Genome-Wide Identification of Osmanthus fragrans Histone Modification Genes and Analysis of Their Expression during the Flowering Process and under Azacytidine and Ethylene Treatments

Histone acetylation and methylation, governed by various histone modification (HM) gene families, are vital for plant biological processes. However, there are limited studies that have explored HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans). We performed genome-wide search and identified 208 OfHMs, encompassing 81 histone methyltransferases (OfHMTs), 51 histone demethylases (OfHDMs), 49 histone acetyltransferases (OfHATs) and 27 histone deacetylases (HDACs). Our comprehensive analysis covered chromosome locations, gene structures, conserved domains, cis-acting elements, phylogenetic comparisons, protein interaction networks and functional enrichment pathways for these gene families. Additionally, tandem and fragment replications were unveiled as contributors to the expansion of OfHMs, with some genes exhibiting positive selection. Furthermore, we examined OfHM expression profiles across various tissues and flowering stages, and under 5′-azacytidine (Aza) and ethylene treatments. Most OfHMs displayed heightened expression in leaves, and were downregulated during the flower opening and senescence stages, including OfPRMTs, OfHDTs, OfHDAs, OfSRTs, OfJMJs and OfHAGs; 75.86% and 80.77% of the differentially expressed OfHMs were upregulated after Aza and ethylene treatments, including OfHAGs, OfHDAs and OfSDGs. This study offers a comprehensive analysis of the OfHM gene family, which indicated their potential involvement in ethylene and Aza responses, and in the flowering process. These findings provide valuable insights into the role of OfHMs in flowering and stress responses.


Introduction
Histone modifications (HMs) are essential for modulating gene expression through modifying chromatin structure and stability, thereby influencing diverse biological processes in plants [1][2][3].These processes include not only biological growth and development, but also the response to stress.The primary types of HMs encompass histone acetylation, Plants 2024, 13, 777 2 of 27 methylation, phosphorylation, ubiquitination, SUMOylation and ADP-ribosylation [4].Among these types, histone acetylation and methylation have been extensively studied [5,6] and are recognized as two pivotal and widespread epigenetic regulatory mechanisms that significantly impact gene expression.
Histone acetylation, under the control of histone acetyltransferases (HATs) and histone deacetylases (HDACs), involves the modification of lysine residues in histone proteins through the addition or removal of acetyl groups [7,8].HAT-induced acetylation promotes the opening of local chromatin, facilitating the binding of transcription factors and increasing gene expression, while HDAC-associated acetylation is often associated with gene repression [9][10][11].Histone methylation, catalyzed by histone demethylases (HDMs) and histone methyltransferases (HMTs), involves the addition or removal of methyl groups from the histone tails [7].Combinatorial binding modules in histone recognizers recognize this modification, ultimately influencing chromatin structure and gene expression [4].The impact of histone methylation on the initiation and inhibition of gene expression is primarily determined by the position of the methylation.For instance, trimethylation of lysine at position 4 on histone H3 (H3K4me3) is linked to the initiation of gene expression, while trimethylation of lysine at position 27 on histone H3 (H3K27me3) is correlated with repressed gene expression [12,13].
The functions of HM genes have been delineated, revealing their involvement in various plant processes [20,21].These processes encompass growth and development, and stress responses, including photomorphogenesis [22], embryo development [23], seed germination and dormancy [2], flowering processes [8], fruit development [15,16,18], stress and defensive responses [3,[16][17][18] and signaling responses to hormones [17].In particular, HM genes play a pivotal role in crucial physiological processes associated with plant flowering, including flower induction, petal senescence and regulation of the flowering period.For instance, members of the HAT family, such as AtHAM1 and AtHAM2, influence flowering time through epigenetic modification of FLOWERING LOCUS C (FLC) and MADS AFFECTING FLOWERING 3/4 (MAF3/4) chromatin through H4K5 acetylation [24].AtHAC1 promotes the Arabidopsis flowering process through epigenetic modifications to FLC upstream factors [25].Within the HDAC family, AtHDA6 and AtHDA9 regulate the flowering locus D/VE (FLD/FVE), osmotically responsive gene 1 (HOS1), nuclear localized protein 22 of the AT hook motif (AHL22) and AGAMOUS-LIKE 9 (AGL9), thus influencing the flowering time of Arabidopsis [26,27].The AtHDA19 complex directly regulates gibberellin (GA) signaling, impacting the expression of FLC and FLOWERING LOCUS T (FT) through a pathway that inhibits flowering [28].In the HDM family, AtJMJ14, AtJMJ15 and AtJMJ18 regulate the flower opening time of Arabidopsis by modulating the trimethylation level of histone H3K4 [29][30][31]; BcJMJ30, a gene that encodes a histone demethylase with a jmjC domain, is involved in the development of pollen and fertilization of rape (Brassica campestris) [32].The trithorax group gene (TrxG) AtSDG25 of the HMT family, which is involved in H3K4 and H3K36 methylation, delays flowering by activating FLC expression [33].However, the roles of HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans), have received limited attention.
O. fragrans, a distinguished member of the Oleaceae family, stands as one of the most renowned and traditional flowers in China.Celebrated for its intense fragrance [34], it is widely embraced in horticulture and landscaping.Beyond its ornamental value, it has found mature applications in the realms of food, cosmetics and medicines [34][35][36].However, despite its versatility, it faces the challenge of a brief flowering period, which typically only lasts 2 to 3 days, limiting its ornamental and economic value [37,38].The role of HMs in regulating the flowering period in O. fragrans remains elusive.This study used bioinformatics to identify HM gene members in the O. fragrans genome, conducting analyses of the gene structure, chromosomal location, phylogenetic comparisons, conserved protein domains, protein-protein interaction networks and functional enrichment.Furthermore, transcriptome data were utilized to analyze gene expression profiles in different tissues (stems, leaves and roots), during the flower opening and senescence processes, and under various treatment agents.These findings provide valuable information on HM genes during the flower opening and senescence stages of O. fragrans, thereby contributing essential information for comprehending the intricacies of flowering and senescence processes and enriching biological theories.Moreover, considering the significant economic importance of O. fragrans, research on OfHM genes holds potential application value in improving its yield and quality.This study could contribute to expanding our comprehensive understanding of plant life activities and provide new theoretical and practical support for plant genetic breeding and biotechnology applications.S1).The OfHMTs exhibited lengths ranging from 258 to 2419 amino acids (aa), with molecular weights (MWs) varying from 29.30 to 276.20 kDa, isoelectric points (pIs) within the range of 4.55 to 9.16, and aliphatic indices within the range of 62.62 to 99.85 (Table S1).Among them, 83.95% of the OfHMTs exhibited an instability index >40, which is indicative of a prevalence of unstable proteins in this gene family (Table S1).The OfHDMs had lengths ranging from 135 to 2136 aa, MWs from 14.79 to 233.73 kDa, pIs from 4.89 to 9.43, and aliphatic indices from 65.85 to 94.89, with 88.24% being classified as unstable proteins (Table S1).The OfHATs exhibited lengths ranging from 105 to 1869 aa, with MWs from 11.73 to 212.34 kDa, pIs from 4.46 to 10.41, and aliphatic indices >60% (Table S1).The majority (67.35%) of them were characterized as unstable proteins (Table S1).OfHDACs, with lengths of 98-659 aa, MWs of 11.47-73.41kDa, pIs of 4.11-10.55,and aliphatic indices ≥48.32, included unstable proteins among the OfSRTs (71.43%),OfHDTs (71.43%), and OfHDAs (47.06%) (Table S1).It is noteworthy that, excluding OfPRMT11, the other OfHMs possessed a grand average of hydropathicity (GRAVY) < 0, signifying hydrophilic properties (Table S1).Furthermore, in addition to OfSDG27 and OfHDA10, the remaining OfHMs lacked protein transmembrane domains (Table S1).

