717 that exhibit no expression (RPKM < 1) were marked with H3K27me3, indicating that this repressive mark regulates a specific set of genes. This is consistent with the 3,991 genes marked by H3K27me3 in callus, as identified by ChIP-chip analysis 5 . Surprisingly, 531 marked genes are associated with active transcription (Fig. 3 b, 3 c and Supplementary Data 5), suggesting either that H3K27me3 is also associated with a distinct transcriptional outcome as reported for other organisms and for Arabidopsis 21 , 54 , 55 , or that it is the result of averaging cells with a distinct feature. Next, we performed GO analysis on the 3,413 H3K27me3-silenced genes. We identified a significantly overrepresented gene group, including 43 miRNA genes, suggesting that H3K27me3 indirectly promotes gene expression by silencing miRNAs, as well as 28 AGAMOUS-like ( AGL ) genes from the 108 MADS-box genes in Arabidopsis. Most striking was the group of TFs, consisting of 467 genes (Supplementary Data 6), out of the 2,192 TFs in Arabidopsis (2.73-fold enrichment, p < 5E-16). This group includes 202 genes classified under the "developmental process" term, many of which are sufficient to direct cell differentiation or de novo organogenesis (Fig. 4 ). One example is the set of TFs required for stomatal differentiation, including SPCH , MUTE , and FAMA 22 , 56 and other epidermal cell differentiation TFs like PROTODERMAL FACTOR 2 , HOMEODOMAIN GLABROUS 2 ( HDG2 ), HDG5 , and MERISTEM LAYER 1 . Another example is the group of abaxial regulatory genes, which have mutual antagonistic interactions with adaxial regulatory genes to establish organ polarity. The abaxial genes are required for specifying cell identity away from the shoot apex side in all lateral organs, for example, the lower side of a leaf. In the callus, genes of this group are marked by H3K27me3 and are silenced (Fig. 4 ), consistent with the view that abaxial cell fate may be the default pattern of differentiation 57 . Remarkably, the genes known to specify adaxial identity, the side close to the shoot apex, for example, the leaf upper side 58 , are marked by H3K4me3 and expressed in the callus (Fig. 4 b). From the list of 3413 genes, we also identified 339 as potentially bivalent genes that are marked with both H3K4me3 and H3K27me and show no expression (RPKM < 1) (Supplementary data 7), that potentially could maintain transcriptional plasticity 59 . Among them, we found 44 genes identified as putative bivalent genes based on ChIP-seq analysis done on Arabidopsis seedlings 50 , thereby confirming the reliability of our analyses. In summary, our results support the hypothesis that keeping many developmental pathways active while maintaining dominant genes silenced to safeguard pluripotent cell identity provides a capacity to rapidly respond to stimuli and regenerate accordingly. However, this strategy requires a mechanism to silence genes upon regenerative stimulus to allow one developmental pathway to dominate and reinforce lineage commitment. emf2 callus exhibits impaired regeneration In the WT callus, 467 genes encoding for TFs are marked by H3K27me3 and silenced, including at least 202 developmental regulators (Supplementary Data 6). To test the prominence of the H3K27me3 mark on callus cell identity and its capacity to regenerate, we produced callus from the PRC2 mutant emf2 -1 (hereafter emf2 ) and analysed its capacity to regenerate (Fig. 5 ). The emf2 mutant was shown to be incapable of forming callus from leaf or cotyledons 5 , probably due to precocious differentiation. To overcome this hindrance, we sowed the mutant and WT directly on callus-inducing media (CIM) to allow the embryonic cells to proliferate, followed by trimming the cotyledons and re-culturing them on CIM. This resulted in emf2 and WT calli, which were phenotypically similar (Fig. 5 a). Next, we tested the callus cell self-identity, i.e., how the callus cells coordinate their inherent cellular programs without any external stimuli. We transferred 100 calli of each genotype, WT, and emf2 , to a hormone-free medium and cultured them in the dark (50 calli) or under light (50 calli) (Fig. 5 b). In the dark, WT calli developed roots, first detected after 13 days. By day 20, all calli had developed roots. This suggests that either the root program is dominant in the absence of hormonal stimuli and light signalling or that the auxin absorbed on CIM remains stable in the dark, thereby promoting root formation. For emf2 calli, root initials were observed on 60% of calli only after day 20. However, these initials failed to elongate further, and all calli began to accumulate dark brown pigment and eventually died (Fig. 5 b). Under light conditions by day 7, 50% of the WT calli and 32% of the emf2 calli accumulated green colour, while the calli of both genotypes remained vigorous and continued to grow in size. At 20 days, 60% of the WT calli and 70% of the emf2 calli developed root initials. From this point onward, the WT calli gained a deep green colour, while all the emf2 calli turned brown and decayed. Both genotypes did not develop shoots during the 47 days of the experiment, indicating that light alone is not a sufficient stimulus to induce de novo shoot meristem formation, although it can drive chloroplast biogenesis. To test the capacity of the emf2 callus to respond to hormonal signals and regenerate accordingly, we cultured the WT and emf2 calli on root or shoot-inducing media (RIM or SIM). For each genotype, 100 calli were cultured on RIM in the dark and 100 calli on SIM under light. After 7 days on RIM, root initials developed on all calli of both genotypes. However, the WT roots elongated and formed lateral roots, while the emf2 roots remained short and eventually decayed, coinciding with the accumulation of a dark tone in the callus (Fig. 5 c). On SIM, green colour accumulated in both genotypes, but it appeared earlier in WT, with 98% of the calli showing green sections by day four, compared to 42% in emf2. By day 15, 96% of the WT calli had regenerated multiple vegetative shoots per callus, whereas only 8% of the emf2 calli had regenerated a single shoot in the reproductive phase (Supplementary Fig. 1). By day 30, all the emf2 calli on SIM had decayed, while the WT calli remained vigorous (Supplementary Fig. 2). These results indicate that without a functional EMF2 complex callus cells fail to acquire new cell identity and regenerate. emf2 callus displays minor changes in gene expression Seedlings of emf2 display reduced H3K27me3 on 54% of the WT-marked genes 60 . To study the impact of EMF2 mutation on transcription in callus, we performed differential gene expression analysis on mRNA-Seq from six-week-old WT and emf2 calli. This analysis yielded surprising results. Out of the 16,646 expressed genes in emf callus (RPKM > 1, Supplementary data 8), only 812 genes exhibited differential expression (p < 0.05): 374 up- and 438 down-regulated genes in emf2 calli as compared to WT, leaving 15,834 genes with similar expression levels (Fig. 6 a Supplementary data 9). These low numbers are surprising, considering that over 2,500 genes were differentially expressed between WT and emf2 seedlings 60 . However, this disparity could be attributed to differences in tissue composition between the seedlings (vegetative in WT and reproductive in emf2). Furthermore, the high expression of VRN2 (RPKM = 7) in both WT and emf2 calli, a gene with functional similarity to EMF2 61 , might compensate for the loss of EMF2 function. Of the 374 emf2 callus up-regulated genes, 131 are silenced (RPKM < 1) in WT. To further investigate how the differentially expressed genes might affect the capacity to regenerate, we performed GO analysis (Fig. 6 b). The most enriched biological processes category for the 374 up-regulated genes was the "transcription factor activity" term, as 76 genes encode for TFs, from which 63 were marked by H3K27me3 in WT callus. Out of the 63 TFs, 14 genes belong to the Type-II MICKC sub-family of the MADS-box TFs, which control flowering transition and development 62 . For example, the SEPALLATA 2 gene required for petals, stamens, and carpels development 63 is marked with H3K27me3 in WT callus and shows no expression (RPKM of 0.06), whereas in emf2 exhibits a high expression level (RPKM of 8.5, Log2 Fold Change = 7). The AGL72 TF, which is involved in floral transition 64 , shows an RPKM value of 9.4 in emf2 and no expression in WT (|log2 Fold Change = 8.5). This is also reflected in the significant enrichment of the "flower development" term (GO:0009908, p < 8.E-07) and is consistent with the emf2 phenotype of flowering upon germination 64 , as well as the regeneration of flower from the emf2 callus (supplementary Fig. 1). Remarkably, ten up-regulated genes in the emf2 calli are explicitly associated with carpel development 65 . For example, SEEDSTICK ( STK ), which is sufficient to induce the transformation of sepals into carpeloid organs and to promote carpel development in the absence of AG activity 66 , showed Log2 Fold Change of 7.4. The enriched GO term "cell differentiation " (GO:00030154, p < 8E-08) stands out because the expression of genes that promote cell differentiation might contribute to the reduced capacity of the emf2 callus to regenerate. Up-regulated genes in this term include TFs involved in xylem fibres differentiation like VASCULAR-RELATED NAC-DOMAIN 3 and 6 and ACAULIS 5 67 , hair cell differentiation ( HDA18 ), and leaf cellular differentiation ( TCP 2, 10 and 17) 68 , suggesting that EMF2 represses those genes in WT callus to prevent cell differentiation, thereby safeguard pluripotency identity. The emf2 callus also exhibits 438 down-regulated genes, potentially an indirect consequence of the upregulation of 76 TF-encoding genes. To test this, we performed GO analysis on the 438 down-regulated genes (Supplementary Table 4) and revealed remarkable enrichment in categories related to photosynthesis, consistent with similar results with emf2 seedlings 69 . For example, 33 of 113 photosynthesis genes (GO:0015979, p < E-31) and 48 of 322 thylakoid genes (GO:0009579, p < E-31) are downregulated in emf2 callus. emf2 exhibits minor changes in H3K4me3 The H3K4 histone methyltransferases antagonize PcG-mediated repression 10 , but how PcGs affect their activity remains unclear. To investigate the impact of the EMF2 mutation on H3K4me3 distribution, we conducted ChIP-seq analysis on emf2 and WT callus using H3K4me3-specific antibodies. In the emf2 callus, the H3K4me3 mark was detected on 16,173 of the genes (Supplementary Data 10), from which 90% (14,562) are expressed (RPKM > 1), demonstrating a positive correlation with expression similar to WT. Next, we analysed the differential levels of enriched H3K4me3 peaks between WT and emf2 calli using edgeR algorithm 70 (Supplementary data 11). Surprisingly, only 214 genes exhibited significantly greater signals in the emf2 callus, from which 109 genes are marked by H3K27me3 in WT (Fig. 6 c, grey triangles). Strikingly, 1,458 genes had weaker signals in the emf2 callus compared to WT, which might stem from the indirect effect of TF and miRNA upregulation. To summarize our analyses, we generated heat maps for WT H3K4me3 marked genes only, sorted by the H3K4me3 signal from the highest to lowest, demonstrating that H3K4me3 displays anti-correlation with H3K27me3 and strong correlation with expression (Fig. 6 d). The emf2 callus exhibits a similar pattern except for the 56 genes that are marked by