Role of methylation in vernalization and photoperiod pathway: a potential flowering regulator?

Abstract Recognized as a pivotal developmental transition, flowering marks the continuation of a plant’s life cycle. Vernalization and photoperiod are two major flowering pathways orchestrating numerous florigenic signals. Methylation, including histone, DNA and RNA methylation, is one of the recent foci in plant development. Considerable studies reveal that methylation seems to show an increasing potential regulatory role in plant flowering via altering relevant gene expression without altering the genetic basis. However, little has been reviewed about whether and how methylation acts on vernalization- and photoperiod-induced flowering before and after FLOWERING LOCUS C (FLC) reactivation, what role RNA methylation plays in vernalization- and photoperiod-induced flowering, how methylation participates simultaneously in both vernalization- and photoperiod-induced flowering, the heritability of methylation memory under the vernalization/photoperiod pathway, and whether and how methylation replaces vernalization/photoinduction to regulate flowering. Our review provides insight about the crosstalk among the genetic control of the flowering gene network, methylation (methyltransferases/demethylases) and external signals (cold, light, sRNA and phytohormones) in vernalization and photoperiod pathways. The existing evidence that RNA methylation may play a potential regulatory role in vernalization- and photoperiod-induced flowering has been gathered and represented for the first time. This review speculates about and discusses the possibility of substituting methylation for vernalization and photoinduction to promote flowering. Current evidence is utilized to discuss the possibility of future methylation reagents becoming flowering regulators at the molecular level.


Introduction
Flowering is an integrated event converging multiple internal and external signals.The internal signals mainly involve a number of f lowering regulators and endogenous phytohormones that inf luence f loral transition.These f lowering regulators are mainly divided into two categories: f loral integrators and characteristic meristematic genes.Floral integrators mainly include four types, namely FLOWERING LOCUS T (FT), CONSTANS-LIKE (COL), SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), and FLOWERING LOCUS C (FLC), and characteristic meristem genes mainly include five types, namely LEAFY (LFY), AGAMOUS-LIKE (AGL), APETALA 1 (AP1), CAULIFLOWER (CAL), and SEPALLATA (SEP).These integrator genes in f lowering pathways trigger f loral transition and activate a range of f loral meristem identity (FMI) genes.The proteins encoded by FMI genes boost f loral development by promoting f lower development genes as well as by repressing AGL24, a promoter of inf lorescence fate [1].The main pathways orchestrating blossoming mediated by the internal and external signals can be divided into several types: the f loral inhibition pathway [2], the autonomous pathway [3], the photoperiod pathway [4], the vernalization pathway [5], the gibberellin (GA) pathway [6], the stress pathway [7] and the aging pathway [8].
Methylation is a crucial concept in epigenetic mechanisms (acetylation, methylation, RNA interference).As a key factor in the evolution and adaptation of plants, methylation is involved in almost every stage of plant development.As the covalent methylation modification of the fifth cytidine site, DNA methylation is the most well-known and studied epigenetic mechanism in plants.CHH, CG, and CHG (where H represents a base, A/T/C) are three DNA methylation contexts.DNA methylation regulates temporal and spatial gene expression and condition-dependent phenotypic traits, including changing f lower symmetry [9] or phenotypic plasticity [10] in different biological processes [11].Histone methylation, as a vital and reversible post-translational modification (PTM), is one of the most important modifications on histone lysine (K) and arginine (R) residues and regulates many crucial biological processes [12].RNA methylation is an important modification in plant development and the abiotic stress response.Methylation on RNA bases, such as N 6 -methyladenosine (m 6 A) and 5-methylcytidine (m 5 C), is the most ubiquitous mRNA modification in eukaryotes [13].The role of methylation in vernalization and photoperiod pathways has been gradually revealed in recent years.
Plant vernalization is an adaptive characteristic acquired in response to chilling temperature, in order to bypass cold seasons and promote selective f lowering in spring when temperatures are more favorable.Some advances have been reviewed on the molecular mechanism of chromatin modification indicating that vernalization, as an epigenetic switch for silencing FLC and five FLC relatives, MADS AFFECTING FLOWERING 1/2/3/4/5 (MAF1/2/3/4/5), promotes Arabidopsis thaliana f lowering [14,15].Alexandre and Hennig discussed progress on the FLC/FLC branch-independent vernalization pathway in Arabidopsis and grasses, and reviewed epigenetic mechanisms of the f lowering promoter AGL24 and inhibitor MAF involved in vernalization of Arabidopsis [16].In many species, duration of the light-dark cycle (photoperiod) strongly affects f lowering.The effects of photoperiod on plants with different light requirements are very different.Notably, the genetic and epigenetic mechanisms (deacetylation and chromatin modification) of seasonal timing of Arabidopsis f lowering have been summarized [17].One review mentioned that chromatin modifiers can regulate photoperiodic f lowering, but the specific chromatin modifiers have not been included [18].The interactive roles of chromatin modification and the circadian clock have been elaborated in the review by Chen and Mas [19].DNA methylation in vernalization has been described [20], but its roles in photoperiod have not been overviewed.Roles of RNA methylation in the vernalization and photoperiod pathways have not been reviewed yet.
In this review we summarize the molecular evidence that histone, DNA, and RNA methylation are involved in f lowering regulation in the vernalization and photoperiod pathways.We expound the intricate molecular regulation network among f lowering genes, methylation (methyltransferases/demethylases), sRNAs and phytohormone and environmental signals (cold and light).Then, we show the molecular evidence that methylation is involved simultaneously in both vernalization-and photoperiodinduced f lowering.The existing evidence for the f lowering regulation of RNA methylation in the vernalization and photoperiod f lowering pathway is collected for the first time.The heritability of methylation state combined with vernalization and photoperiod memory is discussed.Finally, this review speculates that methylation as a f lowering accelerator/inhibitor may substitute for vernalization or photoinduction of f lowering, and considers the feasibility of realizing this substitutability in the future.