Identification and Characterization Analysis of HMs in the O. fragrans Genome
Most OfHM proteins (63.94%) were predicted to be localized in the nucleus (Table S1), and 21.63% of the proteins were identified on the cell membrane, encompassing the endomembrane system, plasma membrane and organelle membrane (Table S1).Furthermore, 11.54% of these proteins were assumed to be located within the chloroplast (Table S1).

Cis-Acting Element Analysis of OfHMs
Promoters are crucial in initiating gene transcription.To explore the potential biological functions and response characteristics of OfHM genes, the promoter sequences were submitted to PlantCARE for the analysis of cis-acting elements.The analysis revealed 24 cis-acting elements involved in light responsiveness, hormone responsiveness (including responses to auxin, GA, SA, MeJA and ABA), physiological stress (such as drought and cold) and growth and development regulation (Figures 4 and 5).The abundance of light response elements was highest at 2259, followed by hormone response (1515), physiological stress elements (917), and growth and development regulation (346) (Figures 4 and 5).Among them, the OfHAC4 gene contained the fewest light-responsive elements, with only 3, while OfJMJ17 had the most (32) (Figures 4 and 5).OfHAC7, OfHDA15, OfJMJ4, OfSDG8, OfSDG46 and OfSDG65 had the fewest hormone-responsive elements (1), while OfHDT4, OfJMJ1 and OfJMJ10 possessed the most response elements (19-20) (Figures 4 and 5).OfHAG12, OfHAG33, OfJMJ6 and OfSDG12 lacked physiological stress response elements, while the others contained 1-10 stress response elements (Figures 4 and 5).Interestingly, 38 OfHMs (18.27%) lacked regulatory cis-acting elements for growth and development, 72 genes (34.62%) had only one cis-acting element, and 54 OfHMs (25.96%) contained two cis-acting elements (Figures 4 and 5).These findings collectively indicate that the expression of OfHMs may be regulated by various cis-acting elements correlated with light responsiveness, hormone responsiveness, physiological stress, and specific growth and development processes.

