JmjC Protein-Mediated Histone Demethylation: Regulating Growth, Development, and Stress Adaptation in Brassica rapa

Abstract

The dynamic regulation of histone methylation is a key mechanism for epigenetic regulation of gene expression. As histone demethylases dependent on divalent iron ions and α-ketoglutarate, the JmjC family plays an important role in plant life activities. Fifty JmjC domain-containing proteins in Arabidopsis thaliana (21) and Brassica rapa (29) are divided into seven distinct groups, with each group endowed with specific functions due to unique structural domains. Some members achieve functional specificity by recognizing specific DNA motifs or interacting with transcription factors, and others exhibit special functional modes due to mutations in their binding sites. By targeting specific genes (such as FLC, FT, WRKY family, PR genes, etc.), they regulate growth and development processes, as well as responses to multiple biotic and abiotic stresses. Focusing on inter-species divergence of Arabidopsis and Brassica, this review summarized JmjC proteins’ structural classification, substrate specificity, and mechanisms, providing a basis for dissecting plant epigenetic networks and guiding Brassica crop breeding desired bolting traits (early or late bolting), high stress resistance and so on.

1. Introduction

Nucleosomes, comprising an octamer of core histones (H2A, H2B, H3, H4) wrapped by 146 base pairs of DNA, constitute the fundamental unit of chromatin [1]. Post-translational modifications (PTMs) at histone N-termini, including methylation, phosphorylation, and ubiquitination, play pivotal roles in regulating gene transcription, maintaining genome stability, and mediating epigenetic inheritance [2,3,4,5,6]. Histone methylation occurs on the guanidino group of arginine (R) or the ε-amino group of lysine (K). The methylation status of histone lysine is dynamically regulated by histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs). Similarly, histone arginine methylation is regulated by protein arginine methyltransferases (PRMTs), which catalyze mono-methylation as well as symmetric or asymmetric di-methylation of arginine [2]. Critically, distinct lysine methylation marks correlate with transcriptional activation or silencing: methylation of H3K4 and H3K36 is associated with gene activation, whereas methylation of H3K9 and H3K27 is linked to gene silencing [2,7].

In eukaryotes, histone lysine demethylation is catalyzed by two major enzyme classes: lysine-specific demethylase 1 (LSD1) and Jumonji C (JmjC) domain-containing demethylases. Arabidopsis harbors four LSD1 homologous genes: FLD, LDL1–3 [8]. LSD1 is a flavin adenine dinucleotide (FAD)-dependent monoamine oxidase capable of demethylating H3K4me1/2 in vivo as well as H3K4me1/2/ and H3K9me1/2 in vitro. Its catalytic mechanism involves using protonated nitrogen as a hydrogen donor, limiting its activity to the removal of mono- and di-methyl groups, but not trimethyl modifications [9]. In contrast, JmjC domain-containing histone demethylases function as Fe (II)-and α-ketoglutarate (α-KG)-dependent dioxygenases. This enzymatic class possesses the capacity to remove mono-, di-, and trimethyl modifications from H3K4, H3K9, H3K27, and H3K36 [10]. The JMJ family in plants constitute a pivotal component of the epigenetic regulatory circuitry, exerting precise governance over four cardinal biological processes—growth and development, abiotic stress adaptation, biotic interplay, and metabolic modulation—through the targeted modulation of specific histone methylation loci.

Brassica, as an important globally cultivated crop, possesses extremely high agricultural and economic value due to multiple uses, including vegetables, oilseeds, and forage. Brassica species exhibit complex genomes with triplicate duplication, providing a new direction for discovering epigenetic mechanisms, especially for the histone-modification-related pathway [11]. Among epigenetic regulators, JmjC domain-containing histone demethylases are core molecules that precisely regulate key flowering time and stress resistance. Their functional characterization and targeted genetic modification offer effective approaches to address production challenges, including premature bolting and continuous cropping obstacles. Meanwhile, they lay the foundation for breaking traditional limitations in precision crop breeding and advancing epigenetic regulation-based breeding, thus playing a pivotal role in Brassica crop improvement. As a fundamental model for epigenetic mechanism research, Arabidopsis has yielded systematic studies on its JmjC family, clarifying catalytic mechanisms and regulatory networks, and providing a classic paradigm for epigenetic research in higher plants. Although Brassica crops, which belong to the same Brassicaceae family as Arabidopsis, exhibit complex genetic backgrounds due to their genomic characteristics, the systematic identification and functional divergence of JmjC family members in core breeding targets such as bolting/flowering and stress resistance remain insufficiently elucidated. Nevertheless, research findings from Arabidopsis can directly provide theoretical and technical support for functional analysis in Brassica plants. In this review, focusing on JmjC proteins in Arabidopsis and Brassica, we explore their structural diversity, regulatory mechanisms, and functions in growth and stress adaptation. Through an emphasis on interspecific differences and functional conservation, it provides a molecular framework for epigenetically enhancing climate resilience and yield in Brassicaceae crops.

2. Key Features and Mutation Impacts of JmjC Proteins in Arabidopsis and B. rapa

The Arabidopsis genome encodes 21 JmjC proteins and B. rapa genome encodes 29 JmjC proteins, phylogenetically classified into seven distinct groups based on domain architecture (Figure 1). The KDM4 group (JMJ11-JMJ13/BrJMJ11-BrJMJ13) features JmjN, JmjC, and zinc finger domains. The KDM5A group (JMJ14-JMJ16, JMJ18, JMJ19/BrJMJ14-BrJMJ16, BrJMJ18, BrJMJ19) and KDM5B group (JMJ17/BrJMJ17) possess JmjN, JmjC, C5HC2 zinc fingers, and FYR domains. The JmjC domain-only group is subdivided into A (JMJ20/BrJMJ20) and B (JMJ30-JMJ32/BrJMJ30-BrJMJ32) subgroups. The JMJD6 group (JMJ21-JMJ22/BrJMJ21-BrJMJ22) contains F-box and JmjC domains. The KDM3 group (JMJ24-JMJ29/BrJMJ24-BrJMJ29) comprises proteins with RING and JmjC domains. Crucially, except for the binding sites of JMJ24 and JMJ28, the other JmjC demethylases harbor conserved binding sites for three Fe (II) ions and two α-KG molecules [12,13]. When the conserved iron-binding sites (His246/JMJ12 (REF6) [14], His293 and Glu295/JMJ13 [15], His397/JMJ14 [16], His381, Glu383 and His407/JMJ16 [17,18], His79 and Glu81/JMJ17 [19], His679 and Asp681/JMJ25 [20], His576 and Asp578/JMJ27 [21], His326/JMJ30 [22], His174/JMJ32 [22]) are mutated to alanine, the histone demethylase activity of these proteins disappears. Mutational analyses targeting conserved Fe (II)-binding residues typically abolish demethylase activity, highlighting their essential role. Notably, JMJ24 retains activity despite Fe-site mutation, as it regulates flowering independently of its histone demethylase activity and may act as a bridge protein in the INO80 complex [23], while JMJ28 likely loses function in vivo upon similar mutation [24]. When mutations occur in the JmjC catalytic domain (Thr345Ala) of BrJMJ18 and between the Zf-C5HC2 domain and the FYRN domain (Tyr633Cys and Leu654Phe), the catalytic activity of BrJMJ18 changes. Collectively, these fifty JmjC demethylases regulate diverse biological processes by catalyzing the removal of methyl groups from specific histone marks (H3K4me1/2/3, H3K9me1/2/3, H3K27me1/2/3, H3K36me1/2/3, H4R3me2s) at target gene loci (Figure 2).