H3K27me3 in WT, acquired the H3K4me3 mark in the emf2 and gained expression (The box at the bottom of the heat maps and Fig. 7 ). Discussion How plant callus cells maintain their pluripotent identity is not fully understood. In this work, we reveal that a callus exhibits a unique gene expression pattern that promotes a high proliferation rate but also maintains the potential to initiate many of the lineages required to develop a mature plant. One mechanism for maintaining pluripotency in mammalian embryonic stem cells is retaining a globally open chromatin state through basal transcription 71 , 72 . We identify a related strategy in callus, where lineage-affiliated genes are kept transcriptionally active, allowing for a rapid response to developmental signals without the need to go through the complex transcription process. However, such a strategy requires a complementary mechanism to prevent precocious differentiation. Gene priming, which keeps genes poised for later activation, may also contribute to the high regenerative competency of plant cells 7 . We propose two independent mechanisms for maintaining callus identity; one is keeping the cell cycle machinery active, which prevents differentiation 73 . This is supported by the observation that upon culturing the callus on media devoid of proliferation-instructive signals, the cells differentiate (Fig. 5 ). The central feature of plant pluripotent stem cells is their slow mitotic rate 38 , 74 , 75 . However, callus cells exhibit highly proliferative characteristics, demonstrating that pluripotency is not necessarily unique to slow-dividing cells; another mechanism involves silencing dominant transcription factors through epigenetic modifications, which are sustained during cell proliferation. Pluripotent cells primarily possess dual capacities: maintaining the cellular identity of less differentiated cells and having the capacity to form multiple cell lineages. This requires the plasticity to reprogram epigenetic states in response to signals, to shut down competing programs, and to establish lineage-specific transcriptional programs. Here, we highlight the crucial role of H3K27 tri-methylation in enabling callus differentiation and regeneration. We further demonstrate that the emf2 callus cannot coordinate these processes effectively. We show that emf2 callus can be established and proliferate in culture, indicating that hormone perception and signal transduction are not compromised. In addition, it indicates that the EMF2 component of the PRC2 is dispensable for cell proliferation, consistent with the view that self-renewal in mammalian ES cells is generally not subjected to repressive epigenetic mechanisms 76 . It is further supported by the highly proliferative callus derived from two transgenic plants expressing the H3.3 K 27 A or H3.15 histone variants, both lacking lysine residue 27, suggesting that reduced levels of H3K27me3 promote cell proliferation 28 , 77 , 78 . We found that only 812 genes are differentially expressed between emf2 and WT. We can propose several scenarios and explanations for the low number of up-regulated genes in emf2 : 1. EMF2 might regulate a small set of genes. However previous study showed 54% reduction in total H3K27me3 in emf2 seedlings compared with WT 60 ; 2. VRN2 might target overlapping genes and largely compensate for the loss of EMF2 function. That scenario highlights the exact set of genes specially regulated by EMF2: marked by H3K27me3 in WT callus and gained the H3K4me3 mark and expression in emf2 ; 3. There is a massive reduction in H3K27me3 in emf2 , but removing this repressive mark does not necessarily activate genes. This phenomenon was demonstrated, for example, in mouse ES cells carrying a mutation in Ezh2 , the HMTase component of PRC2, where the loss of H3K27me3 mark in numerous loci resulted in transcriptional activation of only one-third of the marked loci 79 . Our inability to perform ChIP-seq analysis of H3K27me3 in emf2 callus due to insufficient precipitated DNA for sequencing further supports this scenario. The normal development of emf2 callus, despite the up-regulation of 76 genes encoding for TF, some of which can promote differentiation and organogenesis, suggests that the signal for keeping the cell cycle machinery active is more robust. Yet, the failure to differentiate upon removal of the proliferative signal or in response to regenerative signals (RIM and SIM) suggests that activating lineage-specific gene networks to commit to a single developmental path requires a capacity to turn off pluripotency networks. Several MADS-box TFs were shown to be involved in downregulating the expression of photosynthetic genes to confer flower identity 80 , 81 . Therefore, the enrichment in photosynthesis-related genes among the emf2 -downregulated genes might be the direct effect of MADS-box genes' upregulation in emf2 callus. The activation of the LOB domain 29 ( LBD29 ) TF in the emf2 callus, which was shown to repress genes involved in photosynthesis directly 82 , might also contribute to this enrichment. In Arabidopsis, mutations in EMF2 induce immediate flowering after germination 83 . In general, flowering can be promoted by environmental cues such as long-day photoperiod or low-temperature, leading to the activation of floral integrators genes, FLOWERING LOCUS T ( FT), SOC1, TWIN SISTER OF FT ( TSF ) and LFY 84 . Those TFs induce the transition to flowering, leading to floral meristem specification, followed by the activation of genetic networks comprised mainly of MADS-box genes to specify floral organ identities 85 . In our study, we cultured the callus under conditions that do not promote flowering (under dark at 22C 0 ), and the four integrator genes showed no expression in WT and emf2 calli. However, many downstream MADS-box genes are up-regulated in emf2 , demonstrating that activation of floral organ specification genes is independent of the transition to flowering. This result suggests that the four integrators are required solely to release the suppression and not for direct activation of the downstream MADS-box genes. There is no strong consensus on the homogeneity of callus tissue, and the question of whether callus cells share a similar cellular identity remains open 2 . Our comparative analysis between WT and emf2 calli identified only 324 up-regulated genes, demonstrating the similarity of the two calli and suggesting that the callus produced from embryonic tissue is more unified. This can be tested in the future by performing a transcriptomic analysis at single-cell resolution 86 , with the recognition that it is not a bias-free method. Our approach offers an excellent solution for yielding unbiased results in functional genomic studies, including the study of pluripotency, transcriptome analysis, and chromatin state comparisons across various mutants lacking comparable tissues or organs. Using our experimental system, we could pinpoint the potential genes regulated solely by EMF2, primarily those involved in floral organ specification. A future challenge will be to perform ChIP-seq analysis for EMF2, which could yield data on all the direct targets of EMF2. In summary, we provide insights into H3K27me3 function in establishing pluripotent cell identity in callus by silencing key TFs and contributing to the capacity to initiate new cell lineages. Our findings also provide the starting point for further studies on the genome-wide dynamics of H3K4me3 and H3K27me3 during regeneration. Methods Plant materials and growth conditions Arabidopsis thaliana accession Columbia-0 (Col-0) was used in this work as wild type and the emf2 mutant (ABRC germplasm CS16238) on Col-0 background 64 . For all experiments, seeds were surface sterilized for 10 minutes in 3% Sodium Hypochlorite containing 0.1% Triton X-100 and washed 4 times with DDW before sowing. For the leaf RNA-seq experiment, seeds were sown on Murashige and Skoog (MS) basal medium 87 , and kept under continuous light at 23°C for one week. Seedlings were then transplanted to soil and grown under long-day conditions (16h light/8h dark) at 21°C. Rosette leaves were harvested two weeks after transplanting. Tissue culture To generate callus from Col-0 and emf2 cotyledons, sterilized seeds were sown directly on callus-inducing medium (CIM): Gamborg B5 medium with 0.5g/L MES, 2% dextrose, 0.9% phytagel, supplemented with 2.2µM 2,4-dichlorophenoxyacetic acid and 0.46µM kinetin. Following two days at 4°C, plates were transferred to a continuous light growth room at 23°C. After seven days, cotyledons were trimmed and transferred to a new CIM plate placed in the dark. During the next 5–6 weeks, calli were re-cultured to new CIM plates every 7–10 days. All analyses were done on 6–8 weeks-old calli. Callus internal identity experiments Calli were transferred to Gamborg B5 medium deprived of hormones, and cultured under continuous light or dark conditions. Calli were monitored and photographed under a stereomicroscope. Competency to regenerate tests Calli of WT or emf2 and WT rosette leaves were transferred to either Shoot Inducing Media (SIM): Gamborg B5 medium supplemented with 4.4µM 2-isopentenyl adenine and 0.5µM 1-Naphthaleneacetic acid (NAA), under continuous light conditions or Root Inducing Media (RIM): Gamborg B5 medium supplemented with 0.5µM NAA under dark conditions. RNA extraction To correlate chromatin characteristics with gene expression, tissues from each ChIP experiment were fast-frozen in liquid nitrogen. Samples were ground, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions, including treatment with RNase-free DNase (Qiagen). Each RNA sample was loaded on agarose gel for integrity validation and quantified using NanoDrop. RNA-Sequencing Sequencing was done at the Technion Genome Centre (TGC) according to Illumina's protocols. The TruSeq RNA V2 sample prep kit (RS-122) was used. Total RNA was polyA-selected, followed by fragmentation and random hexamer-primed reverse transcription. Indexed adapters were added, and cDNA was amplified by PCR for 15 cycles. Every four indexed libraries were loaded onto an individual lane of Illumina's HiSeq 2500 system. Chromatin immunoprecipitation ChIP was performed on nuclei isolated from the callus as described 88 , with some modifications. The detailed protocol is found in the Supplementary material file. In short, six-week-old calli samples were harvested from 8 plates. Calli from every two plates were pooled, and 1.5 grams from each pool was placed into a tube and subjected to cross-linking. ChIP validation A comparative semi-quantitative PCR between positive and negative DNA binding sites was performed to validate the ChIP experiment. For each chromatin mark, two sets of primers were designed: positive control (PC) to estimate the positive DNA binding site and negative control (NC) to estimate the negative binding site (Supplementary Table 5). The two sets of primers were tested on the Input DNA sample diluted 1:1,000 and the IP sample. The reaction mix included 7.5µl of GoTaq Green master mix (Promega), 0.4µl of each primer (10µM), and 5.9µl diluted DNA. Along the PCR reaction, samples were taken out from cycle 28 and every 2 cycles and loaded on 2% agarose gel. Chromatin immunoprecipitation sequencing Samples were sequenced at the Technion Genome Centre (TGC) according to Illumina's protocols. The ChIP-seq libraries were prepared using the TruSeq Nano DNA Library Prep Kit (FC-121). ChIP-enriched DNA fragments were size-selected on an agarose gel to enrich for fragments of 200 bp. Indexed adaptors (diluted 1/10) were added, and the library was amplified using PCR for 14 cycles. Every 7–10 indexed libraries were loaded onto an individual lane of Illumina's HiSeq 2500 system for 50 base-pair single-end sequencing. Next Generation Sequencing (NGS) analysis Each NGS analysis included quality control using FastQC 89 and adaptor trimming using cutadapt 71 . NGS reads were mapped to the Arabidopsis thaliana reference transcriptome TAIR10 using TopHat 2 90 for RNA-Seq and Bowtie2 91 for ChIP-Seq. Integrative Genomics Viewer (IGV) was used for visualizing the data 92 . Biological replication consistency was measured using Spearman correlation of BAM files in deepTools 93 . RNA-Seq samples with p > 0.96 and ChIP-Seq samples with p > 0.88 were merged. Declarations Author Contributions Conceptualization: L.E.W. 