Cis-Acting Element Analysis of OfHMs
Promoters are crucial in initiating gene transcription.To explore the potential biological functions and response characteristics of OfHM genes, the promoter sequences were submitted to PlantCARE for the analysis of cis-acting elements.The analysis revealed 24 cis-acting elements involved in light responsiveness, hormone responsiveness   As shown in Figure 6b, a syntenic map of the OfHMs and AtHMs was constructed to elucidate their potential evolutionary relationships.A total of 87 segmental duplications of OfHMs and AtHMs were identified (83 pairs of genes shown in Chrs, Figure 6a) (Table S3).The identified pairs consisted of 6 PRMTs, 44 SDGs, 2 HDMAs,
duplication, we calculated the Ka/Ks values for these gene pairs.It was found that 11 pairs of HMs, namely AtPRMT13 and OfPRMT5, AtPRMT13 and OfPRMT10, AtSDG6 and OfSDG24, AtSDG6 and OfSDG43, AtSDG13 and OfSDG48, AtSDG8 and OfSDG62, AtSDG5 and OfSDG64, AtJMJ28 and OfJMJ2, AtJMJ18 and OfJMJ3, AtJMJ17 and OfJMJ6, and AtJMJ11 and OfJMJ12, exhibited Ka/Ks values < 1 (Table S3), indicating purifying selection during evolution.A pair of HMs, namely AtSDG13 and OfSDG18, had a Ka/Ks value of 1 (Table S3), suggesting neutral selection during evolution.For 20 pairs of HMs, that is, 9 pairs of SDGs, 1 pair of HDMAs, 7 pairs of JMJs, 2 pairs of HAGs and 1 pair of HDAs, the Ka/Ks value was > 1 (Table S3), indicated that they had undergone positive selection.The importance of gene duplication in the generation of new genes and functions is evident, with segmental and tandem duplications serving as primary drivers during the expansion of gene family [39].This study investigated the amplification of the OfHM genes through an analysis of gene duplication events.The findings revealed that 89 OfHMs resulted from duplication events, covering 3 pairs of tandem duplicate genes (i.e., OfJMJ23 and OfJMJ24, OfSDG49 and OfSDG50, and OfHDT1 and OfHDT2) and 86 pairs of segmental duplications, with 70 pairs located on Chrs (Figure 6a).To assess the selective pressure on OfHM gene duplication during O. fragrans evolution, this study calculated the non-synonymous (Ka)/synonymous (Ks) ratios for homologous gene pairs.Among them, 44 pairs of OfHM genes exhibited a Ka/Ks ratio < 1 (Table S2), suggesting purifying selection and evolutionary conservation of functions.On the contrary, 11 pairs of OfHM genes, including OfHDMA1 and OfHDMA12, OfHAC1 and OfHAC2, OfHAC2 and OfHAC4, OfHAC3 and OfHAC4, OfHAC1 and OfHAC7, OfJMJ8 and OfJMJ18, OfJMJ26 and OfJMJ14, OfHAG17 and OfHAG35, OfSDG36 and OfSDG17, OfSDG45 and OfSDG18, and OfSDG57 and OfSDG65, displayed a Ka/Ks ratio > 1 (Table S2), indicating positive selection during evolution.
As shown in Figure 6b, a syntenic map of the OfHMs and AtHMs was constructed to elucidate their potential evolutionary relationships.A total of 87 segmental duplications of OfHMs and AtHMs were identified (83 pairs of genes shown in Chrs, Figure 6a) (Table S3).The identified pairs consisted of 6 PRMTs, 44 SDGs, 2 HDMAs, 16 JMJs, 3 HAGs, 1 SRT, 9 HDAs and 6 HDTs.To assess the selection pressure during duplication, we calculated the Ka/Ks values for these gene pairs.It was found that 11 pairs of HMs, namely AtPRMT13 and OfPRMT5, AtPRMT13 and OfPRMT10, AtSDG6 and OfSDG24, AtSDG6 and OfSDG43, AtSDG13 and OfSDG48, AtSDG8 and OfSDG62, AtSDG5 and OfSDG64, AtJMJ28 and OfJMJ2, AtJMJ18 and OfJMJ3, AtJMJ17 and OfJMJ6, and AtJMJ11 and OfJMJ12, exhibited Ka/Ks values < 1 (Table S3), indicating purifying selection during evolution.A pair of HMs, namely AtSDG13 and OfSDG18, had a Ka/Ks value of 1 (Table S3), suggesting neutral selection during evolution.For 20 pairs of HMs, that is, 9 pairs of SDGs, 1 pair of HDMAs, 7 pairs of JMJs, 2 pairs of HAGs and 1 pair of HDAs, the Ka/Ks value was > 1 (Table S3), indicated that they had undergone positive selection.

Functional Enrichment Analysis of OfHMs
An enrichment analysis was performed to uncover the potential biological functions of the OfHMs.The functions of all HM-modified genes were determined based on Gene Ontology (GO) categories, encompassing biological processes, molecular functions and cellular components (Figure 7a).In terms of biological processes, these genes exhibited enrichment in pathways such as methylation, macromolecule methylation, HM, protein alkylation, protein methylation and protein methylation.Regarding cellular components, the genes were more abundant in heterochromatin, the SWI/SNF complex and chromatin pathways.For molecular functions, enrichment was observed in pathways such as (N-)methyltransferase activity, histone methyltransferase activity, and protein methyltransferase activity.However, the histone acetylation modification genes did not exhibit enrichment within the relevant metabolic pathways.
Furthermore, analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways found that the genes associated with histone methylation modifications are enriched in the lysine degradation and transcription machinery pathways (Figure 7b).On the contrary, the histone acetylation modification genes were more abundant in pathways such as the viral life cycle of HIV-1, the metabolism of nicotinate and nicotinamide, mitochondrial biogenesis, arginine biosynthesis and basal transcription factors (Figure 7c).Consequently, these OfHM genes are presumed to play a variety of roles in cellular metabolism.