3. The H3K4me Demethylation Function of JmjC Proteins

In plants, the dynamic regulation of gene expression involves H3K4 methyltransferases and demethylases. H3K4 methylation exists in three states: mono-/me1, di-/me2, and tri-/me3 [25]. These marks exhibit distinct genomic distributions: H3K4me1 is enriched within transcribed regions, biased towards the 3′ end. H3K4me2/3 localize to the 5′ end of transcribed regions and promoter regions, with H3K4me3 positioned downstream of H3K4me2 [25,26,27]. H3K4 methylation is catalyzed by a class of Complex Proteins Associated with Set (COMPASS) or COMPASS-like complexes. In Arabidopsis, the COMPASS-like complex contains seven potential H3K4 methyltransferases, including five Trithorax-like (ATX1-ATX5) and two ATX-related (ATXR3/7) proteins [28]. Seven JmjC histone demethylases including JMJ14-JMJ18, JMJ28, and JMJ29 can demethylate H3K4. Among them, JMJ14, JMJ16, and JMJ17 demethylate H3K4me1/2/3 [16,17,19,29], JMJ18 targets H3K4me2/3 [30], whereas JMJ11, JMJ15, JMJ28, and JMJ29 specifically demethylate H3K4me3 [29,31,32,33]. JMJ14 specifically recognizes H3K4me3, with its JmjC-Helix-C5HC2 catalytic domain serving as the functional core underlying substrate recognition. Specifically, the guanidino groups of Glu-285 (in the jumonji domain) and Glu-516 (in the C5HC2 domain) form hydrogen bond and salt bridge interactions with H3R2, while Asp-312 in the jumonji domain establishes two hydrogen bonds with the backbone amide group and side-chain amide group of H3Q5, respectively [34]. These enzymes play a critical role in regulating diverse processes, including flowering time, circadian rhythms, salt tolerance, leaf senescence, dehydration stress response, seed germination, cotyledon greening, immune responses and gene silence in plants (Table 1).

JMJ14, JMJ15, and JMJ18, as H3K4me demethylases, regulate flowering by targeting key regulators FLOWERING LOCUS C (FLC) or FLOWERING LOCUS T (FT). At the FLC gene locus, H3K4me and H3K36me promote transcription and delay plant flowering [35,36,37,38], while H3K9me and H3K27me methylation inhibit gene transcription and promote earlier flowering [36,37,38,39]; H3K36me3 and H3K27me3 act antagonistically at the FLC locus [40]. Among them, JMJ15 and JMJ18 inhibit FLC gene expression by reducing H3K4me3 modification at the transcription start site (TSS), the first exon, and introns of FLC, thereby promoting flowering [30,31]. In jmj18 knockout mutants, the plants show minor flowering delay without significant AtFLC, CONSTANS (CO), or AtFT expression changes during vegetative growth compared to wild-type plants [30]. In the FT gene locus, increased H3K4me and H3K36me modifications promote an increase in FT expression and lead to earlier flowering, whereas increased H3K9me and H3K27me modifications inhibit FT gene expression and result in delayed flowering [37,41,42,43]. In the jmj14 mutant, the plants exhibit accelerated flowering accompanied by significantly increased FT and TWIN SISTER OF FT (TSF) expression. Under long-day conditions for 10 days, JMJ14 suppresses FT and TSF in vascular tissues by removing H3K4me2/3 at their TSS. However, another study under long-day conditions for 10 days reported significantly increased FT, LEAFY (LFY), SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and APETALA1 (AP1) expression in jmj14 mutants without significant H3K4me2/3 changes at the FT locus [16]. This discrepancy is presumably mainly due to experimental microenvironmental interference with FT rhythmic expression, and functional compensation or residual JMJ14 activity from the mutant genetic background. Notably, under short-day conditions for 57 days, jmj14 mutant showed elevated H3K4me3 at the FT TSS and first exon of the FT locus [16]. JMJ14 also forms functional redundancy with ELF6, and the two jointly target the chromatin of the flowering-promoting FT gene [29]. Furthermore, JMJ17 regulates flowering, though its potential role via H3K4me3 demethylation remains unclear.

Both JMJ14 and JMJ29, exhibiting circadian rhythm, modulate the core clock genes expression of CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL 1 (LHY), and PSEUDO-RESPONSE REGULATOR 9 (PRR9) by catalyzing the removal of H3K4me3 marks at their loci. The jmj29 mutant displays a shortened circadian period compared to the wild type [32,44]. In the jmj14 mutant, at Zeitgeber Time 0 and 24, the H3K4me3 modifications at the CCA1 and LHY loci are elevated, and the expression levels of CCA1 and LHY are increased [44]. In the jmj29 mutant, the circadian rhythms of CCA1 and PRR9 are altered. JMJ29 interacts with ELF3 and binds to the EE motif (AAAATATCT) in the promoters of CCA1 and PRR9, removing the H3K4me3 marks at these loci, thereby ensuring low expression of CCA1 and PRR9 in the evening [32].

JMJ15 enhances salt tolerance by removing the H3K4me3 marks on WRKY DNA-BINDING PROTEIN 46 (WRKY46) and WRKY70. JMJ15 gain-of-function mutant show increased salt tolerance, and loss-of-function mutants are more sensitive to salt stress [45]. JMJ15 directly binds to H3K4me3-marked promoters and coding regions of WRKY46 and WRKY70, demethylating these regions, which reduces RNA polymerase II occupancy and suppresses their expression under salt stress [46].

JMJ16 suppresses leaf senescence by removing H3K4me3 methylation marks from the WRKY53 and SENESCENCE-ASSOCIATED GENE 201 (SAG201). SAG201 expression increases during leaf senescence and is induced by auxin [17]. JMJ16 demethylates H3K4me3 on senescence-associated genes (SAGs) in an age-dependent manner, with WRKY53 and SAG201 being its targets. JMJ16 combines the transcription start sites of WRKY53 and SAG201, removes the H3K4me3 marks from these genes, suppresses their expression in mature leaves, and prevents premature aging of plants [17].

JMJ17 modulates dehydration tolerance via OPEN STOMATA 1 (OST1), a key regulator of ABA-mediated stomatal closure [19,47]. JMJ17 loss-of-function mutants exhibit enhanced dehydration tolerance and ABA hypersensitivity, while overexpressors are dehydration-sensitive [19]. Under dehydration stress or ABA treatment, JMJ17 binds OST1 chromatin, removes H3K4me3, and modulates OST1 mRNA abundance [19]. Notably, although some key regulators are shared by salt and dehydration stress, JMJ15, which functions in response to salt stress, does not participate in the dehydration stress response [19].