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Supplementary Files SupplementaryTable12and5.docx Supplementary Table 1, 2 and 5 SupplementaryTable31950developmentgenesinwtcallus.xlsx Supplementary Table 3 SupplementaryTable4438emf2downregulatedgenes.xlsx Supplementary Table 4 SupplementaryData1.xlsx Supplementary Data 1 SupplementaryData2.xlsx Supplementary Data 2 SupplementaryData3.xlsx Supplementary Data 3 SupplementaryData4.xlsx Supplementary Data 4 SupplementaryData5.xlsx Supplementary Data 5 SupplementaryData6.xlsx Supplementary Data 6 SupplementaryData7.xlsx Supplementary Data 7 SupplementaryData8.xlsx Supplementary Data 8 SupplementaryData9.xlsx Supplementary Data 9 SupplementaryData10.xlsx Supplementary Data 10 SupplementaryData11.xlsx Supplementary Data 11 SupplementaryFigures.docx Supplementary Figures 1 and 2 Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-5582331","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":393626397,"identity":"db36e44d-c5b4-4878-9842-f1bee5ebdcab","order_by":0,"name":"Leor Eshed Williams","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-2766-2608","institution":"Hebrew University of Jerusalem","correspondingAuthor":true,"prefix":"","firstName":"Leor","middleName":"Eshed","lastName":"Williams","suffix":""},{"id":393626398,"identity":"305bacff-f662-40dd-9d95-c6e1738f2c80","order_by":1,"name":"Tali Mandel","email":"","orcid":"","institution":"MIGAL Galilee Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Tali","middleName":"","lastName":"Mandel","suffix":""},{"id":393626399,"identity":"f0af85eb-060a-4a11-96df-8c676de5490e","order_by":2,"name":"udi Landau","email":"","orcid":"","institution":"Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"udi","middleName":"","lastName":"Landau","suffix":""},{"id":393626400,"identity":"cd8a1d65-16aa-444e-baba-0c5b08b541aa","order_by":3,"name":"Tommy Kaplan","email":"","orcid":"https://orcid.org/0000-0002-1892-5461","institution":"The Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Tommy","middleName":"","lastName":"Kaplan","suffix":""},{"id":393626401,"identity":"514d2d35-62ab-43be-b611-c663fdf9c6b1","order_by":4,"name":"Yotam Cohen","email":"","orcid":"","institution":"Hebrew University of Jerusalem","correspondingAuthor":false,"prefix":"","firstName":"Yotam","middleName":"","lastName":"Cohen","suffix":""}],"badges":[],"createdAt":"2024-12-04 20:35:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5582331/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5582331/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74646346,"identity":"23176287-d3ca-4fd7-9bb4-be1b759b9347","added_by":"auto","created_at":"2025-01-24 09:56:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":460855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWT callus exhibits enrichment in genes involved in diverse developmental pathways\u003c/strong\u003e. Six-week-old Arabidopsis calli, derived from cotyledons and leaves of three-week-old plants, were subjected to mRNA-seq analysis in three replicates. \u003cstrong\u003ea\u003c/strong\u003e Scatter plot of differential gene expression between \u003cem\u003eemf2\u003c/em\u003e and WT callus. Red dots represent \u003cem\u003eemf2\u003c/em\u003e-upregulated genes (FDR \u0026lt; 0.05), blue dots represent emf2-downregulated genes (FDR \u0026lt; 0.05), and black dots indicate genes with no significant change in expression. \u003cstrong\u003eb\u003c/strong\u003e Gene Ontology (GO) enrichment analysis of callus-expressed genes: within each GO category, white bars represent the total number of genes in the GO category, black bars indicate the number of callus-expressed genes (RPKM \u0026gt; 1), and red bars show the number of callus-expressed genes upregulated compared to leaf tissue. The y-axis represents the percentage of genes within each GO category. The q-value, annotated above each bar, indicates the statistical significance of enrichment for the respective GO term. Enriched GO terms include nucleic acid metabolic process (GO:0090304), cell cycle (GO:0007049), multicellular organismal development (GO:0007275), transcription (GO:0006350), and response to endogenous stimulus (GO:0009719). \u003cstrong\u003ec\u003c/strong\u003eDetailed Gene Ontology (GO) analysis for the 1,950 callus-expressed genes within the multicellular organismal development (GO:0007275) term. For each pathway, the number of callus-expressed genes relative to the total number of genes in the term is indicated. The complete list of genes associated with each term is provided in Supplementary Table 3.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/8672ce9a03e25aed90c45753.png"},{"id":74646343,"identity":"a79186a7-3938-461c-9ad1-41a1d33780a2","added_by":"auto","created_at":"2025-01-24 09:56:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":223704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eH3K4me3 enrichment correlates with gene expression in the callus. a\u003c/strong\u003e Profile of normalized H3K4me3 signal across the Transcription Start Site (TSS) for genes categorized into five groups based on their expression levels in callus tissue: Silent genes with no detectable expression and four quantiles based on expression levels. The x-axis represents the distance from the TSS in kilobases (kb), and the y-axis represents the normalized H3K4me3 signal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Venn diagram showing the overlap between callus-expressed genes and callus H3K4me3-marked genes. The number of genes in each category is indicated within the diagram. On the right, a histogram displays the distribution of genes according to their RPKM values for H3K4me3 non-marked genes (orange) and all expressed genes (black). On the left, a histogram shows the distribution of H3K4me3 log(Q-value) for H3K4me3-marked, non-expressed genes (blue) and all H3K4me3-marked genes. \u003cstrong\u003ec\u003c/strong\u003e Integrated Genome Viewer (IGV) tracks of mRNA-seq and ChIP-seq signals: Examples of genes showing distinct correlations between H3K4me3 enrichment (blue) and mRNA expression levels (black). The tracks display H3K4me3 ChIP-seq signals in the upper panels and corresponding mRNA-seq signals in the lower panels. Gene models are shown below, with red arrows indicating the transcriptional direction.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/e5e7f99c5c466c1ce7e5856c.png"},{"id":74646344,"identity":"b6cb1086-e80c-424a-b39d-bf9f7e38d1d7","added_by":"auto","created_at":"2025-01-24 09:56:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":188916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe H3K27me3 mark in the callus is associated with the transcriptional repression of a specific set of genes. a \u003c/strong\u003eH3K27me3 exhibits a broad enrichment domain across gene bodies, from -0.5 kb upstream of the transcription start site (TSS) to 0.5 kb downstream of the transcription end site (TES). The ChIP-seq profiles are shown for all Arabidopsis genes, divided into the following categories: silent genes, further classified into H3K27me3 non-marked (black) and H3K27me3 marked (red), and expressed genes, categorised into high (blue), medium (green), and low (yellow) expression levels. \u003cstrong\u003eb\u003c/strong\u003e Venn diagram shows the overlap between callus non-expressed genes and callus H3K27me3-marked genes, with the number of genes in each category indicated. On the right, a histogram displays the distribution of gene expression levels (RPKM values) for expressed H3K27me3-marked genes (red) and all expressed genes (black). \u003cstrong\u003ec\u003c/strong\u003e Integrative Genomics Viewer (IGV) tracks illustrate mRNA-seq and ChIP-seq signals, providing examples of silenced developmental genes marked by H3K27me3 and a case where H3K27me3-marked genes are expressed, representing an exception to the trend.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/6756bfcf1c73c80846edea35.png"},{"id":74645048,"identity":"c77cc15d-5565-4152-9097-3a527e00c39e","added_by":"auto","created_at":"2025-01-24 09:48:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135895,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenes sufficient to direct differentiation or de novo organogenesis are silenced in callus\u003c/strong\u003e. IGV genome browser screenshot for mRNA and ChIP-seq data of H3K4me3 and H3K27me3: \u003cstrong\u003ea\u003c/strong\u003e Examples of transcription factors sufficient to promote differentiation processes, including stomata (SPEECHLESS and FAMA), trichome (GLABRA1), and fibre (NST1, NST3, and MYB85) development. These genes are marked by H3K27me3 (red) and are not expressed. \u003cstrong\u003eb\u003c/strong\u003e The antagonistic adaxial and abaxial identity genes exhibit distinct H3K27me3 and H3K4me3 landscapes. In the upper panel, adaxial polarity-specifying genes (e.g., REVOLUTA, PHABULOSA, CORONA, and PHAVOLUTA) are marked by H3K4me3 (blue) and actively expressed (black) in callus. In the lower panel, YABBY and KANADI genes, known for promoting abaxial specification, are silenced and marked by H3K27me3 (red).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/0146fcdfaad8db04b6b6d689.png"},{"id":74645051,"identity":"e2843f0e-f31b-4ec2-9fda-282d8bfbc7db","added_by":"auto","created_at":"2025-01-24 09:48:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1268699,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eemf2 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ecallus exhibits impaired regeneration capacity\u003c/strong\u003e. \u003cstrong\u003ea\u003c/strong\u003e On the left are seedlings of WT and \u003cem\u003eemf2\u003c/em\u003e. On the right is a callus generated from cotyledons of WT and \u003cem\u003eemf2\u003c/em\u003e seeds, which were sown directly onto a callus-inducing medium (CIM), trimmed on day six, and re-cultured on CIM. \u003cstrong\u003eb\u003c/strong\u003e Cell self-identity test: Five-week-old WT or \u003cem\u003eemf2\u003c/em\u003e calli were transferred to B5 medium without hormones and cultured either in the dark or under light conditions. Representative images were captured on days seven and 25. \u003cstrong\u003ec\u003c/strong\u003e Regeneration test: Five-week-old WT or \u003cem\u003eemf2\u003c/em\u003e calli were transferred to root-inducing medium (RIM) or shoot-inducing medium (SIM). Representative images were captured on day 25 (on the left bottom side of the plate on the right upper side).