Prediction of Interactions of OfHM Proteins
For a more comprehensive understanding of the biological interactions involving the OfHM proteins, we performed a protein interaction network analysis.As illustrated in Figure 8, 93 proteins from 11 HM-related groups, consisting of 33 OfSDGs, 18 OfHAGs,

Expression Analysis of OfHMs in Different Tissues and Flowering Stages
To gain insights into the responsiveness of OfHMs during the flower opening and senescence stages, as well as under ethylene and Aza treatments, we examined their expression profiles utilizing publicly available transcriptome data [40].Among the identified OfHMs, 182 genes with expression levels (fragments per kilobase of exon model per million mapped fragments values (FPKM)max > 1) were recognized across various tissues and flowering stages, comprising 11 diverse gene families (Figures S2 and S3).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis of OfHM Genes
To validate the reliability of the OfHM expression profiles derived from the RNA-seq data, we determined the expression levels of six OfHMs during the flower opening and senescence stages using qRT-PCR.The results revealed that the expression patterns of these OfHMs closely resembled those obtained in the RNA-seq analysis (Figure 12).Specifically, the expression of these genes, OfJMJ19, OfSRT3, OfSDG55, OfHDT7, OfHDMA4 and OfHAG9, showed a decreasing trend during flower opening and senescence (Figure 12a-f).Furthermore, the R 2 value reached 0.72 through linear fitting between the qRT-PCR and FPKM data (Figure 12g), indicating a high degree of reliability in the OfHM gene profiles.Therefore, our transcriptome-based analysis of OfHM gene expression levels demonstrates high reproducibility and reliability, providing a robust reference for further studying the roles of OfHMs.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis of OfHM Genes
To validate the reliability of the OfHM expression profiles derived from the RNA-seq data, we determined the expression levels of six OfHMs during the flower opening and senescence stages using qRT-PCR.The results revealed that the expression patterns of these OfHMs closely resembled those obtained in the RNA-seq analysis (Figure 12).Specifically, the expression of these genes, OfJMJ19, OfSRT3, OfSDG55, OfHDT7, OfHDMA4 and OfHAG9, showed a decreasing trend during flower opening and senescence (Figure 12a-f).Furthermore, the R 2 value reached 0.72 through linear fitting between the qRT-PCR and FPKM data (Figure 12g), indicating a high degree of reliability in the OfHM gene profiles.Therefore, our transcriptome-based analysis of OfHM gene expression levels demonstrates high reproducibility and reliability, providing a robust reference for further studying the roles of OfHMs.

Discussion
Numerous previous studies have highlighted the pivotal role of HMs in plant growth and development processes, along with responses to both abiotic and biotic stresses, through the nuanced modulation of gene transcription [14,16].Consequently, an increasing number of investigations have focused on understanding the functions of modifying enzymes in various plant species.Although substantial progress has been made in some model plants [7,8,16], this information has not been reported for O. fragrans until now.Here, we conducted a comprehensive characterization of OfHMs, providing insights into their gene location, conserved domains, phylogenetic relationships, gene expansion, cis-acting elements and gene structure.Furthermore, we analyzed the expression patterns of OfHMs during flower opening and senescence, and under stress treatments.The findings presented here contribute to the growing knowledge in this field and provide a basis for further research on O. fragrans.

Discussion
Numerous previous studies have highlighted the pivotal role of HMs in plant growth and development processes, along with responses to both abiotic and biotic stresses, through the nuanced modulation of gene transcription [14,16].Consequently, an increasing number of investigations have focused on understanding the functions of modifying enzymes in various plant species.Although substantial progress has been made in some model plants [7,8,16], this information has not been reported for O. fragrans until now.Here, we conducted a comprehensive characterization of OfHMs, providing insights into their gene location, conserved domains, phylogenetic relationships, gene expansion, cisacting elements and gene structure.Furthermore, we analyzed the expression patterns of OfHMs during flower opening and senescence, and under stress treatments.The findings presented here contribute to the growing knowledge in this field and provide a basis for further research on O. fragrans.
The uneven distribution of OfHMs in the genomes, as observed in Figure 6a, aligns with findings in citrus [16] and apple [8].This pattern of uneven chromosome distribution suggests that these changes occurred prior to species differentiation.