JMJ17 facilitates seed germination by removing the H3K4me3 mark on ABA INSENSITIVE 5 (ABI5), an integrator of hormone signaling in regulating seed germination [48]. WRKY40, an ABA repressor, not only inhibits ABI5 expression but also binds to the W-box [TGACC (A/T)] in the promoter of its target genes [49,50]. After binding to the plus domain of JMJ17, WRKY40 recognizes the G-box sequence in the ABI5 promoter, recruits JMJ17 to the ABI5 promoter, and removes the H3K4me3 mark, thereby repressing ABI5 gene expression. Meanwhile, WRKY40 inhibits the transcriptional activation of ELONGATED HYPOCOTYL 5 (HY5), interacts with HY5, and binds to the G-box site in the ABI5 promoter to induce ABI5 expression [50]. When ABA is applied, ABA suppresses the activity of WRKY40, preventing it from binding to HY5 and thus promoting HY5 activation of ABI5 gene expression [50]. Therefore, JMJ17-WRKY40 and WRKY40-HY5 interactions coregulate germination [50].

JMJ17 promotes cotyledon greening during de-etiolation by removing the H3K4me3 mark from the promoter of PROTOCHLOROPHYLLIDE OXIDOREDUCTASE C (PORC), a tetrapyrrole biosynthesis (TPB) rate-limiting enzyme that catalyzes the conversion of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). JMJ17 can bind to the promoters of TPB genes, including HEMA1, CHLI1, and PORC [51]. In darkness, JMJ17 interacts with PHYTOCHROME INTERACTING FACTOR1 (PIF1), facilitating its demethylation of H3K4me3 at the PORC promoter to suppress PORC expression, which helps prevent excessive Pchlide accumulation in etiolated seedlings that could cause photo-oxidative damage upon light exposure [51]. During dark-to-light transition, light-induced transition triggers JMJ17 dissociation from target TPB gene promoters (without degradation), relieving repression to activate gene expression, thereby promoting Pchlide-to-Chlide conversion, chlorophyll biosynthesis, cotyledon greening and photomorphogenesis [51].

In Arabidopsis, JMJ14 and JMJ28 mediate the regulation of immune responses through remarkably distinct mechanisms. JMJ14 functions as a positive regulator of plant immunity, executing its role by positively modulating the expression of defense-related genes and the accumulation of the pipecolic acid (Pip) signaling system [52]. Specifically, upon local infection or activation of systemic acquired resistance (SAR), JMJ14 can promote the H3K4me3 enrichment and transcription of key defense genes in the salicylic acid (SA) and Pip pathways, including PATHOGENESIS-RELATED GENE 1 (PR1), AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), and FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1); meanwhile, it negatively regulates the expression of SUPPRESSOR OF NPR1-1 INDUCIBLE 1 (SNI1), a negative repressor of SAR, to maintain immune sensitivity [52]. Moreover, the regulatory effect of JMJ14 on SAR is dependent on the accumulation of Pip in systemic leaves, and exogenous application of Pip can partially rescue the SAR defect in jmj14 loss-of-function mutants [52]. Furthermore, JMJ14 is integrated into the SA-Pip synergistically enhanced defense amplification loop, which ensures the robust activation of defense responses even under conditions of low SA concentrations [52]. As a consequence, the jmj14 mutant displays a significant reduction in local resistance against the bacterial pathogen Pseudomonas syringae pv. Tomato (Pst) DC3000, coupled with a complete impairment of SAR [52]. In contrast, JMJ28 acts as a negative regulator of plant immunity. It recognizes the specific “YTGCAG” DNA motif within the genome via a plant-specific WRC domain, interacts with the core component RbBP5-like (RBL) of the COMPASS-like complex via its C-terminal, recruiting the ATX1/2-COMPASS-like complex to deposit H3K4me modifications on target genes (encompassing fungal resistance-associated genes such as PR5 and WRKY33) and consequently suppressing the hyperactivation of immune-related genes [33]. The jmj28 mutant exhibits substantially enhanced resistance to two necrotrophic fungal pathogens, Verticillium dahliae and Botrytis cinerea [33]. Additionally, functional redundancy exists between the JMJ28-ATX1/2-COMPASS complex and other COMPASS-like complexes in the regulation of H3K4 methylation [33].

JMJ14 regulates gene silencing primarily by modulating the epigenetic state of transgenes, collaborating with specific proteins, and mediating the transmission of silencing signals. It can specifically inhibit CG, CHG, and CHH methylation of transgenes (with minimal impact on the methylation of endogenous genes), promote the production of aberrant RNAs to support RDR6-mediated Post-transcriptional Gene Silencing (PTGS). Mutations in jmj14 lead to increased transgene methylation and reduced aberrant RNAs, thereby inhibiting PTGS; they also decrease the binding efficiency of RNA polymerase II to reduce transgene transcription, and its regulation relies on the DOMAINS REARRANGED METHYLTRANSFERASE 2(DRM2)-CHROMOMETHYLASE 3 (CMT3) pathway [53,54]. JMJ14 needs to bind to NAC domain containing protein 50 (NAC050) and NAC52 via its C-terminal FYRN/FYRC domains, and together they localize to transgene regions to maintain low methylation levels, which facilitates PTGS. The sgs1 mutation (a neomorphic nac52 allele) mimics this regulatory defect and additionally downregulates SUPPRESSOR OF GENE SILENCING 3 (SGS3) to enhance the inhibition of PTGS; meanwhile, TRB proteins can bind to specific DNA motifs to recruit JMJ14, assisting it in removing the H3K4me3 activation mark, and cooperate with PRC2 to enhance silencing [55,56,57,58]. In terms of signal transmission, JMJ14 is essential; jmj14 mutants cannot receive or transmit 21-nt/22-nt small interfering RNAs (siRNAs) systemic silencing signals, and it acts downstream of RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and ARGONAUTE 4 (AGO4), contributing to signal response through chromatin modification [59].

Table 1. H3K4 histone demethylase function.

Gene	Interaction	Target Gene	Function
JMJ14		FT	Flowering time [29]
	NAC050/052, TRBs		Gene silence [56,57,58]
		PR1, ALD1, FMO1, SNI1	Immune responses [52]
		CCA1, LHY	Circadian rhythms [44]
JMJ15		FLC	Flowering time [31]
		WRKY46, WRKY70	Salt tolerance [45,46]
JMJ16		WRKY53, SAG201	Leaf Senescence [17]
JMJ17		OST1	Dehydration stress [19]
	WRKY40, HY5	ABI5	Seed germination [50]
	PIF1	TPBs	De-etiolation [51]
JMJ18		AtFLC	Flowering time [30]
JMJ28	RBL	PR5, WRKY33	Immune responses [33]
JMJ29	ELF3	CCA1, PRR9	Circadian rhythms [32]

Table 1. H3K4 histone demethylase function.