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/85d4daf27024a11946ae9f1b.png"},{"id":74645042,"identity":"129b5c46-45b9-4d19-b96a-4f82ebaad890","added_by":"auto","created_at":"2025-01-24 09:48:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":454745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eemf2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e callus exhibits minor alterations in gene expression and H3K4me3 acquisition, with an enrichment of upregulated genes encoding transcription factors. a\u003c/strong\u003e Scatter plot of differential gene expression between \u003cem\u003eemf2\u003c/em\u003e and WT callus. Red dots represent \u003cem\u003eemf2\u003c/em\u003e-upregulated genes (FDR \u0026lt; 0.05), blue dots represent \u003cem\u003eemf2\u003c/em\u003e-downregulated genes (FDR \u0026lt; 0.05), and black dots indicate genes with no significant change in expression. \u003cstrong\u003eb \u003c/strong\u003eGene Ontology (GO) biological processes enriched in differentially expressed genes in \u003cem\u003eemf2\u003c/em\u003e compared to WT callus. Red bars: GO terms for genes upregulated in \u003cem\u003eemf2\u003c/em\u003e. Blue bars: GO terms for genes downregulated in \u003cem\u003eemf2\u003c/em\u003e. The x-axis represents fold enrichment. In the middle: Euler diagram showing significant enrichment (P = 5.32e-16) of transcription factor (TF)-encoding genes (76, gray) among \u003cem\u003eemf2\u003c/em\u003eupregulated genes (374, red). The P-value was calculated using a hypergeometric test with the following data: 2,192 TFs out of 29,420 genes in Arabidopsis (based on TAIR) and 76 TFs out of the 374 \u003cem\u003eemf2\u003c/em\u003e upregulated genes. The number of genes in each category is indicated. On the right: Pie chart showing the distribution of TF families among \u003cem\u003eemf2\u003c/em\u003e upregulated genes. The most significant proportion belongs to the MADS family. \u003cstrong\u003ec\u003c/strong\u003e Scatter plot showing differential H3K4me3 modifications between \u003cem\u003eemf2\u003c/em\u003e and WT calli. Red dots: Significant enrichment of the H3K4me3 mark. Blue dots: Significant reduction of the H3K4me3 mark. Grey triangles highlight 109 genes marked by H3K27me3 in WT. Black dots represent genes with no significant change. \u003cstrong\u003ed \u003c/strong\u003eH3K4me3 anti-correlates with H3K27me3 and correlates with gene expression. Heatmaps of WT H3K4me3-marked genes sorted by H3K4me3 signal strength (highest to lowest). Panels show H3K4me3 and H3K27me3 ChIP-seq occupancy (-0.5 kb to +1.5 kb for H3K4me3, -0.5 kb to +5 kb for H3K27me3) and mRNA-seq for WT and \u003cem\u003eemf2\u003c/em\u003ecallus. The box at the bottom represents the 56 genes that acquired the H3K4me3 mark in the \u003cem\u003eemf2\u003c/em\u003e callus (In WT, showing strong H3K27me3 signal, no H3K4me3 signal, and no expression; in \u003cem\u003eemf2\u003c/em\u003e, high H3K4me3 signal and expression).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/88fb1f3ddbebb1a1632845af.png"},{"id":74645055,"identity":"5493ce3e-683c-43f9-ab17-c9055a634607","added_by":"auto","created_at":"2025-01-24 09:48:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":379660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIGV tracks showing H3K27me3, H3K4me3, and mRNA profiles for selected genes. a \u003c/strong\u003eLeft: A gene in WT marked by H3K27me3 (red), with no H3K4me3 enrichment and no expression in both WT and \u003cem\u003eemf2\u003c/em\u003e. Middle: Genes in WT without H3K27me3 enrichment, marked by H3K4me3, and expressed in WT and \u003cem\u003eemf2\u003c/em\u003e. Right: Genes in WT marked by H3K27me3 and H3K4me3 (potential bivalent genes) with no expression, which in \u003cem\u003eemf2\u003c/em\u003egain H3K4me3 enrichment and are expressed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Examples of genes that gained expression in \u003cem\u003eemf2\u003c/em\u003e callus. Left: Genes marked by H3K27me3 in WT, showing no H3K4me3 enrichment or expression, which in \u003cem\u003eemf2\u003c/em\u003eacquire H3K4me3 and are expressed. Right: Example of genes that gained expression in \u003cem\u003eemf2\u003c/em\u003e without acquiring H3K4me3.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/8575b1673952f4b7fe7e57dd.png"},{"id":88559329,"identity":"6b4ff5b6-2c0e-4d3a-9d94-ac943d0576cd","added_by":"auto","created_at":"2025-08-07 17:33:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4872358,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/9763b778-bc9a-40c2-9ea8-fe0d56ce5044.pdf"},{"id":74645045,"identity":"e1740f51-5e14-45b1-8296-ee0485c7bec0","added_by":"auto","created_at":"2025-01-24 09:48:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":30754,"visible":true,"origin":"","legend":"Supplementary Table 1, 2 and 5","description":"","filename":"SupplementaryTable12and5.docx","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/76f8355a4ae748a49e11f68b.docx"},{"id":74646679,"identity":"6cec6c1c-9a41-4e9f-94f2-d4c145f0e363","added_by":"auto","created_at":"2025-01-24 10:04:01","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":469842,"visible":true,"origin":"","legend":"Supplementary Table 3","description":"","filename":"SupplementaryTable31950developmentgenesinwtcallus.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/9b2867fa74823d4442caa1ba.xlsx"},{"id":74646345,"identity":"2f9a34d7-5b67-4937-ad4d-0e198ddda743","added_by":"auto","created_at":"2025-01-24 09:56:01","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":46773,"visible":true,"origin":"","legend":"Supplementary Table 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10\u003c/p\u003e","description":"","filename":"SupplementaryData10.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/5c52498a55dfd09bf9599f7d.xlsx"},{"id":74645078,"identity":"82fc34a9-911c-4420-b52f-dda2b2c92d68","added_by":"auto","created_at":"2025-01-24 09:48:02","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":170871,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data 11\u003c/p\u003e","description":"","filename":"SupplementaryData11.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/c56b410796dc1b37b92e1a28.xlsx"},{"id":74645071,"identity":"ce52fe28-d28d-4889-a4c2-4dca0547ab85","added_by":"auto","created_at":"2025-01-24 09:48:02","extension":"docx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":230094,"visible":true,"origin":"","legend":"Supplementary Figures 1 and 2","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5582331/v1/b79771a41ee4f51bf3ae31b4.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCertain plant cells retain plasticity and are capable, under appropriate stimuli such as wounding or hormone stimulation, to switch their developmental program, re-enter the cell cycle, and form a mass of less-differentiated pluripotent cells, termed callus\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. These cells acquire the competence to rapidly respond to diverse stimuli and adopt new fates accordingly\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, to regenerate organs that often differ from their respective origins. For example, auxin induces root regeneration, while a low auxin/cytokinin ratio promotes shoot induction\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Although numerous studies have investigated competency acquisition during callus formation, highlighting the significant role of epigenetic regulation in this process\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, the mechanisms governing callus cell identity as proliferative, pluripotent cells with a high regenerative capacity remain elusive.\u003c/p\u003e \u003cp\u003eThe remarkable process of acquiring new cell fates and forming new lineages during organ regeneration requires the establishment of specific gene expression patterns and a cellular memory system to transmit these patterns through cell divisions to maintain cellular identity \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCentral to controlling developmental gene expression during cell fate transitions is the tri-methylation of histone H3 at lysine 27 (H3K27me3), a repressive chromatin modification catalysed by the Polycomb Repressive Complex 2 (PRC2)\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe PRC2 was first elucidated in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e by identifying mutants exhibiting homeotic conversions due to ectopic expression of the \u003cem\u003eHox\u003c/em\u003e genes\u003csup\u003e\u003cb\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/b\u003e\u003c/sup\u003e. PRC2 is a four-core subunit complex, highly conserved across higher organisms\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. It is the only known complex with H3K27me3 methyltransferase activity, and its importance in numerous organisms is highlighted by the severe developmental defects and even lethality caused by mutation in PRC2 subunits\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The subunits of the Drosophila PRC2, each encoded by a single-copy gene, include the catalytic component Enhancer of zeste (E(z)); the non-catalytic partners, Suppressor of zeste 12 (Su(z)12) and Extra sex combs (Esc), which directly contact E(Z) and are indispensable for the methyltransferase activity; and the Nucleosome remodelling factor 55 kDa (Nurf55)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the Arabidopsis thaliana model plant, the PRC2 core subunits, with the exception of the Esc homolog FERTILISATION INDEPENDENT ENDOSPERM (FIE), are encoded by multigene families and assemble in various combinations to form three distinct PRC2 complexes, named after their respective Su(z)12 homolog components: the \u003cem\u003eEMBRYONIC FLOWER2\u003c/em\u003e (\u003cem\u003eEMF2\u003c/em\u003e), the \u003cem\u003eFERTILISATION INDEPENDENT SEED\u003c/em\u003e (\u003cem\u003eFIS2\u003c/em\u003e), and the \u003cem\u003eVERNALISATION 2\u003c/em\u003e (\u003cem\u003eVRN2\u003c/em\u003e) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Each complex controls various processes and developmental programs with some redundancy, including cell fate specification, embryogenesis, vegetative growth, and flowering time\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. EMF2 silences genes associated with floral induction and reproductive development to ensure sufficient vegetative growth before flowering. Accordingly, \u003cem\u003eEMF2\u003c/em\u003e mutations in Arabidopsis trigger flowering immediately upon germination, bypassing the vegetative growth phase\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe genomic distribution of H3K27me3 in plants is dynamic and strongly correlates with\u003c/p\u003e \u003cp\u003etranscriptional repression, impacting hundreds of genes, many of which play roles in development\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. ChIP-Seq analyses across diverse plant species have revealed extensive H3K27me3 reprogramming during biotic stress responses\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and abiotic stress responses\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, cell differentiation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e, and developmental processes. For instance, the temporal and sequential expression of genes controlling cell fate switch during flower morphogenesis requires the differential deposition or removal of H3K27 methylation on numerous genes \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Other prominent examples include H3K27me3 redistribution during the differentiation of stem cells at the shoot apical meristem\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e or, conversely, during the acquisition of a pluripotent state, both in the male gametophyte\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e and callus formation\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eH3K27me3 deposition was also shown to be essential for callus formation from leaf tissue. This is evidenced by the impaired callus formation observed in Arabidopsis leaf explants of PRC2 mutants, including \u003cem\u003eCURLY LEAF\u003c/em\u003e (\u003cem\u003eCLF\u003c/em\u003e), \u003cem\u003eSWINGER\u003c/em\u003e (\u003cem\u003eSWN\u003c/em\u003e), and \u003cem\u003eEMF2\u003c/em\u003e\u003csup\u003e5\u003c/sup\u003e. Further support comes from defective callus formation in the hypocotyls of the \u003cem\u003eHTR15\u003c/em\u003e mutant, which encodes a histone H3 variant lacking the lysine 27 residue\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, we reveal a mechanism underlying callus cell identity, characterized by the expression of numerous genes from diverse developmental pathways while simultaneously silencing dominant genes that drive differentiation or organogenesis, thereby maintaining pluripotency. We demonstrate that callus derived from the \u003cem\u003eEMF2\u003c/em\u003e mutant exhibits severely impaired regenerative capacity, suggesting that the EMF2 complex is crucial for silencing pluripotency networks to enable commitment to a single developmental pathway. Furthermore, we emphasise the advantages of using callus as a model system for studying transcriptional regulation, as it minimises the averaging effects typical of other systems\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePluripotent callus cells express genes from multiple developmental pathways.