Evolution and Amplification Analyses of OfHM Gene Family
The construction of phylogenetic trees, aligning genes across diverse botanical species, provides profound insights into the intricate tapestry of evolutionary interrelations among genes [42].In this study, four phylogenetic trees, i.e., HMTs, HDMs, HATs and HDACs, were constructed, utilizing all members of the HM gene families from both O. fragrans and A. thaliana (Figure 1).The observed clustering pattern for each category of OfHMs is consistent with observations documented in other species, as reported in previous studies [7,8,14,17].However, there are exceptions, exemplified by the clustering of HDAs on a particular branch, excluding AtHDT4 (Figure 1d).This divergence might be attributed to the partial matching of their protein sequences.
The structure of genes and conserved motifs are pivotal in shaping the evolution of gene families.In our study, different gene families within the OfHMs showcased conserved typical domains, as illustrated in Figures 2 and 3 [7,8,14,17].Notable examples include the presence of a PRMT5/MT domain in the OfPRMTs (Figure 2a), a characteristic SET domain in the OfSDGs (Figure 2b), and cupin_RmlC-like superfamily/JmjC and SWIRM conserved domains in the OfJMJs and OfHDMAs, respectively (Figure 2c).Furthermore, the domains AT, HAT_KAT11 and MOZ-SAS were identified in the OfHAGs, OfHACs and OfHAMs (Figure 3a,b), while the HDAC1, SIR2 and NPL domains were contained in the OfHDAs, OfSRTs and OfHDTs, respectively (Figure 3c).Additionally, various genes within each gene family harbored additional structural domains (Figures 2 and 3).For instance, the I-type OfSDGs featured SET domains; the II-type OfSDGs included AWS domains; the III-type OfSDGs, excluding OfSDG5 (9), encompassed zf-HC5HC2H and FYRC/PWWP domains; and the V-type OfSDGs typically contained SRR/WIYLD and Pre-SET domains; and most of the VI/VII-type OfSDGs included rubis-subs-bind/AWS/TPR domains (Figure 2b).This conservation of domains within the same family/subfamily implies shared features across species.Furthermore, OfHMs with distinct structures and conserved motifs exhibited clustering at considerable distances, whereas those with analogous structures and motifs tended to cluster close together (Figures 2 and 3).This clustering pattern is consistent with the observations in other species [8,16], indicating a potential correlation among phylogeny, gene structure and protein motifs.However, these correlations require validation through additional experiments.
Segmental and tandem duplications serve as fundamental mechanisms driving the expansion of gene families [39].In our study, 89 OfHMs were identified as products of duplication events, and 86 HM segmental duplications existed between the OfHMs and AtHMs (Figure 6b).This discovery aligns with results in apple [8,41], where 67 pairs of MdHMs were reported.Most of the OfHM gene pairs exhibited a Ka/Ks ratio < 1 (Table S2), indicative of purifying selection and evolutionary conservation of their functions.In particular, 11 OfHM gene pairs, including 1 OfHDMA, 4 OfHACs, 2 OfJMJs, 1 OfHAG and 3 OfSDGs, exhibited a Ka/Ks ratio > 1 (Table S2).However, the majority of pairs of AtHM and OfHM genes displayed a Ka/Ks ratio > 1 (Table S3), indicating positive selection during evolution [43].These results diverged from most studies [8,17,41].For instance, gene pairs duplicated within apple, millet (Setaria italica), or between them and Arabidopsis generally exhibited Ka/Ks ratios < 1 (Table S3).These gene pairs with Ka/Ks ratios greater than 1 may have played a pivotal role in species adaptive evolution or in response to specific environmental stresses.Collectively, the OfHMs have undergone tandem and segmental Plants 2024, 13, 777 20 of 27 duplications, contributing significantly to the expansion of the OfHMs and their subsequent structural and functional diversification.

OfHMs Are Involved in the Flowering Process and the Induction of Aza and Ethylene Responses
HM genes play a crucial role in the orchestration of plant growth and development [14,16].The functional enrichment analysis revealed that OfHMs were enriched in lysine degradation, transcription machinery, viral life cycle of HIV-1, nicotinate and nicotinamide metabolism, mitochondrial biogenesis, arginine biosynthesis and basal transcription factors pathways (Figure 7b,c).The promoter region of the OfHM genes contained elements that respond to plant hormones, stress, light, and growth and development (Figures 4 and 5), indicating their involvement in light responses, hormone responses, physiological stress, and the regulation of growth and development.
The expression patterns of genes provide vital information for the exploration of gene function [44,45].Across three different tissues (roots, stems and leaves), 83 OfHMs exhibited differential expression patterns, with the majority of genes (73.49%) exhibiting increased expression in leaves (Figure 9).However, it should be noted that certain studies have observed nearly ubiquitous expression of TaHMs during the development stages of wheat grains, with numerous genes displaying elevated expression levels in specific layers of grain tissue [46], thus indicating potential species-specific differences.

Identification and Chromosome Location of OfHM Gene Family
To identify members of the HM gene family in O. fragrans, various genomic data, including whole-genome data, CDS data, protein sequences and the General Feature Format version 3 (GFF3) file, were obtained from the National Center for Biotechnology Information (NCBI) database (PRJNA679852) [62].Two methods were employed to identify members within the OfHM gene family.Firstly, hidden Markov Model (HMMER) files corresponding to specific domains (PF00583, 00850, 00856, 01853, 02146, 02373, 04433, 05185, 08214 and 09247) were acquired from the Pfam database (http://pfam.sanger.ac.uk/, accessed on 25 December 2023), following the approach of previous studies [14,16].Additionally, an HMMER file was constructed using protein sequences encoded by four AtHDT genes, i.e., AtHDT1-4 (At3g44750, At5g22650, At5g03740 and At2g27840), obtained from the TAIR database (http://www.arabidopsis.org/,accessed on 25 December 2023).Subsequently, these 11 HMMER files served as queries to search the O. fragrans genome using HMMER3.0,with an E-value threshold set to <0.00001 [63].Secondly, a total of 102 AtHM protein sequences [7] were retrieved from the TAIR website.A BLASTP alignment search was performed using these AtHM sequences as query sequences, with an expected value (E value) set at 0.00001 to identify candidate OfHMs.After eliminating redundant sequences, all candidate protein sequences were analyzed through the SMART database (http://smart.embl.de/,accessed on 28 December 2023) and the Conserved Domain Search (CDD) database (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi,accessed on 28 December 2023).Throughout this process, genes lacking known conserved domains were systematically excluded, resulting in the final identification of the OfHM genes.The OfHM genes, including HMTs (SDGs and PRMTs), HDMs (HDMAs and JMJs), HATs (HAGs, HAMs, HACs and HAFs) and HDACs (HDAs, SRTs and HDTs), were named according to their Chr orders, following the convention of a previous study [16].
The chromosomal location information for the OfHM genes were extracted from the O. fragrans genome, and the MG2C online tool (http://mg2c.iask.in/mg2c_v2.1/,accessed on 31 December 2023) was utilized to visually represent their chromosomal locations.