Gene	Interaction	Target Gene	Function
JMJ14		FT	Flowering time [29]
	NAC050/052, TRBs		Gene silence [56,57,58]
		PR1, ALD1, FMO1, SNI1	Immune responses [52]
		CCA1, LHY	Circadian rhythms [44]
JMJ15		FLC	Flowering time [31]
		WRKY46, WRKY70	Salt tolerance [45,46]
JMJ16		WRKY53, SAG201	Leaf Senescence [17]
JMJ17		OST1	Dehydration stress [19]
	WRKY40, HY5	ABI5	Seed germination [50]
	PIF1	TPBs	De-etiolation [51]
JMJ18		AtFLC	Flowering time [30]
JMJ28	RBL	PR5, WRKY33	Immune responses [33]
JMJ29	ELF3	CCA1, PRR9	Circadian rhythms [32]

4. The H3K9me Demethylation Function of JmjC Proteins

H3K9 can undergo monomethylation (me1), dimethylation (me2), or trimethylation (me3). H3K9me2 serves as a key epigenetic mark associated with heterochromatin formation and transcriptional silencing [60], often coupled with DNA methylation in repetitive sequences and transposons via pathways like RNA-directed DNA methylation (RdDM) and de novo DNA methylation [61]. RNA-directed DNA methylation is guided by 24-nt siRNAs that recognize complementary DNA sequences in the genome. RdDM involves two core stages: in the first stage, RNA Polymerase IV (Pol IV) transcribes single-stranded RNA, converted to double-stranded RNA (dsRNA) by RDR2 and diced into 24-nt siRNAs by DICER-LIKE 3 (DCL3); in the second stage, siRNAs guide AGO4 to recruit DNA methyltransferase DRM2 for cytosine methylation [62]. DNA methylation is maintained at CG sites by METHYLTRANSFERASE 1 (MET1), at CHG sites by CMT3, and at asymmetric CHH sites by DRM2 [62]. In total, eight JmjC-containing histone demethylases, JMJ16, JMJ24, IBM1/JMJ25, JMJ26–JMJ30 target H3K9me, with IBM1, JMJ27, and JMJ28 demethylating H3K9me1/2 [21,63,64], JMJ29 demethylating H3K9me2 and JMJ16 and JMJ30 demethylating H3K9me3 [65,66]. These H3K9 demethylases critically regulate RdDM, RNA silencing, stomatal development, flowering time, drought response, callus formation, trichome development, disease resistance, and meiosis (Table 2).

JMJ24 and IBM1 modulate RdDM by affecting RDR2. JMJ24 interacts with and ubiquitinates CMT3, degrading it to regulate CHG methylation and H3K9me2 levels at the target promoters [23]. Positioned upstream, JMJ24 interacts with RDR2 and regulates the expression of SDC, SOLTR, and ATSN1 [67]. IBM1 demethylates H3K9me2 on RDR2 and DCL3, suppressing their expression, thereby affecting the biosynthesis of siRNA and the RdDM pathway, and interfering with the regulation of gene expression [68].

The IBM1 gene produces two transcripts: the full-length JmjC-containing IBM-L and the truncated IBM-S [62]. INCREASE IN BONSAI METHYLATION 2 (IBM2, via BAH and RRM domain), ASI1-IMMUNOPRECIPITATED PROTEIN 1(AIPP1) and ENHANCED DOWNY MILDEW 2 (EDM2, via PHD domain), form a complex (IBM2-AIPP1-EDM2) that recognizes heterochromatin (H3K9me2) and repeat elements within the large 7th intron of IBM1, promoting distal polyadenylation of IBM-L and suppressing gene body CHG methylation, particularly in longer genes [62,69,70]. IBM1, along with IBM2, AIPP1 and EDM2 prevents the silencing of thousands of genes by antagonizing CMT3 and KYP-mediated H3K9me2/CHG methylation [68,70,71,72]. The RdDM pathway/CHH methylation does not regulate IBM1 expression, and the CHG methylation of the large intron of IBM1 is strongly dependent on KYP activity [73]. IBM1 plays a crucial role in maintaining the normal expression of endogenous genes and preventing the accumulation of abnormal RNA, thereby indirectly influencing the occurrence of PTGS [53].

IBM1 regulates stomatal development. The functional expression of IBM1 relies on the upstream chromatin regulatory factor EDM2, which promotes the distal polyadenylation of IBM1 mRNA to generate the long transcript IBM1-L [74]. By demethylating H3K9me2, IBM1 inhibits CHG site methylation mediated by the DNA methyltransferase CMT3, thereby preventing abnormal hypermethylation in the gene body regions of ERECTA (ER), ERECTA-LIKE 1(ERL1), ERL2—key receptor kinase genes in the stomatal development signaling pathway that inhibit the excessive proliferation of stomatal lineage cells [74]. When IBM1 function is lost, the transcriptional levels of ER family genes decrease due to CHG hypermethylation, which weakens their inhibitory effect on the proliferation of stomatal lineage cells. This leads to a significant increase in the number of stomatal lineage cells and causes stomatal development defects [74]. Moreover, this regulatory process is dependent on the H3K9me2 methyltransferases KYP and CMT3 [74].

JMJ27 and JMJ28 regulate flowering by removing methylation marks on H3K9me2 of FLC or CO. JMJ28 interacts with FLOWERING BHLH (FBH1/2/3/4) activators, is recruited by FBH to the CO locus, reduces H3K9me2 levels at its proximal promoter and first exon, and activates CO gene expression. JMJ28 overexpression accelerates flowering under both long-day and short-day conditions [64]. The flowering delay is more pronounced in jmj28 under long-day conditions compared to short-day conditions CONSTANS-LIKE 2 (COL2), COL5, MYB DOMAIN PROTEIN 30 (MYB30), TARGET OF FLC AND SVP1 (TFS1), AGAMOUS-LIKE 6 (AGL6), and REVEILLE 2 (RVE2) are also targets of JMJ28, though how JMJ28 targets these genes is still unclear [64]. In jmj27 mutants, H3K9me2 modification increases at the FLC promoter, repressing FLC and increasing FT/SOC1 expressions, leading to early flowering that is more pronounced under short-day conditions than long-day conditions [21]. The jmj24 mutant shows a significant early flowering phenotype under short-day conditions, and when the iron-binding site is mutated from histidine to alanine, the flowering time of the jmj24 mutant is similar to the wild type [21,24,33]. JMJ24 regulates flowering independently of its histone demethylase activity. Compared with atx4/5 double mutants and jmj24 single mutants, atx4/5jmj24 triple mutants flower even earlier, suggesting that JMJ24 may have other activities besides acting as a bridge protein in the INO80 complex [24].

JMJ27 and JMJ29 enhance drought tolerance by removing H3K9me2 methylation marks on RD20, GALACTINOL SYNTHASE 2 (GOLS2), or ETHYLENE-RESPONSIVE ELEMENT BINDING FACTOR 15 (ERF15). ERF15 positively regulates ABA responses [75], and ABA regulates stomatal closure [76]. GOLS2 and RD20 are positive regulators of stomatal closure and drought stress responses [77,78]. JMJ27 interacts with the 26S proteasome regulatory particle non-ATPase 1a (RPN1a), binding to GALACTINOL SYNTHASE 2 (GOLS2) and RESPONSIVE TO DESICCATION 20 (RD20) promoters, significantly reduces H3K9me2 levels, and activates their expression [79]. Long-chain non-coding RNAs (lncRNAs) DANA2 interact with ERF84, and both are upregulated under drought stress. ERF84 is recruited to the JMJ29 promoter, inducing JMJ29 expression. JMJ29 then associates with ERF15 and GOLS2, removes H3K9me marks, and activates their expression, enhancing drought resistance [80].