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo enhance our understanding of pluripotency and regenerative competence, we compared the transcriptomes of six-week-old Arabidopsis calli derived from cotyledons, which exhibit a high capacity for regeneration, with those of leaves from three-week-old plants, which in Arabidopsis demonstrate little to no capacity for direct regeneration\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. A total of 16,703 genes were expressed in callus and 16,694 in leaves (RPKM\u0026thinsp;\u0026gt;\u0026thinsp;1) (Supplementary Data 1 and 2), with 12,109 genes exhibiting significant differential expression (cutoff of: |log2FC| \u0026gt; 0.6 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Supplementary Data 3), demonstrating the substantial dissimilarity between cells comprising the callus and the leaf (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). As expected, Gene Ontology (GO) analysis revealed that the 6223 genes downregulated in calli (i.e. upregulated in leaves) were significantly enriched for photosynthesis-related biological processes (p\u0026thinsp;\u0026lt;\u0026thinsp;E-76), and plastid and thylakoid-related cellular components (p\u0026thinsp;\u0026lt;\u0026thinsp;E-69), (Supplementary Table\u0026nbsp;1). This reflects the function of the leaf as a photosynthesis factory and indicates that the leaf cells are differentiated\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Selected GO-enriched terms for the 5,886 up-regulated callus genes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb (full terms list in Supplementary Table\u0026nbsp;2). Out of the 3425 genes in the nucleic acid metabolic term (GO:0090304), 2574 are expressed in callus (p\u0026thinsp;\u0026lt;\u0026thinsp;8.07E-75), suggesting high production of nucleic acids required for DNA synthesis in the highly proliferative cells. This is consistent with the enrichment of cell cycle genes, with 316 out of 423 genes involved in the cell cycle process (GO:0007049). The enrichment in response to endogenous stimulus (GO:0009719) was also predictable as the callus is cultured on media supplemented with the phytohormones auxin and cytokinin. However, what caught our attention is the significant enrichment in multicellular organismal development genes (GO:0007275). This was unexpected since callus is considered an unorganized, less differentiated mass of cells and has been shown to be enriched for root developmental genes\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. A detailed GO analysis on the 1950 callus-developmental genes (Supplementary Table\u0026nbsp;3) confirmed that 339 genes are related to root development (GO:0048364; 432 genes), including the root marker genes \u003cem\u003eWUSCHEL RELATED HOMEOBOX 5 (WOX5)\u003c/em\u003e, \u003cem\u003eSHORT ROOT\u003c/em\u003e (\u003cem\u003eSHR\u003c/em\u003e) and \u003cem\u003eSOMBRERO\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Most striking was the significant enrichment of genes participating in many other developmental pathways, including shoot, leaf, flower, embryo, and seed development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). For example, out of the 282 genes in the leaf development term (GO:0048366), 197 (70%) are expressed in callus, including key transcription factors (TFs) essential for leaf morphogenesis such as \u003cem\u003eASYMMETRIC LEAVES 1\u003c/em\u003e (\u003cem\u003eAS1\u003c/em\u003e), \u003cem\u003eCUP-SHAPED COTYLEDON 2\u003c/em\u003e (\u003cem\u003eCUC2\u003c/em\u003e), \u003cem\u003eAUXIN RESPONSE FACTOR 3/\u003c/em\u003eand \u003cem\u003e4\u003c/em\u003e (\u003cem\u003eARF3/4\u003c/em\u003e) and the five members of the Class \u003cem\u003eIII HD-ZIP\u003c/em\u003e genes\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Another example is the activation of meristem development genes (135 out of 174 in the term - GO:0048507), from which many are participating in shoot and inflorescent meristem development, like \u003cem\u003eKNOTTED1-LIKE HOMEOBOX GENE 6\u003c/em\u003e (\u003cem\u003eKNAT6\u003c/em\u003e) and \u003cem\u003eKNAT1\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e. Remarkably, the callus does not express genes specific to stem cells at the shoot apical meristem, such as \u003cem\u003eCLAVATA3\u003c/em\u003e (\u003cem\u003eCLV3\u003c/em\u003e), \u003cem\u003eAINTEGUMENTA-LIKE 7\u003c/em\u003e, and \u003cem\u003eINHIBITOR OF GROWTH 1\u003c/em\u003e\u003csup\u003e38,39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of numerous genes from diverse developmental programs in the callus may enable a rapid response to stimuli and facilitate de novo organogenesis without requiring the release of transcriptional silencing or undergoing the complex, multistep transcription process. In this scenario, we hypothesized that genes encoding for TFs, that can independently induce differentiation or de novo organogenesis must be silenced to maintain callus cell pluripotency and identity. Indeed, genes that can induce cell differentiation into stomata, trichomes, xylem tracheary cells, root hair, and others are not expressed (RPKM\u0026thinsp;\u0026lt;\u0026thinsp;1) in the callus. One example is the \u003cem\u003eSPEECHLESS\u003c/em\u003e (\u003cem\u003eSPCH\u003c/em\u003e), \u003cem\u003eMUTE\u003c/em\u003e, and \u003cem\u003eFAMA\u003c/em\u003e TFs regulating stomatal development and guard cell differentiation\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Key TFs that are sufficient to trigger \u003cem\u003ede novo\u003c/em\u003e organogenesis, for instance, the \u003cem\u003eWUSCHEL (WUS\u003c/em\u003e) gene, that its expression alone in callus, leaves, or roots, induces shoot meristem formation\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, the \u003cem\u003eLEAFY\u003c/em\u003e (\u003cem\u003eLFY\u003c/em\u003e) that can direct flower formation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003eAGAMOUS\u003c/em\u003e (\u003cem\u003eAG\u003c/em\u003e), that its miss-expression leads to carpel formation\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e are also not expressed in the callus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eH3K4me3 enrichment in the callus correlates with gene expression.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAcquiring new cell fates and maintaining cell identity rely on the establishment of lineage-specific transcriptional programs, which are primarily regulated by epigenetic mechanisms\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. To characterize the H3K4me3 and H3K27me3 epigenetic marks, which are associated with transcriptional activation and repression, respectively\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, we performed ChIP-seq analyses on nuclei isolated from callus using H3K4me3- and H3K27me3-specific antibodies. In total, 16,161 genes were found to be marked by H3K4me3 (Supplementary Data 4), with an enrichment typically covering\u0026thinsp;~\u0026thinsp;800bp of the gene, starting at the transcription start site (TSS) and peaking within ~\u0026thinsp;200bp downstream to the TSS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), consistent with the hallmark of H3K4me3 at gene TSSs\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The H3K4me3 coverage profile across the TSS for all expressed genes, categorized into four expression level quantiles, demonstrates a strong correlation between H3K4me3 and gene activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), suggesting its consistent role in transcription. Overlapping the callus-expressed genes with the H3K4me3-marked genes reveals three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb): 14,502 expressed genes marked by H3K4me3, accounting for 87% of all expressed genes, indicating that H3K4me3 functions as a general mechanism rather than a regulator of a specific gene set; 2,201 expressed genes-not marked by H3K4me3, indicating that the presence of the H3K4me3 mark is not imperative for gene expression. However, as this analysis was performed at a population level, some genes may show mixed signals, with H3K4me3 and transcription co-occurring in some cells but absent in others. This averaging dilutes the H3K4me3 signal below the cutoff, while expression levels remain above it; 1659 non-expressed-marked by H3K4me3 genes, including known bivalent genes like \u003cem\u003eFLOWERING LOCUS C\u003c/em\u003e, \u003cem\u003eNGATHA3\u003c/em\u003e and \u003cem\u003eBLADE ON PETIOLE 1\u003c/em\u003e, which are marked with both H3K4me3 and H3K27me3 and silenced\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, suggesting that some genes within this group are potential candidates for bivalency. Other genes in this group might be marked with H3K4me3 to be poised for later expression, as was suggested for zebrafish \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. An example for genes in each group is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eThe H3K27me3 mark in the callus is associated with a specific set of silent genes\u003c/h2\u003e \u003cp\u003eMapping the H3K27me3 mark in the callus reveals enrichment across the gene body of silenced genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), consistent with other reports\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. However, only 3413 genes out of the 12,717 that exhibit no expression (RPKM\u0026thinsp;\u0026lt;\u0026thinsp;1) were marked with H3K27me3, indicating that this repressive mark regulates a specific set of genes. This is consistent with the 3,991 genes marked by H3K27me3 in callus, as identified by ChIP-chip analysis\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Surprisingly, 531 marked genes are associated with active transcription (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and