Physicochemical Characteristics Examination, Phylogenetic Tree Elaboration, and Gene Structure Research
ProtParam (https://web.expasy.org/protparam/,accessed on 13 January 2024) was employed for the analysis of the physical and chemical properties of proteins.Deep TMHMM (https://dtu.biolib.com/DeepTMHMM,accessed on 12 January 2024) was used to predict the transmembrane structure of the OfHM proteins.Additionally, the BUSCA website (http://busca.biocomp.unibo.it/,accessed on 13 January 2024) was used to predict their subcellular localization.
In conducting the phylogenetic analysis, molecular evolutionary genetics analysis (MEGA) v11.0 [64] was used to explore the phylogenetic relationships of HMs between O. fragrans and A. thaliana.The protein sequences of AtHMs and OfHMs were aligned by the MUSCLE program with default parameters.Subsequently, the resulting multiple sequence alignment files were utilized to construct phylogenetic trees with the neighbor-joining (NJ) or maximum likelihood method, with bootstrap values determined through 1000 times.
The TBtools software v2.061 [65] was employed to determine the positions of untranslated regions (UTRs), introns, CDSs and domains within the O. fragrans genome annotation file (gff3), and to visualize the gene structure.For the prediction of conserved motifs in OfHMs, the MEME website (https://meme-suite.org/meme/tools/meme, accessed on 30 December 2023) was utilized with the specified parameter motif = 20 and any number of repetitions in the site distribution.Subsequently, the identified motifs were visualized using the TBtools software [65].The CDD database was employed to predict the conserved domains of OfHMs, and the results were visualized using the TBtools software [65].

Cis-Acting Regulatory Element Analysis of OfHM Genes
The 2000 bp base sequence upstream of the start codon (ATP) for each member of the HM gene family was extracted as the promoter region using TBtools [65].Subsequently, Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 January 2024) [66] was employed to predict cis-acting regulatory elements within these promoter regions.The identified cis-acting regulatory elements were then visualized using the TBtools software [65].

Tandem Duplication and Synteny Analyses
Tandem duplication and synteny relationships were explored.Tandem duplication of OfHMs genes was identified according to their physical locations in individual Chrs within the O. fragrans genome.Specifically, genes located within a 200 kb region on the Chr, exhibiting > 70% identity, were defined as tandem duplication genes [39].Furthermore, synteny blocks among different O. fragrans Chrs were identified using the multiple collinearity scan (MCScanX) toolkit [67].

Protein-Protein Interaction Network Construction and Functional Enrichment Analysis of OfHM Genes
A BLASTP analysis was utilized to identify the homologous genes of OfHMs and AtHMs.Interaction pairs among the OfHMs were determined using the STRING database v12.0 (http://string-db.org,accessed on 21 January 2024).For this analysis, the network type was configured as the full STRING network, where the edges represent both functional and physical protein associations; the meaning of network edges was adjusted to reflect the confidence in thickness, indicative of the strength of the data support; and a minimum required interaction score of high confidence (>0.70) was applied to ensure the reliability of the identified interactions.The visualization of gene pairs with high confidence scores was achieved using Cytoscape v3.6.1 (http://www.cytoscape.org/,accessed on 22 January 2024).Furthermore, GO and KEGG enrichment analyses of histone methylation and acetylation genes were performed using the resources available at https://www.omicsshare.com/(accessedon 22 January 2024), respectively.

Plant Materials and Treatment
The experimental material for this study was a healthy and well-growing O. fragrans 'Liuyejingui' (OFL) tree located on the campus of Huazhong Agricultural University in Wuhan, Hubei, China (114 • 21 ′ W, 30 • 29 ′ N), as detailed by Chen et al. [62].Various tissues, including roots, stems and leaves, were collected from the same tree, alongside samples from different stages of flowering (S1: linggeng stage, characterized by closed flower buds; S2: initial flowering stage, where flowers exhibit a slight opening with a petal angle of less than 45 • ; S3: early stage of full flowering, with flowers partially open and petal angles ranging from 45 to 90 • ; S4: full stage of flowering, where petals are fully expanded; S5: late stage of full flowering, marked by petals slightly losing turgor and darker pollen color; and S6: stage of abscission, during which petals lose turgor and undergo natural abscission) [62].Detached branches that carried floral buds at stage S1 were treated with 200 mL of 10 mM 5 ′ -azacytidine (Aza) or 500 mg L −1 ethephon (dissolved in ddH 2 O).In contrast, as a control, flowering branches were sprayed with 200 mL of ddH 2 O alone [40].The spray treatment was repeated at 3-day intervals, specifically targeting flowers at stages S1, S3 and S5.Three independent biological replicates were performed for each treatment.