JMJ29 regulates trichome development by removing the H3K9me2 mark at the GLABRA 3 (GL3) gene locus. The core trichome initiation regulatory factors GLABRA1 (GL1), GL3, and TRANSPARENT TESTA GLABRA1 (TTG1) form a MWB complex and activate the downstream trichome activator GL2 expression to regulate trichome development [81,82]. In the jmj29 mutant, H3K9me2 modification levels are increased at the GL2/3 introns and GL3 TSS, significantly reducing GL2/3 expression. JMJ29 directly binds near the GL3 TSS, removes H3K9me2, and activates GL3 expression to promote trichome development [66].

JMJ30 promotes callus formation by removing H3K9me3 from LATERAL ORGAN BOUNDARIES DOMAIN (LBD16) and LBD19 and increasing H3K36me3 marks. LBDs are activated during cell dedifferentiation, promoting callus formation [83]. AUXIN RESPONSE FACTOR 7 (ARF7), ARF19, and ARABIDOPSIS TRITHORAX-RELATED 2 (ATXR2) are recruited to the LBD promoters, leading to the recruitment of H3K36me3 at the promoter [83]. JMJ30 directly interacts with auxin signaling components ARF7 and ARF19 and removes H3K9me3 marks from LBD16 and LBD29 promoters. JMJ30 forms a JMJ30-ARF-ATXR2 complex through interaction with ATXR2. Both epigenetic marks, H3K9me3 and H3K36me3, at the LBD16 and LBD29 promoter regions activate LBD gene expression and promote the conversion of leaf tissue into callus [65].

JMJ25/IBM1 and JMJ27 participate in defense against Pseudomonas syringae by removing H3K9me2 from PR1, PR2, FRUCTOKINASE 1 (FRK1), or WRKY25. Post-inoculation with Pst DC3000, the expression of PR1, PR2, and FRK1 is elevated in wild-type but abolished in the ibm1 mutant [84]. IBM1 can remove the H3K9me2 modification from these genes to activate their expression [84]. When IBM1 is mutated, the expression of these genes is reduced. WRKY25 is involved in regulating the expression of defense genes and contains a W-box domain [TGACC (A/T)] in the promoter region [21,85]. Post-inoculation with Pst DC3000, the expression of PR1, PR3, PR4, and PR5 in the jmj27 mutant is reduced by 4–5 times, while WRKY25 expression is significantly increased [21]. This indicates JMJ27 responds to Pst DC3000 by removing the H3K9me2 modification from the W-box domain of the WRKY25 promoter [21].

JMJ16, IBM1, and JMJ27 modulate meiosis by removing H3K9me2/3 modifications from meiotic genes. IBM1 and JMJ27 cooperatively mediate the removal of H3K9me2 methylation marks from essential crossover genes, thereby ensuring their proper expression to regulate meiosis. In the ibm1jmj27 double mutant, ectopic H3K9me2 modifications are augmented, accompanied by a significant downregulation in the expression of a subset of meiotic genes. Notably, the H3K9me2 levels on SHOC1 and ZIP4 genes are substantially elevated [86]. Furthermore, IBM1 and JMJ27 physically interact with multiple cohesin cofactors PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5s) and exert regulatory effects on male meiosis and gene expression in a manner independent of H3K9 demethylation [86]. In JMJ16, H3K4 and H3K9 occupy similar positions, but H3K4me3 is nearer the histone tail terminus than H3K9me3, enabling specific recognition of H3K4me3 [18]. The C5HC2-ZF and Helica domains sterically hinder the JMJ16 catalytic domain from accessing H3K9me3 [18]. Upon binding of MMD1 to the FYR domain of JMJ16, the interaction between JMJ16′s catalytic domain and FYR domain is disrupted, triggering conformational changes in both the catalytic domain and the ZF + HD (zinc finger + helix) domain. This conformational rearrangement alleviates spatial hindrance, thereby enabling JMJ16 to specifically recognize and demethylate H3K9me3. JMJ16 interacts with MALE MEIOCYTE DEATH 1 (MMD1) to remove the H3K9me3 modification from the CAP-D3 gene, promoting meiotic chromosome condensation [18].

Table 2. H3K9 histone demethylase function.

Gene	Interaction	Target Gene	Function
JMJ16	MMD1	CAP-D3	Meiotic chromosome condensation [18]
JMJ24	RDR2	SDC, SOLTR, ATSN1	RdDM [67]
	CMT3	QQS, SDC	CHG methylation and H3K9me2 [23]
IBM1/JMJ25		ASI1, EDM2, AIPP1	Gene silencing [62,69,70]
		RDR2, DCL3	RdDM, siRNA production [68]
		ER, ERL1, ERL2	Stomatal development [74]
		PR1-2, FRK1, WRKY25	Defense response [84]
	PDS5s	SHOC1, ZIP4, etc.	Male meiosis [86]
JMJ27		FLC	Flowering time [21]
	RPN1a	GOLS2, RD20	Drought tolerance [79]
	PDS5s	SHOC1, ZIP4. etc.	Male meiosis [86]
		PR1-5, WRKY25	Defense response [21]
JMJ28	FBHs	CO	Flowering time [64]
JMJ29		GL3	Trichome development [66]
		ERF15, GOLS2	Drought tolerance [80]
JMJ30	ARF7, ARF19, ATXR2	LBD16, LBD29	Callus formation [65]

Table 2. H3K9 histone demethylase function.

Gene	Interaction	Target Gene	Function
JMJ16	MMD1	CAP-D3	Meiotic chromosome condensation [18]
JMJ24	RDR2	SDC, SOLTR, ATSN1	RdDM [67]
	CMT3	QQS, SDC	CHG methylation and H3K9me2 [23]
IBM1/JMJ25		ASI1, EDM2, AIPP1	Gene silencing [62,69,70]
		RDR2, DCL3	RdDM, siRNA production [68]
		ER, ERL1, ERL2	Stomatal development [74]
		PR1-2, FRK1, WRKY25	Defense response [84]
	PDS5s	SHOC1, ZIP4, etc.	Male meiosis [86]
JMJ27		FLC	Flowering time [21]
	RPN1a	GOLS2, RD20	Drought tolerance [79]
	PDS5s	SHOC1, ZIP4. etc.	Male meiosis [86]
		PR1-5, WRKY25	Defense response [21]
JMJ28	FBHs	CO	Flowering time [64]
JMJ29		GL3	Trichome development [66]
		ERF15, GOLS2	Drought tolerance [80]
JMJ30	ARF7, ARF19, ATXR2	LBD16, LBD29	Callus formation [65]

5. The H3K27me Demethylation Function of JmjC Proteins

H3K27 can undergo three types of methylation modifications, namely H3K27me1, H3K27me2, and H3K27me3. Among them, H3K27me1 is enriched in the heterochromatin regions near centromeres [87], while H3K27me2/3 is predominantly distributed in gene bodies [88]. Polycomb Repressive Complex 2 (PRC2) is responsible for catalyzing and maintaining H3K27me3 modifications. In contrast, the PRC1 complex recognizes H3K27me3 and catalyzes histone H2Aub modification, which promotes chromatin compaction and inhibits transcription initiation complexes recruitment, thereby repressing gene transcription. In Arabidopsis, five JmjC histone demethylases JMJ11/ELF6, REF6, JMJ13, JMJ30, and JMJ32 can remove H3K27 methylation. Specifically, ELF6 and REF6 can demethylate H3K27me2/3, while JMJ13, JMJ30, and JMJ32 target H3K27me3 for demethylation [14,15,22,89]. REF6 possesses four C-terminal tandem Cys2His2 zinc finger domains (4 x ZnF-C2H2) recognizing the “CTCTGYTY” DNA motif (Y = T/C), directing it to specific genomic regions for H3K27me3 removal [90]. REF6 mutation elevates global H3K27me3 levels and represses endogenous genes. REF6 regulates 2836 genes, but not all contain the CTCTGYTY motif, and only 15% of the CTCTGYTY motifs are REF6-bound, suggesting additional recognition sequences [90]. REF6 co-localizes with BRM at CTCTGYTY motifs, facilitating BRM recruitment, but its own genomic targeting is BRM-independent [91]. DNA CHG methylation prevents REF6 binding, and REF6 tends to bind to low methylation regions [92]. H3K27me2/3 histone demethylases play significant regulatory roles in multiple biological processes, including organ boundary formation, leaf senescence, lateral root formation, seed germination and dormancy, growth arrest, heat memory, flowering time, and self-fertilization (Table 3).