Supplementary Data 5), suggesting either that H3K27me3 is also associated with a distinct transcriptional outcome as reported for other organisms and for Arabidopsis\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, or that it is the result of averaging cells with a distinct feature. Next, we performed GO analysis on the 3,413 H3K27me3-silenced genes. We identified a significantly overrepresented gene group, including 43 miRNA genes, suggesting that H3K27me3 indirectly promotes gene expression by silencing miRNAs, as well as 28 \u003cem\u003eAGAMOUS-like\u003c/em\u003e (\u003cem\u003eAGL\u003c/em\u003e) genes from the 108 MADS-box genes in Arabidopsis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMost striking was the group of TFs, consisting of 467 genes (Supplementary Data 6), out of the 2,192 TFs in Arabidopsis (2.73-fold enrichment, p\u0026thinsp;\u0026lt;\u0026thinsp;5E-16). This group includes 202 genes classified under the \"developmental process\" term, many of which are sufficient to direct cell differentiation or \u003cem\u003ede novo\u003c/em\u003e organogenesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). One example is the set of TFs required for stomatal differentiation, including \u003cem\u003eSPCH\u003c/em\u003e, \u003cem\u003eMUTE\u003c/em\u003e, and \u003cem\u003eFAMA\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e and other epidermal cell differentiation TFs like \u003cem\u003ePROTODERMAL FACTOR 2\u003c/em\u003e, \u003cem\u003eHOMEODOMAIN GLABROUS 2\u003c/em\u003e (\u003cem\u003eHDG2\u003c/em\u003e), \u003cem\u003eHDG5\u003c/em\u003e, and \u003cem\u003eMERISTEM LAYER 1\u003c/em\u003e. Another example is the group of abaxial regulatory genes, which have mutual antagonistic interactions with adaxial regulatory genes to establish organ polarity. The abaxial genes are required for specifying cell identity away from the shoot apex side in all lateral organs, for example, the lower side of a leaf. In the callus, genes of this group are marked by H3K27me3 and are silenced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), consistent with the view that abaxial cell fate may be the default pattern of differentiation\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Remarkably, the genes known to specify adaxial identity, the side close to the shoot apex, for example, the leaf upper side\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, are marked by H3K4me3 and expressed in the callus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFrom the list of 3413 genes, we also identified 339 as potentially bivalent genes that are marked with both H3K4me3 and H3K27me and show no expression (RPKM\u0026thinsp;\u0026lt;\u0026thinsp;1) (Supplementary data 7), that potentially could maintain transcriptional plasticity\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. Among them, we found 44 genes identified as putative bivalent genes based on ChIP-seq analysis done on Arabidopsis seedlings\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, thereby confirming the reliability of our analyses.\u003c/p\u003e \u003cp\u003eIn summary, our results support the hypothesis that keeping many developmental pathways active while maintaining dominant genes silenced to safeguard pluripotent cell identity\u003c/p\u003e \u003cp\u003eprovides a capacity to rapidly respond to stimuli and regenerate accordingly. However, this strategy requires a mechanism to silence genes upon regenerative stimulus to allow one developmental pathway to dominate and reinforce lineage commitment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eemf2\u003c/b\u003e \u003cb\u003ecallus exhibits impaired regeneration\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the WT callus, 467 genes encoding for TFs are marked by H3K27me3 and silenced, including at least 202 developmental regulators (Supplementary Data 6). To test the prominence of the H3K27me3 mark on callus cell identity and its capacity to regenerate, we produced callus from the PRC2 mutant \u003cem\u003eemf2\u003c/em\u003e-1 (hereafter \u003cem\u003eemf2\u003c/em\u003e) and analysed its capacity to regenerate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The \u003cem\u003eemf2\u003c/em\u003e mutant was shown to be incapable of forming callus from leaf or cotyledons \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, probably due to precocious differentiation. To overcome this hindrance, we sowed the mutant and WT directly on callus-inducing media (CIM) to allow the embryonic cells to proliferate, followed by trimming the cotyledons and re-culturing them on CIM. This resulted in \u003cem\u003eemf2\u003c/em\u003e and WT calli, which were phenotypically similar (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we tested the callus cell self-identity, i.e., how the callus cells coordinate their inherent cellular programs without any external stimuli. We transferred 100 calli of each genotype, WT, and \u003cem\u003eemf2\u003c/em\u003e, to a hormone-free medium and cultured them in the dark (50 calli) or under light (50 calli) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). In the dark, WT calli developed roots, first detected after 13 days. By day 20, all calli had developed roots. This suggests that either the root program is dominant in the absence of hormonal stimuli and light signalling or that the auxin absorbed on CIM remains stable in the dark, thereby promoting root formation. For \u003cem\u003eemf2\u003c/em\u003e calli, root initials were observed on 60% of calli only after day 20. However, these initials failed to elongate further, and all calli began to accumulate dark brown pigment and eventually died (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eUnder light conditions by day 7, 50% of the WT calli and 32% of the \u003cem\u003eemf2\u003c/em\u003e calli accumulated green colour, while the calli of both genotypes remained vigorous and continued to grow in size. At 20 days, 60% of the WT calli and 70% of the \u003cem\u003eemf2\u003c/em\u003e calli developed root initials. From this point onward, the WT calli gained a deep green colour, while all the \u003cem\u003eemf2\u003c/em\u003e calli turned brown and decayed. Both genotypes did not develop shoots during the 47 days of the experiment, indicating that light alone is not a sufficient stimulus to induce \u003cem\u003ede novo\u003c/em\u003e shoot meristem formation, although it can drive chloroplast biogenesis.\u003c/p\u003e \u003cp\u003eTo test the capacity of the \u003cem\u003eemf2\u003c/em\u003e callus to respond to hormonal signals and regenerate accordingly, we cultured the WT and \u003cem\u003eemf2\u003c/em\u003e calli on root or shoot-inducing media (RIM or SIM). For each genotype, 100 calli were cultured on RIM in the dark and 100 calli on SIM under light. After 7 days on RIM, root initials developed on all calli of both genotypes. However, the WT roots elongated and formed lateral roots, while the \u003cem\u003eemf2\u003c/em\u003e roots remained short and eventually decayed, coinciding with the accumulation of a dark tone in the callus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). On SIM, green colour accumulated in both genotypes, but it appeared earlier in WT, with 98% of the calli showing green sections by day four, compared to 42% in \u003cem\u003eemf2.\u003c/em\u003e By day 15, 96% of the WT calli had regenerated multiple vegetative shoots per callus, whereas only 8% of the \u003cem\u003eemf2\u003c/em\u003e calli had regenerated a single shoot in the reproductive phase (Supplementary Fig.\u0026nbsp;1). By day 30, all the \u003cem\u003eemf2\u003c/em\u003e calli on SIM had decayed, while the WT calli remained vigorous (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eThese results indicate that without a functional EMF2 complex callus cells fail to acquire new cell identity and regenerate.\u003c/p\u003e \u003cp\u003e \u003cb\u003eemf2\u003c/b\u003e \u003cb\u003ecallus displays minor changes in gene expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeedlings of \u003cem\u003eemf2\u003c/em\u003e display reduced H3K27me3 on 54% of the WT-marked genes\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. To study the impact of \u003cem\u003eEMF2\u003c/em\u003e mutation on transcription in callus, we performed differential gene expression analysis on mRNA-Seq from six-week-old WT and \u003cem\u003eemf2\u003c/em\u003e calli. This analysis yielded surprising results. Out of the 16,646 expressed genes in \u003cem\u003eemf\u003c/em\u003e callus (RPKM\u0026thinsp;\u0026gt;\u0026thinsp;1, Supplementary data 8), only 812 genes exhibited differential expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05): 374 up- and 438 down-regulated genes in \u003cem\u003eemf2\u003c/em\u003e calli as compared to WT, leaving 15,834 genes with similar expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea Supplementary data 9).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese low numbers are surprising, considering that over 2,500 genes were differentially expressed between WT and \u003cem\u003eemf2\u003c/em\u003e seedlings\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. However, this disparity could be attributed to differences in tissue composition between the seedlings (vegetative in WT and reproductive in \u003cem\u003eemf2).\u003c/em\u003e Furthermore, the high expression of \u003cem\u003eVRN2\u003c/em\u003e (RPKM\u0026thinsp;=\u0026thinsp;7) in both WT and \u003cem\u003eemf2\u003c/em\u003e calli, a gene with functional similarity to \u003cem\u003eEMF2\u003c/em\u003e\u003csup\u003e61\u003c/sup\u003e, might compensate for the loss of EMF2 function.\u003c/p\u003e \u003cp\u003eOf the 374 \u003cem\u003eemf2\u003c/em\u003e callus up-regulated genes, 131 are silenced (RPKM\u0026thinsp;\u0026lt;\u0026thinsp;1) in WT. To further investigate how the differentially expressed genes might affect the capacity to regenerate, we performed GO analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The most enriched biological processes category for the 374 up-regulated genes was the \"transcription factor activity\" term, as 76 genes encode for TFs, from which 63 were marked by H3K27me3 in WT callus. Out of the 63 TFs, 14 genes belong to the Type-II MICKC sub-family of the MADS-box TFs, which control flowering transition and development\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. For example, the \u003cem\u003eSEPALLATA 2\u003c/em\u003e gene required for petals, stamens, and carpels development\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e is marked with H3K27me3 in WT callus and shows no expression (RPKM of 0.06), whereas in \u003cem\u003eemf2\u003c/em\u003e exhibits a high expression level (RPKM of 8.5, Log2 Fold Change\u0026thinsp;=\u0026thinsp;7). The \u003cem\u003eAGL72\u003c/em\u003e TF, which is involved in floral transition \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, shows an RPKM value of 9.4 in \u003cem\u003eemf2\u003c/em\u003e and no expression in WT (|log2 Fold Change\u0026thinsp;=\u0026thinsp;8.5). This is also reflected in the significant enrichment of the \"flower development\" term (GO:0009908, p\u0026thinsp;\u0026lt;\u0026thinsp;8.E-07) and is consistent with the \u003cem\u003eemf2\u003c/em\u003e phenotype of flowering upon germination\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, as well as the regeneration of flower from the \u003cem\u003eemf2\u003c/em\u003e callus (supplementary Fig.