Identification of OfHM Expression Profiles Using High-Throughput Sequencing
Total RNA was extracted using TRIzol reagent (Invitrogen Co., Carlsbad, CA, USA) following the manufacturer's protocol.RNA-seq libraries were prepared using the SMARTer cDNA synthesis kit (Clontech Laboratories, Mountain View, CA, USA), and subsequently processed on the MGI-SEQ 2000 platform (Frasergen Bioinformatics Co., Ltd., Wuhan, Hubei, China).Genes with FPKMmax values greater than 1 were selected from the O. fragrans samples.The FPKM data were then converted into TPM (transcripts per kilobase of exon model per million mapped reads) data, and the limma package [69] was applied to screen for differentially expressed genes (DEGs) between groups.The default filtering threshold for DEGs was set at |log2FoldChange| > 1 and q-value < 0.05.The generated heatmaps were visualized using the heatmap tool on https://cloud.oebiotech.com(accessed on 31 January 2024).This visualization process involved Z-score normalization and clustering of the row data, ensuring an accurate representation of the underlying patterns and trends within the dataset.

qRT-PCR Analysis of OfHM Genes
A total of 6 OfHMs, selected for their high expression from different gene families, underwent assessment of their expression levels at various stages of flowering using qRT-PCR.The qRT-PCR was performed using the Applied Biosystems 7500 Sequence Detection System (ABI7500; Thermo Fisher Scientific, Inc., Waltham, MA, USA).The primers sequences for qRT-PCR were designed using Prime Premier 5 (Table S5).The qRT-PCR reaction mixture, with a total volume of 15 µL, included 2 µL of cDNA, 0.8 µL of each forward and reverse primers, 10 µL of SYBR Mix and 6.4 µL of ddH 2 O.The expression level of OfRAN1 served as the reference, and qRT-PCR amplification was carried out under the following conditions: 94 • C for 30 s, followed by 40 cycles of 94 • C for 10 s and 60 • C for 30 s. Gene relative expression levels were calculated using the 2 −∆∆CT method [70], with each analysis comprising four replicates.The normalized log2 fold change for each gene was calculated using both RNA-seq and qRT-PCR data, with S1 data serving as the reference.Subsequently, linear fitting was applied to the RNA-seq and qRT-PCR datasets, and the correlation index (R 2 ) was computed.

Conclusions
In this study, we systematically identified HM genes within the O. fragrans genome, providing comprehensive insights into their chromosomal location, gene structure, phylogenetic relationships and protein-protein interactions.Utilizing high-throughput RNA-seq data, we examined the expression patterns of these genes across the flower opening and senescence stages, various tissues, and in response to specific treatments.The comprehensive findings derived from our research offer a thorough understanding of OfHMs, contributing not only to the study of biological theory, but also laying the groundwork for future in-depth analyses.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants13060777/s1, Figure S1: The chromosomal localization of histone modification genes in Osmanthus fragrans; Figure S2: Heatmaps illustrating the expression of histone methylation genes from O. fragrans across various tissues and flowering stages; Figure S3: Heatmaps illustrating the expression of histone acetylation genes from O. fragrans across various tissues and the flowering stages; Table S1: Detailed information and physicochemical properties of OfHMs; Table S2: The Ka/Ks values of segmental and tandem duplications between OfHMs; Table S3: The Ka/Ks values of segmental duplications between OfHMs and AtHMs; Table S4: Differentially expressed OfHMs detected under Aza or ethylene treatment; Table S5: Gene primers used in the study.

Figure 1 .
Figure 1.Phylogenetic analysis of histone modification (HM) genes between O. fragrans and Arabidopsis.(a) Histone methyltransferases (HMTs).Triangle symbols represent SET domain group (SDG) genes, and square symbols represent protein arginine methyltransferase (PRMT) genes.Solid symbols represent O. fragrans genes and hollow symbols represent Arabidopsis genes.I-VII represent the different types of SDG genes, and "a" and "b" represent different types of PRMT genes.(b) Histone demethylases (HDMs).Circle symbols represent SWIRM and C-terminal domain (HDMA) genes, and square symbols represent JmjC domain protein family (JMJ) genes.Solid symbols represent O. fragrans genes, and hollow symbols represent Arabidopsis genes.Different colored squares represent the various types of JMJ genes.(c) Histone demethylases (HDMAs) and (d) histone deacetylases (HDACs).Solid circles represent O. fragrans genes and hollow circles denote Arabidopsis genes.Different colors represent genes from different gene families.