REF6 participates in organ boundary formation by recognizing the CTCTGYTY in the CUP-SHAPED COTYLEDON 1 (CUC1) to remove H3K27me3 methylation. CUC1, CUC2, and CUC3 are homologous genes are involved in regulating the formation of boundaries between the cotyledons and other organs [93]. REF6 strongly binds to the CTCTGYTY motif in the first intron of CUC1, but binds weakly to CUC3, leading to the demethylation of H3K27me3 at both loci, activating the expression of CUC1 and CUC3, and regulating organ boundary formation [90]. Although CUC2 lacks the CTCTGYTY motif, but contains the CTCTGT motif in vitro, REF6 binds both CUC1 and CUC2 in vitro, with higher affinity for CUC1 [94].

The loss of REF6 increases H3K27me3 levels across Senescence-associated genes (SAGs) and suppresses premature transcriptional activation of SAGs.REF6 directly binds to the CTGYTY motif in the promoter of NONYELLOWING (NYE1/2) through its zinc finger domain, upregulating the expression of NYE1/2 [95]. This promotes chlorophyll degradation during senescence and regulates the overall leaf senescence process [95]. REF6 also coordinately regulates Senescence-Associated Genes (SAGs) related to hormone signals such as ethylene (ETHYLENE INSENSITIVE 2 (EIN2), ORE1, NAC-LIKE, ACTIVATED BY AP3/PI (NAP)) and jasmonic acid (PHYTOALEXIN DEFICIENT 4 (PAD4)), as well as those associated with nutrient recycling (PYRUVATE ORTHOPHOSPHATE DIKINASE (PPDK), NAC DOMAIN CONTAINING PROTEIN 3 (AtNAC3), NAC TRANSCRIPTION FACTOR-LIKE 9 (NTL9)) [95]. Eventually, it becomes a key epigenetic regulator for the initiation of leaf senescence in Arabidopsis.

REF6 binds to the CTGYTY motif in the promoters of PIN-FORMED (PIN1, PIN3, PIN7), removing H3K27me3 methylation to activate the expression of these genes, thereby promoting lateral root primordia (LRP) and lateral root tips (LRS) formation. PIN1 overexpression rescues the lateral root defect in the ref6 mutant, demonstrating the role of PIN1 in lateral root development [96].

REF6 mediates seed dormancy by removing H3K27me3 methylation from the ABA catabolic genes CYP707A1 and CYP707A3. REF6 specifically recognizes and binds to the CTCTGYTY motif (where Y represents C or T) in CYP707A1 and CYP707A3 through its zinc finger domain. Subsequently, it exerts H3K27 demethylase activity to remove H3K27me3 at the loci of these two genes, thereby relieving chromatin silencing and activating their expression [97,98]. Loss-of-function mutations in CYP707A1 and CYP707A3 enhance seed dormancy and increase seed endogenous ABA levels [99,100]. REF6 deficiency results in elevated ABA levels in seeds, and CYP707A1 overexpression rescues the dormancy phenotype in ref6 mutants [97,101]. In mature seeds, although the expression and binding ability of REF6 are reduced due to chromatin condensation, the previously established low H3K27me3 state at CYP707A1/CYP707A3 locus can be maintained, which prevents excessive accumulation of ABA and ensures the normal regulation of seed dormancy [98].

REF6 participates in seed germination by removing H3K27me3 methylation from hormones signaling and cell wall-loosening genes. REF6 activates seed germination through the gibberellin (GA), ABA, and auxin signaling pathways, is a positive regulator of phyB-mediated seed germination in Arabidopsis [101]. In the ref6 mutant, the expression levels of auxin-related signaling genes PIN3, PIN7, ARABIDOPSIS P-GLYCOPROTEIN 4 (ABCB4), and cell wall-loosening genes EXPANSIN A1 (EXPA1), EXPA9, and EXPA10 are both downregulated [101]. All these genes contain the CTCTGYTY motif. REF6 targets the TSS and first exons of PIN3, PIN7, and ABCB4, as well as the first exons of EXPA1, EXPA9, and EXPA10, removing H3K27me3 methylation and activating the transcription of auxin signaling and cell wall loosening genes [101].

JMJ30 removes the H3K27me3 modification at SNF1-related protein kinase 2.8 (SnRK2.8) and BRASSINAZOLE-RESISTANT 1 (BZR1), participating in growth arrest through the ABA and Brassinosteroid (BR) pathways. After seedling germination, plants stop growing under adverse conditions [102]. JMJ30 and JMJ32 mediate post-germination growth arrest during seedling establishment (2–3 days post-germination) [103]. SnRK2.8, encoded a kinase, activates and elevates ABI3 expression to induce plant growth arrest [103,104]. ABA application induces ABI3 recognition of the RY[A(G/T) TAAC (C/T)] motif in JMJ30, promoting its expression. JMJ30 removes the H3K27me3 modification from the SnRK2.8 promoter, activating SnRK2.8 expression, which in turn activates ABI3 to control JMJ30/JMJ32-mediated growth arrest [103]. In the post-germination phase, JMJ30 and JMJ32 antagonize ABA and BR signaling [103,105,106]. BR inhibits JMJ30 expression but not JMJ32 [106]. ABA application prompts JMJ30 removes H3K27me3 from the BZR1 locus, activating BZR1 expression, transcriptionally activating BR-responsive genes, and promoting plant development [106].

REF6 participates in heat response by removing the H3K27me3 marks from HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2), GIBBERELLIN 20 OXIDASE 2 (GA20OX2), BASIC HELIX-LOOP-HELIX 87 (bHLH87) and JMJ30 leading to HSP22 and HSP17.6C activation. ELF6, REF6, JMJ30, and JMJ32 play a redundant role in heat memory [107]. REF6 forms a feedback loop along with HSFA2 to participate in cross-generational temperature memory. Under prolonged 30 °C induction, the expression of HSFA2 increases, triggering REF6/BRM expression via recognition of heat shock elements (HSEs) in their promoters [108]. Concurrently, REF6 removes the H3K27me3 mark on HSFA2 by binding its CTCTGYTY motif, thereby activating the expression of HSFA2 [108]. REF6 and PHYTOCHROME INTERACTING FACTOR 4 (PIF4) cooperatively regulate the activation of BASIC HELIX-LOOP-HELIX 87 (bHLH87) [109]. At 28 °C, REF6 removes the H3K27me3 mark from bHLH87 and PIF4 activates its expression [109]. JMJ30 mRNA and protein levels remain unchanged at 28 °C [22]. The regulation of thermomorphogenesis by REF6 depends on the GA signal. REF6 promotes GA synthesis by activating GA20ox2, and exogenous GA treatment can partially restore the heat-sensitive phenotype of the ref6 mutant [109]. Post-heat shock treatment, the quadruple mutant jmjq (jmj30jmj32elf6ref6) exhibits lower survival than single mutants (ref6, elf6, jmj13, jmj30, jmj32) or jmj30jmj32 double mutants [107]. After heat acclimation and heat shock treatment, the acquired thermotolerance of jmjq is reduced [107]. JMJ30 is induced pre-heat acclimation and during heat activation, it removes the H3K27me3 marks from HSP22 and HSP17.6C, inducing their expression [107].