\u0026nbsp;1). Remarkably, ten up-regulated genes in the \u003cem\u003eemf2\u003c/em\u003e calli are explicitly associated with carpel development\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. For example, \u003cem\u003eSEEDSTICK\u003c/em\u003e (\u003cem\u003eSTK\u003c/em\u003e), which is sufficient to induce the transformation of sepals into carpeloid organs and to promote carpel development in the absence of AG activity\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, showed Log2 Fold Change of 7.4. The enriched GO term \"cell differentiation \" (GO:00030154, p\u0026thinsp;\u0026lt;\u0026thinsp;8E-08) stands out because the expression of genes that promote cell differentiation might contribute to the reduced capacity of the \u003cem\u003eemf2\u003c/em\u003e callus to regenerate. Up-regulated genes in this term include TFs involved in xylem fibres differentiation like \u003cem\u003eVASCULAR-RELATED NAC-DOMAIN 3\u003c/em\u003e and \u003cem\u003e6\u003c/em\u003e and \u003cem\u003eACAULIS 5\u003c/em\u003e\u003csup\u003e67\u003c/sup\u003e, hair cell differentiation (\u003cem\u003eHDA18\u003c/em\u003e), and leaf cellular differentiation (\u003cem\u003eTCP\u003c/em\u003e 2, 10 and 17)\u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, suggesting that EMF2 represses those genes in WT callus to prevent cell differentiation, thereby safeguard pluripotency identity.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eemf2\u003c/em\u003e callus also exhibits 438 down-regulated genes, potentially an indirect consequence of the upregulation of 76 TF-encoding genes. To test this, we performed GO analysis on the 438 down-regulated genes (Supplementary Table\u0026nbsp;4) and revealed remarkable enrichment in categories related to photosynthesis, consistent with similar results with \u003cem\u003eemf2\u003c/em\u003e seedlings\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. For example, 33 of 113 photosynthesis genes (GO:0015979, p\u0026thinsp;\u0026lt;\u0026thinsp;E-31) and 48 of 322 thylakoid genes (GO:0009579, p\u0026thinsp;\u0026lt;\u0026thinsp;E-31) are downregulated in \u003cem\u003eemf2\u003c/em\u003e callus.\u003c/p\u003e \u003cp\u003e \u003cb\u003eemf2\u003c/b\u003e \u003cb\u003eexhibits minor changes in H3K4me3\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe H3K4 histone methyltransferases antagonize PcG-mediated repression\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, but how PcGs affect their activity remains unclear. To investigate the impact of the \u003cem\u003eEMF2\u003c/em\u003e mutation on H3K4me3 distribution, we conducted ChIP-seq analysis on \u003cem\u003eemf2\u003c/em\u003e and WT callus using H3K4me3-specific antibodies. In the \u003cem\u003eemf2\u003c/em\u003e callus, the H3K4me3 mark was detected on 16,173 of the genes (Supplementary Data 10), from which 90% (14,562) are expressed (RPKM\u0026thinsp;\u0026gt;\u0026thinsp;1), demonstrating a positive correlation with expression similar to WT. Next, we analysed the differential levels of enriched H3K4me3 peaks between WT and \u003cem\u003eemf2\u003c/em\u003e calli using edgeR algorithm\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e (Supplementary data 11). Surprisingly, only 214 genes exhibited significantly greater signals in the \u003cem\u003eemf2\u003c/em\u003e callus, from which 109 genes are marked by H3K27me3 in WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, grey triangles). Strikingly, 1,458 genes had weaker signals in the \u003cem\u003eemf2\u003c/em\u003e callus compared to WT, which might stem from the indirect effect of TF and miRNA upregulation.\u003c/p\u003e \u003cp\u003eTo summarize our analyses, we generated heat maps for WT H3K4me3 marked genes only, sorted by the H3K4me3 signal from the highest to lowest, demonstrating that H3K4me3 displays anti-correlation with H3K27me3 and strong correlation with expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The \u003cem\u003eemf2\u003c/em\u003e callus exhibits a similar pattern except for the 56 genes that are marked by H3K27me3 in WT, acquired the H3K4me3 mark in the \u003cem\u003eemf2\u003c/em\u003e and gained expression (The box at the bottom of the heat maps and Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHow plant callus cells maintain their pluripotent identity is not fully understood. In this work, we reveal that a callus exhibits a unique gene expression pattern that promotes a high proliferation rate but also maintains the potential to initiate many of the lineages required to develop a mature plant. One mechanism for maintaining pluripotency in mammalian embryonic stem cells is retaining a globally open chromatin state through basal transcription \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. We identify a related strategy in callus, where lineage-affiliated genes are kept transcriptionally active, allowing for a rapid response to developmental signals without the need to go through the complex transcription process. However, such a strategy requires a complementary mechanism to prevent precocious differentiation. Gene priming, which keeps genes poised for later activation, may also contribute to the high regenerative competency of plant cells\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe propose two independent mechanisms for maintaining callus identity; one is keeping the cell cycle machinery active, which prevents differentiation \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. This is supported by the observation that upon culturing the callus on media devoid of proliferation-instructive signals, the cells differentiate (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The central feature of plant pluripotent stem cells is their slow mitotic rate\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e,\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. However, callus cells exhibit highly proliferative characteristics, demonstrating that pluripotency is not necessarily unique to slow-dividing cells; another mechanism involves silencing dominant transcription factors through epigenetic modifications, which are sustained during cell proliferation. Pluripotent cells primarily possess dual capacities: maintaining the cellular identity of less differentiated cells and having the capacity to form multiple cell lineages. This requires the plasticity to reprogram epigenetic states in response to signals, to shut down competing programs, and to establish lineage-specific transcriptional programs. Here, we highlight the crucial role of H3K27 tri-methylation in enabling callus differentiation and regeneration. We further demonstrate that the \u003cem\u003eemf2\u003c/em\u003e callus cannot coordinate these processes effectively.\u003c/p\u003e \u003cp\u003eWe show that \u003cem\u003eemf2\u003c/em\u003e callus can be established and proliferate in culture, indicating that hormone perception and signal transduction are not compromised. In addition, it indicates that the EMF2 component of the PRC2 is dispensable for cell proliferation, consistent with the view that\u003c/p\u003e \u003cp\u003eself-renewal in mammalian ES cells is generally not subjected to repressive epigenetic mechanisms\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. It is further supported by the highly proliferative callus derived from two transgenic plants expressing the \u003cem\u003eH3.3\u003c/em\u003e\u003csup\u003e\u003cem\u003eK\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003eA\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eH3.15\u003c/em\u003e histone variants, both lacking lysine residue 27, suggesting that reduced levels of H3K27me3 promote cell proliferation\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe found that only 812 genes are differentially expressed between \u003cem\u003eemf2\u003c/em\u003e and WT. We can propose several scenarios and explanations for the low number of up-regulated genes in \u003cem\u003eemf2\u003c/em\u003e: 1. EMF2 might regulate a small set of genes. However previous study showed 54% reduction in total H3K27me3 in \u003cem\u003eemf2\u003c/em\u003e seedlings compared with WT \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e; 2. VRN2 might target overlapping genes and largely compensate for the loss of EMF2 function. That scenario highlights the exact set of genes specially regulated by EMF2: marked by H3K27me3 in WT callus and gained the H3K4me3 mark and expression in \u003cem\u003eemf2\u003c/em\u003e; 3. There is a massive reduction in H3K27me3 in \u003cem\u003eemf2\u003c/em\u003e, but removing this repressive mark does not necessarily activate genes. This phenomenon was demonstrated, for example, in mouse ES cells carrying a mutation in \u003cem\u003eEzh2\u003c/em\u003e, the HMTase component of PRC2, where the loss of H3K27me3 mark in numerous loci resulted in transcriptional activation of only one-third of the marked loci\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Our inability to perform ChIP-seq analysis of H3K27me3 in \u003cem\u003eemf2\u003c/em\u003e callus due to insufficient precipitated DNA for sequencing further supports this scenario.\u003c/p\u003e \u003cp\u003eThe normal development of \u003cem\u003eemf2\u003c/em\u003e callus, despite the up-regulation of 76 genes encoding for TF, some of which can promote differentiation and organogenesis, suggests that the signal for keeping the cell cycle machinery active is more robust. Yet, the failure to differentiate upon removal of the proliferative signal or in response to regenerative signals (RIM and SIM) suggests that activating lineage-specific gene networks to commit to a single developmental path requires a capacity to turn off pluripotency networks.\u003c/p\u003e \u003cp\u003eSeveral MADS-box TFs were shown to be involved in downregulating the expression of photosynthetic genes to confer flower identity \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Therefore, the enrichment in photosynthesis-related genes among the \u003cem\u003eemf2\u003c/em\u003e-downregulated genes might be the direct effect of MADS-box genes' upregulation in \u003cem\u003eemf2\u003c/em\u003e callus. The activation of the \u003cem\u003eLOB domain 29\u003c/em\u003e (\u003cem\u003eLBD29\u003c/em\u003e) TF in the \u003cem\u003eemf2\u003c/em\u003e callus, which was shown to repress genes involved in photosynthesis directly\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e, might also contribute to this enrichment.\u003c/p\u003e \u003cp\u003eIn Arabidopsis, mutations in \u003cem\u003eEMF2\u003c/em\u003e induce immediate flowering after germination \u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. In general, flowering can be promoted by environmental cues such as long-day photoperiod or low-temperature, leading to the activation of floral integrators genes, \u003cem\u003eFLOWERING LOCUS T\u003c/em\u003e (\u003cem\u003eFT), SOC1, TWIN SISTER OF FT\u003c/em\u003e (\u003cem\u003eTSF\u003c/em\u003e) and \u003cem\u003eLFY\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Those TFs induce the transition to flowering, leading to floral meristem specification, followed by the activation of genetic networks comprised mainly of MADS-box genes to specify floral organ identities \u003csup\u003e\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e\u003c/sup\u003e. In our study, we cultured the callus under conditions that do not promote flowering (under dark at 22C\u003csup\u003e0\u003c/sup\u003e), and the four integrator genes showed no expression in WT and \u003cem\u003eemf2\u003c/em\u003e calli. However, many downstream MADS-box genes are up-regulated in \u003cem\u003eemf2\u003c/em\u003e, demonstrating that activation of floral organ specification genes is independent of the transition to flowering. This result suggests that the four integrators are required solely to release the suppression and not for direct activation of the downstream MADS-box genes.