Figure 1 .
Figure 1.Phylogenetic analysis of histone modification (HM) genes between O. fragrans and Arabidopsis.(a) Histone methyltransferases (HMTs).Triangle symbols represent SET domain group (SDG) genes, and square symbols represent protein arginine methyltransferase (PRMT) genes.Solid symbols represent O. fragrans genes and hollow symbols represent Arabidopsis genes.I-VII represent the different types of SDG genes, and "a" and "b" represent different types of PRMT genes.(b) Histone demethylases (HDMs).Circle symbols represent SWIRM and C-terminal domain (HDMA) genes, and square symbols represent JmjC domain protein family (JMJ) genes.Solid symbols represent O. fragrans genes, and hollow symbols represent Arabidopsis genes.Different colored squares represent the various types of JMJ genes.(c) Histone demethylases (HDMAs) and (d) histone deacetylases (HDACs).Solid circles represent O. fragrans genes and hollow circles denote Arabidopsis genes.Different colors represent genes from different gene families.

Figure 4 .
Figure 4. Analysis of cis-acting elements in the promoters of histone methylation modification genes.(a) OfHMTs and (b) OfHDMs.The figure on the left shows the different cis-acting elements, which are represented by boxes in different columns.The picture on the right provides the statistics regarding the number of each of the four kinds of cis-acting elements in the OfHM promoters.

Figure 5 .
Figure 5. Analysis of cis-acting elements in the promoters of histone acetylation modification genes.(a) OfHATs and (b) OfHDACs.The figure on the left shows the different cis-acting elements, which are represented by boxes in different columns.The picture on the right provides the statistics regarding the number of each of the four kinds of cis-acting elements in the OfHM promoters.

Figure 5 .
Figure 5. Analysis of cis-acting elements in the promoters of histone acetylation modification genes.(a) OfHATs and (b) OfHDACs.The figure on the left shows the different cis-acting elements, which are represented by boxes in different columns.The picture on the right provides the statistics regarding the number of each of the four kinds of cis-acting elements in the OfHM promoters.

Figure 6 .
Figure 6.Gene collinearity analysis.(a) Synteny of OfHMs genes in the O. fragrans genome.Colored lines connecting two genes indicate syntenic regions.(b) Synteny of HM genes between O. fragrans and Arabidopsis genomes.Colored lines connecting two genes indicate syntenic regions.

Figure 7 .
Figure 7. Functional enrichment analysis of OfHM genes.(a) GO enrichment analysis of histone methylation genes.The top 20 pathways with a gene number greater than 5 are shown.The x-axis represents -log10(p-value) and the y-axis represents the enriched GO terms.(b) KEGG analysis of histone methylation genes; (c) KEGG analysis of histone acetylation genes.In these analyses, the colors and sizes of the dots correspond to the significance (p) and the number of genes, respectively.

Figure 7 .
Figure 7. Functional enrichment analysis of OfHM genes.(a) GO enrichment analysis of histone methylation genes.The top 20 pathways with a gene number greater than 5 are shown.The x-axis represents -log10(p-value) and the y-axis represents the enriched GO terms.(b) KEGG analysis of histone methylation genes; (c) KEGG analysis of histone acetylation genes.In these analyses, the colors and sizes of the dots correspond to the significance (p) and the number of genes, respectively.

Figure 8 .
Figure 8. Interaction analysis of OfHM proteins.Circles represent genes, and lines represent

Figure 8 .
Figure 8. Interaction analysis of OfHM proteins.Circles represent genes, and lines represent OfHM gene pairs that may interact.

Figure 11 .
Figure 11.Heatmaps depicting the expression patterns of differentially expressed OfHMs in response to Aza or ethylene treatment.(a) After Aza treatment; (b) after ethylene treatment.The differently colored squares represent the genes, with red and blue representing high and low expression levels.

Figure 11 .
Figure 11.Heatmaps depicting the expression patterns of differentially expressed OfHMs in response to Aza or ethylene treatment.(a) After Aza treatment; (b) after ethylene treatment.The differently colored squares represent the genes, with red and blue representing high and low expression levels.

Figure 12 .
Figure 12.The expression levels of OfHMs were validated utilizing quantitative real-time polymerase chain reaction (qRT-PCR).(a) OfJMJ19; (b) OfSRT3; (c) OfSDG55; (d) OfHDT7; (e) OfHDMA4; and (f) OfHAG9.The x-axis represents the different flowering stages, including S1, S2, S3, S4 and S6.The right and left y-axes represent the fragments per kilobase of exon model per million mapped fragments (FPKM) values and the relative expression levels (REL), respectively.The values are presented as the mean ± standard deviation.(g) A linear fitting analysis was performed on the qRT-PCR and FPKM data.The x-axis represents the qRT-PCR data, and the y-axis represents the FPKM data.

Figure 12 .
Figure 12.The expression levels of OfHMs were validated utilizing quantitative real-time polymerase chain reaction (qRT-PCR).(a) OfJMJ19; (b) OfSRT3; (c) OfSDG55; (d) OfHDT7; (e) OfHDMA4; and (f) OfHAG9.The x-axis represents the different flowering stages, including S1, S2, S3, S4 and S6.The right and left y-axes represent the fragments per kilobase of exon model per million mapped fragments (FPKM) values and the relative expression levels (REL), respectively.The values are presented as the mean ± standard deviation.(g) A linear fitting analysis was performed on the qRT-PCR and FPKM data.The x-axis represents the qRT-PCR data, and the y-axis represents the FPKM data.