ELF6, REF6, JMJ30, and JMJ32 regulate flowering by removing the H3K27me3 methylation from FLC. The elf6 mutant flowers significantly earlier than the wild-type under short-day conditions, and slightly earlier under long-day conditions [29,110]. When the conserved valine in the JmjC domain of ELF6 is mutated to alanine, the elf6-5 mutant exhibits a semi-embryo phenotype, affecting the epigenetic reprogramming of AtFLC expression during reproductive development [89]. The elf6-5 mutants exhibit an early-flowering phenotype, and the early-flowering phenotype of the next generation becomes more pronounced after vernalization [89]. The ref6 mutant shows delayed flowering, which is associated with the vernalization pathway and inhibition of the AtFLC gene [110]. Different mutation sites of REF6 cause varying flowering times, with ref6^G flowering later than ref6-1/3. Unlike the REF6 regulatory pathway in Arabidopsis, the brref6 mutant of Chinese cabbage delays flowering by downregulating BrFTa expression and affecting the transcription of genes related to BrGA, BrGA1 and BrGA20ox1 [111]. Exogenous application of GA3 can restore this phenotype [111]. The jmj13 mutant flowers earlier under long-day conditions regardless of temperature (16 °C or 28 °C), and under short-day conditions at high temperature (28 °C), but not at low temperatures (16 °C or 22 °C) [15]. JMJ13 specifically recognizes H3K27me3 through hydrogen bonds and hydrophobic interactions with H3R26, H3S28, and H3P30. H3R26 forms salt bridges and hydrogen bonds with JMJ13’s D236, and H3R28 forms side chain hydrogen bonds with JMJ13’s D296. H3P30 stacks parallel to JMJ13’s Phe179 aromatic ring, leading to hydrophobic stacking and CH-π interactions. The specific stacking of H3P30 (N + 3) and Phe179 determines the specificity of the H3K27me3 receptor [15]. This phenotype may be H3K27me3-independent, as the temperature pathway (AtFLM, AtSVP) and photoperiod pathway (AtGI, AtCO) gene expression is significantly reduced in the jmj13 mutant without significant H3K27me3 changes [15]. AtJMJ30 directly binds to AtFLC TSS and removes the methyl group from the H3K27me3 mark on AtFLC, thereby suppressing flowering [22]. The jmj30jmj32 double mutant flowers earlier under long-day and high-temperature conditions (28 °C) [22]. However, the flowering time of the jmj30 mutant does not significantly differ from wild type [112]. In AtJMJ30 overexpressing plants, the expression of AtSOC1 and AtFT is reduced, leading to delayed flowering [112,113]. The five H3K27me3 demethylases redundantly regulate flowering time. Relative to the wild type, both elf6-3ref6^C and elf6-3ref6^Cjmj13^G exhibit delayed flowering [114]. Notably, no significant discrepancy in flowering time is observed between the jmj30-2jmj32-1elf6-3ref6^Cjmj13^G pentuple mutant and elf6-3ref6^Cjmj13^G [114]. These findings indicate that AtJMJ30, along with its functional ablation, fails to exacerbate the developmental impairments of elf6-3ref6^Cjmj13^G [114].

ELF6 and JMJ13 antagonistically regulate pistil and stamen development to control self-fertility. The elf6 mutant, has shorter pistil cells and overall pistil length, with pollen positioned above the anther, enhancing self-pollination and fertility [115]. The jmj13 mutant has shorter stamens, longer pistils, and reduced fertility, with potential abortion even in later flowers. JASMONATE-ZIM-DOMAIN PROTEIN 7 (JAZ7), SMALL AUXIN UP RNA 26 (SAUR26) and ARABINOGALACTAN PROTEINS (AGPs) are silenced in the flower buds of the jmj13 mutant, suggesting they may be the targets of JMJ13 [115]. The ref6 mutant shows no fertility change [115]. Fertility of the jmj30 and jmj32 mutants is currently unreported.

Table 3. H3K27 histone demethylase function.

Gene	Target Gene	Function
AtJMJ11/AtELF6		Self-fertility [115]
	AtFLC	Flowering time [110]
BrELF6	BrFLC3	Flowering time [111]
AtREF6	CUCs	Organ boundary formation [90]
	NYE1/2, EIN2, ORE1, NAP, AtNNAC3, NTL9, LOX1, PAD4, PPDK	Leaf senescence [95]
	PIN1/3/7	Lateral root formation [96]
	PIN3/7, ABCB4, EXPA1/9/10	Seed germination [98,101]
	CYP707A1, CYP707A3	Seed dormancy [97]
	HSFA2, bHLH87, GA20OX2	Heat response [108,109]
	AtFLC	Flowering time [110]
BrREF6	BrFTa, BrGA1, BrGA20OX2	Flowering time [111]
JMJ13		Flowering time [15]
	JAZ7, SAUR26, AGPs	Self-fertility [115]
JMJ30	FLC	Flowering time [22]
	HSP22, HSP17.6C	Heat memory [107]
	SnRK2.8, BZR1	Growth arrest [103]
JMJ32	FLC	Flowering time [22]

Table 3. H3K27 histone demethylase function.

Gene	Target Gene	Function
AtJMJ11/AtELF6		Self-fertility [115]
	AtFLC	Flowering time [110]
BrELF6	BrFLC3	Flowering time [111]
AtREF6	CUCs	Organ boundary formation [90]
	NYE1/2, EIN2, ORE1, NAP, AtNNAC3, NTL9, LOX1, PAD4, PPDK	Leaf senescence [95]
	PIN1/3/7	Lateral root formation [96]
	PIN3/7, ABCB4, EXPA1/9/10	Seed germination [98,101]
	CYP707A1, CYP707A3	Seed dormancy [97]
	HSFA2, bHLH87, GA20OX2	Heat response [108,109]
	AtFLC	Flowering time [110]
BrREF6	BrFTa, BrGA1, BrGA20OX2	Flowering time [111]
JMJ13		Flowering time [15]
	JAZ7, SAUR26, AGPs	Self-fertility [115]
JMJ30	FLC	Flowering time [22]
	HSP22, HSP17.6C	Heat memory [107]
	SnRK2.8, BZR1	Growth arrest [103]
JMJ32	FLC	Flowering time [22]

6. The H3K36me Demethylation Function of JmjC Proteins

H3K36 methylation comprises three modification states: monomethylation (me1), dimethylation (me2), and trimethylation (me3), with dimethylation and trimethylation being the predominant forms associated with gene activation [116]. H3K36me2 is enriched at the 3′ end of genes, while H3K36me3 primarily marks the 5′ half of the gene body [88,117]. SET DOMAIN GROUP 4 (SDG4) catalyzes H3K36me1, SDG4/SDG8 catalyze H3K36me2/3 methylation, and SDG25 catalyzes the deposition of H3K36me2. AtJMJ30 and BrJMJ18 are dual-specificity demethylases that target both H3K36me2 and H3K36me3 [112,118]. BrJMJ18 and JMJ30 play a role in regulating flowering time; JMJ30 also plays a role in regulating the circadian clock and promoting leaf conversion into callus tissue (see the H3K9 section) (Table 4).