\u003c/p\u003e \u003cp\u003eThere is no strong consensus on the homogeneity of callus tissue, and the question of whether callus cells share a similar cellular identity remains open \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Our comparative analysis between WT and \u003cem\u003eemf2\u003c/em\u003e calli identified only 324 up-regulated genes, demonstrating the similarity of the two calli and suggesting that the callus produced from embryonic tissue is more unified. This can be tested in the future by performing a transcriptomic analysis at single-cell resolution \u003csup\u003e\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e, with the recognition that it is not a bias-free method. Our approach offers an excellent solution for yielding unbiased results in functional genomic studies, including the study of pluripotency, transcriptome analysis, and chromatin state comparisons across various mutants lacking comparable tissues or organs. Using our experimental system, we could pinpoint the potential genes regulated solely by EMF2, primarily those involved in floral organ specification. A future challenge will be to perform ChIP-seq analysis for EMF2, which could yield data on all the direct targets of EMF2.\u003c/p\u003e \u003cp\u003eIn summary, we provide insights into H3K27me3 function in establishing pluripotent cell identity in callus by silencing key TFs and contributing to the capacity to initiate new cell lineages. Our findings also provide the starting point for further studies on the genome-wide dynamics of H3K4me3 and H3K27me3 during regeneration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eArabidopsis thaliana accession Columbia-0 (Col-0) was used in this work as wild type and the \u003cem\u003eemf2\u003c/em\u003e mutant (ABRC germplasm CS16238) on Col-0 background \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor all experiments, seeds were surface sterilized for 10 minutes in 3% Sodium Hypochlorite containing 0.1% Triton X-100 and washed 4 times with DDW before sowing.\u003c/p\u003e \u003cp\u003eFor the leaf RNA-seq experiment, seeds were sown on Murashige and Skoog (MS) basal medium \u003csup\u003e\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u003c/sup\u003e, and kept under continuous light at 23\u0026deg;C for one week. Seedlings were then transplanted to soil and grown under long-day conditions (16h light/8h dark) at 21\u0026deg;C. Rosette leaves were harvested two weeks after transplanting.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTissue culture\u003c/h3\u003e\n\u003cp\u003eTo generate callus from Col-0 and \u003cem\u003eemf2\u003c/em\u003e cotyledons, sterilized seeds were sown directly on callus-inducing medium (CIM): Gamborg B5 medium with 0.5g/L MES, 2% dextrose, 0.9% phytagel, supplemented with 2.2\u0026micro;M 2,4-dichlorophenoxyacetic acid and 0.46\u0026micro;M kinetin. Following two days at 4\u0026deg;C, plates were transferred to a continuous light growth room at 23\u0026deg;C. After seven days, cotyledons were trimmed and transferred to a new CIM plate placed in the dark. During the next 5\u0026ndash;6 weeks, calli were re-cultured to new CIM plates every 7\u0026ndash;10 days. All analyses were done on 6\u0026ndash;8 weeks-old calli.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCallus internal identity experiments\u003c/h2\u003e \u003cp\u003eCalli were transferred to Gamborg B5 medium deprived of hormones, and cultured under continuous light or dark conditions. Calli were monitored and photographed under a stereomicroscope.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCompetency to regenerate tests\u003c/h3\u003e\n\u003cp\u003eCalli of WT or \u003cem\u003eemf2\u003c/em\u003e and WT rosette leaves were transferred to either Shoot Inducing Media (SIM): Gamborg B5 medium supplemented with 4.4\u0026micro;M 2-isopentenyl adenine and 0.5\u0026micro;M 1-Naphthaleneacetic acid (NAA), under continuous light conditions or Root Inducing Media (RIM): Gamborg B5 medium supplemented with 0.5\u0026micro;M NAA under dark conditions.\u003c/p\u003e\n\u003ch3\u003eRNA extraction\u003c/h3\u003e\n\u003cp\u003eTo correlate chromatin characteristics with gene expression, tissues from each ChIP experiment were fast-frozen in liquid nitrogen. Samples were ground, and total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's instructions, including treatment with RNase-free DNase (Qiagen). Each RNA sample was loaded on agarose gel for integrity validation and quantified using NanoDrop.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRNA-Sequencing\u003c/h2\u003e \u003cp\u003eSequencing was done at the Technion Genome Centre (TGC) according to Illumina's protocols. The TruSeq RNA V2 sample prep kit (RS-122) was used. Total RNA was polyA-selected, followed by fragmentation and random hexamer-primed reverse transcription. Indexed adapters were added, and cDNA was amplified by PCR for 15 cycles. Every four indexed libraries were loaded onto an individual lane of Illumina's HiSeq 2500 system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation\u003c/h2\u003e \u003cp\u003eChIP was performed on nuclei isolated from the callus as described \u003csup\u003e\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e\u003c/sup\u003e, with some modifications. The detailed protocol is found in the Supplementary material file. In short, six-week-old calli samples were harvested from 8 plates. Calli from every two plates were pooled, and 1.5 grams from each pool was placed into a tube and subjected to cross-linking.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eChIP validation\u003c/h2\u003e \u003cp\u003eA comparative semi-quantitative PCR between positive and negative DNA binding sites was performed to validate the ChIP experiment. For each chromatin mark, two sets of primers were designed: positive control (PC) to estimate the positive DNA binding site and negative control (NC) to estimate the negative binding site (Supplementary Table\u0026nbsp;5). The two sets of primers were tested on the Input DNA sample diluted 1:1,000 and the IP sample. The reaction mix included 7.5\u0026micro;l of GoTaq Green master mix (Promega), 0.4\u0026micro;l of each primer (10\u0026micro;M), and 5.9\u0026micro;l diluted DNA. Along the PCR reaction, samples were taken out from cycle 28 and every 2 cycles and loaded on 2% agarose gel.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eChromatin immunoprecipitation sequencing\u003c/h2\u003e \u003cp\u003eSamples were sequenced at the Technion Genome Centre (TGC) according to Illumina's protocols. The ChIP-seq libraries were prepared using the TruSeq Nano DNA Library Prep Kit (FC-121). ChIP-enriched DNA fragments were size-selected on an agarose gel to enrich for fragments of 200 bp. Indexed adaptors (diluted 1/10) were added, and the library was amplified using PCR for 14 cycles. Every 7\u0026ndash;10 indexed libraries were loaded onto an individual lane of Illumina's HiSeq 2500 system for 50 base-pair single-end sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eNext Generation Sequencing (NGS) analysis\u003c/h2\u003e \u003cp\u003eEach NGS analysis included quality control using FastQC\u003csup\u003e\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e\u003c/sup\u003e and adaptor trimming using cutadapt\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. NGS reads were mapped to the Arabidopsis thaliana reference transcriptome TAIR10 using TopHat 2 \u003csup\u003e90\u003c/sup\u003e for RNA-Seq and Bowtie2 \u003csup\u003e91\u003c/sup\u003e for ChIP-Seq.\u0026nbsp;Integrative Genomics Viewer (IGV) was used for visualizing the data \u003csup\u003e\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u003c/sup\u003e. Biological replication consistency was measured using Spearman correlation of BAM files in deepTools \u003csup\u003e\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e\u003c/sup\u003e. RNA-Seq samples with p\u0026thinsp;\u0026gt;\u0026thinsp;0.96 and ChIP-Seq samples with p\u0026thinsp;\u0026gt;\u0026thinsp;0.88 were merged.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: L.E.W. (lead) and T.M; Performed the experiments: T.M (lead), and Y.C; Performed the genomic data curation, analysis, and visualisation: U.L and T.M; Wrote the original manuscript: L.E.W; contributed to genomic data analysis T.K;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWhite, P. R. Potentially Unlimited Growth of Excised Plant Callus in an Artificial Nutrient. \u003cem\u003eAmerican Journal of Botany\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 59-64 (1939). https://doi.org:10.2307/2436709\u003c/li\u003e\n\u003cli\u003eIkeuchi, M., Sugimoto, K. \u0026amp; Iwase, A. 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Yet, the mechanisms underlying their pluripotent state remain elusive. We propose a strategy that involves two independent mechanisms to endow callus cells with pluripotency: (1) maintaining a unique transcriptional profile, characterised by the expression of genes from diverse developmental pathways that allows rapid response to developmental cues; (2) preventing premature differentiation through H3K27 methylation-mediated silencing of key transcription factors such as \u003cem\u003eWUCHEL\u003c/em\u003e and \u003cem\u003eSPEECHLESS\u003c/em\u003e. This strategy relies on a mechanism to silence the pluripotency network upon regenerative stimuli, enabling a single developmental pathway to dominate.\u003c/p\u003e \u003cp\u003eOur study reveals that the EMF2 complex, a key regulator of H3K27 tri-methylation, plays a crucial role in this process. Callus derived from the \u003cem\u003eemf2\u003c/em\u003e mutant, deficient in H3K27me3, exhibits severely impaired regeneration. Comparative analyses of chromatin states and transcription profiles between wild-type and \u003cem\u003eemf2\u003c/em\u003e calli revealed that the loss of \u003cem\u003eEMF2\u003c/em\u003e leads to upregulation of key transcription factors in callus, and identified the genes regulated solely by EMF2. Our findings suggest that suppressing pluripotency networks through H3K27me3 is essential for executing specific developmental programs to ensure effective regeneration.\u003c/p\u003e","manuscriptTitle":"H3K27me3 epigenetic mark crucial for callus cell identity and regeneration capacity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-24 09:47:56","doi":"10.21203/rs.3.rs-5582331/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"330998bb-fb4c-4286-b642-5113d7d1e343","owner":[],"postedDate":"January 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":41955455,"name":"Biological sciences/Developmental biology/Pluripotency"},{"id":41955456,"name":"Biological sciences/Plant sciences/Plant development/Cell fate"},{"id":41955457,"name":"Biological sciences/Developmental biology/Reprogramming"}],"tags":[],"updatedAt":"2025-08-07T17:25:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-24 09:47:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5582331","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5582331","identity":"rs-5582331","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}