BrJMJ18 regulates flowering time in a temperature-dependent manner by removing the H3K36me2/3 modification at the BrFLC3 locus. At 22 °C, BrJMJ18^Par can directly bind to the gene region of BrFLC3 and inhibit BrFLC3 expression by removing the H3K36me2/3 modification, thereby promoting flowering [118]. Under high temperatures (29 °C and above), BrJMJ18^Par undergoes three amino acid mutations (T345A, Y633C, and L654F), which mutations lead to the dissociation of its binding ability to BrFLC3 and a reduction in its demethylase activity towards H3K36me2/3, then the levels of H3K36me2/3 in the BrFLC3 gene region increase, and BrFLC3 expression is upregulated, resulting in delayed flowering, preventing premature flowering of plants which would otherwise affect yield and quality [118]. In contrast, the BrJMJ18 alleles of other subspecies do not carry such mutations and thus cannot effectively regulate BrFLC3 expression under high temperatures [118].

In Arabidopsis, the nuclear interaction between JMJ30 and EARLY FLOWERING MYB PROTEIN (EFM)—both of which directly bind the AtFT locus, with JMJ30’s binding to this locus being partially EFM-dependent—enables JMJ30 to specifically demethylate the H3K36me2 mark at AtFT, thereby repressing AtFT expression and inhibiting flowering [112]. JMJ30 may indirectly regulate the H3K36 methylation of circadian clock genes to participate in clock regulation. The expression of JMJ30 exhibits circadian rhythmicity (peaking in the evening), and both its loss of function or overexpression can affect the expression period of core oscillator genes (such as CCA1, LHY and TOC1) [113,119]. At 27 °C, the jmj30 mutant displays the largest difference in the circadian period length compared to the wild type, exhibiting a short-cycle phenotype [120]. JMJ30 overexpressing plants also display a phenotype of shortened circadian period, and the expression of endogenous [113]. JMJ30 and LUX ARRHYTHMO (LUX) act in parallel pathways to inhibit PSEUDO-RESPONSE REGULATOR 7 (PRR7) expression, but no significant changes in H3K36me1/2/3 modifications at PRR7 are observed [120].

Table 4. H3K36 histone demethylase function.

Gene	Target Gene	Function
JMJ30	EFM, FT	Flowering time [112]
	CCA1, LHY, TOC1	circadian clock [113,120]
BrJMJ18	BrFLC3	Flowering time [118]

Table 4. H3K36 histone demethylase function.

Gene	Target Gene	Function
JMJ30	EFM, FT	Flowering time [112]
	CCA1, LHY, TOC1	circadian clock [113,120]
BrJMJ18	BrFLC3	Flowering time [118]

7. The H4R3me2s Demethylation Function of JmjC Proteins

H4R3me2s represents a repressive chromatin state and exhibits crosstalk with other histone marks. Mutations in Protein Arginine Methyltransferase 5 (AtPRMT5) result in decreased H4R3me2 at the FLC chromatin, correlating with reduced levels of H3K9me3 and H3K27me3. The JMJD6 group, the smallest subgroup within the JmjC family, comprises JMJ21 and JMJ22, and these two (JMJ21 and JMJ22) are most similar to JMJ20 in Group IV of the JmjC domain-only A subfamily.

JMJ20 and JMJ22 remove H4R3me2s from the GIBBERELLIN 3-OXIDASE 1 (GA3OX1) gene to influence seed germination. JMJ20 and JMJ22 affect seed germination, while JMJ21 does not. Following red light pulse treatment, the germination rate of jmj20jmj22 mutants is lower compared to the wild type [121]. JMJ20 and JMJ22 act as H4R3me2s histone arginine demethylases, removing the methylation from the GA biosynthesis gene GA3OX1 and acting as positive regulators of seed germination in the PHB-PIF5 pathway [121].

8. Conclusions and Outlook

Through systematic comparative analysis of JmjC proteins in Arabidopsis and B. rapa, this study clearly reveals the conservation and species-specific divergence patterns of histone demethylation mechanisms in Brassicaceae plants, thereby holding significant theoretical research value and evolutionary biological significance (Figure 3). The JmjC domain-containing proteins in Arabidopsis and B. rapa are both classified into seven evolutionary clades. Beyond conserving their Fe²⁺/α-ketoglutarate-dependent catalytic activity, members of the ELF6, REF6, and JMJ18 subfamilies within the JmjC protein family exhibit conserved and specific functions in flowering time regulation, each precisely modulating plant flowering progression [13,30,89,110,111,118]. However, species-specific divergence characteristics are particularly prominent, driven by genome triplication. The JmjC family has undergone expansion in B. rapa, with 29 members identified (compared to 21 in Arabidopsis), accompanied by adaptive functional remodeling. For instance, BrJMJ18 has evolved into a demethylase specifically acting on H3K36me2/3, influencing BrFLC3 expression through a temperature-dependent regulatory mechanism to adapt to environmental stresses during crop cultivation. The regulatory pathways of BrREF6 and BrELF6 for flowering time also exhibit obvious differences from those in Arabidopsis, focusing on the specific regulation of GA synthesis genes and BrFLC3, respectively, which reflects the unique epigenetic regulatory strategies formed in Brassica during long-term domestication [12,13,30,89,110,111,118].

From an evolutionary perspective, such “functional divergence within a conserved framework” represents a key epigenetic strategy for Brassicaceae plants to adapt to complex environments and crop domestication processes: conservation ensures the stability of core growth and development pathways, guaranteeing the orderly progression of basic life activities. The species-specific divergence endows Brassica with greater environmental adaptability and agronomic trait plasticity. The copy number amplification and substrate specificity switching of the JmjC protein family not only intuitively reflect the remodeling effect of genome duplication events on the epigenetic regulatory network but also provide a molecular basis for the directional selection of key agronomic traits. Such as flowering time optimization and stress tolerance enhancement, during the domestication of Brassica from wild to cultivated varieties, highlight the core value of epigenetic mechanisms in plant adaptive evolution and crop genetic breeding.

Taken together, future studies should further integrate comparative genomics and evolutionary biology perspectives to thoroughly dissect the divergence patterns of the JmjC family during the whole-genome triplication event in B. rapa. The classification analysis of JmjC should be performed to discover the specific molecular pathways underlying the neofunctionalization of functionally redundant genes. Moreover, molecular markers and gene editing technologies can be developed based on the epigenetic regulatory characteristics of JmjC proteins, paving a new way for breeding high-yield and high-quality Brassica crops.