Methylation is involved in vernalization-induced flowering
A large proportion of plants need continuous low temperature (vernalization) to fulfill their crucial development transition so that plants enter reproductive growth from vegetative growth.Especially for the absolute vernalization plants, low temperature is a must.Recent studies show that methylation may play an important role in the vernalization-induced f lowering pathway.Histone methylation modification, DNA methylation and RNA methylation may alter when f lowering plants are undergoing vernalization.

Histone methylation of lysine and arginine pathways in the vernalization pathway and the potential substitution of histone lysine methylation for vernalization
The histone methylation sites that have been identified occur chief ly on the residues of lysine and arginine [21], which covalently modify H3 and H4 histones to affect vernalization.For lysine (Fig. 1a), FLC, as the main locus of histone modifications, converges various signals of f lowering.In Arabidopsis, Polycomb Repressive Complex 2 (PRC2), required for vernalization, can methylate vernalized genes near the histone region.PRC2 catalyzes histone 3 lysine 27 trimethylation (H3K27me3) to inhibit gene expression (Fig. 1a).In addition, cold (vernalization) induces plant homologous domain (PHD)-PRC2, a modified PRC2 with PHD proteins (VIN1/3/5, VRN5), to trigger high levels of H3K27me3 and stable epigenetic repression at FLC locus [22][23][24] (Fig. 1a).Interestingly, vernalization makes it possible to hold a steady memory of the long-term cooling effect after rewarming [18], which allows PHD-PRC2 to continue to output H3K27me3 and epigenetic inhibition [25].Eventually even PHD-PRC2 and H3K27me3 cover the entire FLC locus [25,26].This is the main reason that FLC remains silent in the growth of plants from now on.PRC2, prior to FLC silencing by vernalization, binds specifically to long non-coding RNAs (lncRNAs) to form PRC2-lncRNAs, which are recruited to a specific target, i.e.FLC [22] (Fig. 1a).Polycomb of PRC2 silences target genes by modifying histone, particularly methylating histone (H3K27me3, H3K36me3, and H3K4me3), and thus polycomb is widely involved in pivotal life processes such as development, proliferation, and differentiation.COOLAIR is transcribed antisense from the FLC locus induced by cold.Antisense RNA is a specific type of non-coding RNA (ncRNA) modulating genetic activity in cells at multiple levels [27].Growing evidence reveals the functionality and biological relevance of ncRNAs.According to its function, plant ncRNA is divided into two types: regulatory and structural ncRNAs.The regulatory ncRNAs mainly include lncRNA, circular RNA (circRNA), and small RNAs (sRNAs) containing microRNA (miRNA), short interfering RNA (siRNA), and PIWI-interacting RNA (piRNA); the structural ncRNAs includes ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA) and the like [28][29][30].The lncRNA COOLAIR, which notably causes polycomb silencing, has been found to boost transcriptional shutdown of FLC in Arabidopsis [5,22,26,31,32].Further, at the FLC locus, COOLAIR-mediated demethylation of H3K36me3 and trimethylation of H3K27 coordinate to silence FLC, which in parallel regulate blossoming during vernalization [5,22,26] (Fig. 1a).In addition, plants with a mutation of the group-III WRKY transcription factor WRKY63 exhibit an early-f lowering phenotype [31,33].Interestingly, WRKY63 directly activates FLC under non-vernalization, but indirectly represses FLC by inducing two lncRNAs, COOLAIR and COLDAIR, during vernalization, and in this process the reduction of H3K27me3 occurs [31] (Fig. 1a).Another study has shown that COOLAIR-mediated FLC repression during vernalization is significantly related to cell size [32].However, some reports showed that COOLAIR only participates in FLC repression at room temperature, and does not mediate FLC regulation during vernalization [34,35] (Fig. 1a).A later study found that the vernalization-triggered COOLAIR mechanism requires C-repeat (CRT)/dehydration-responsive elements (DREs) at the 3 -end of FLC and CRT/DRE-binding factors (CBFs) [34].During vernalization, CBFs can bind to the CRT/DRE of FLC in vitro/vivo, resulting in an increase in COOLAIR transcripts, while mutation of CBFs leads to a serious COOLAIR defect, but shows an almost normal vernalization response [34] (Fig. 1a).Hence, COOLAIR is probably not a necessity for vernalization.This is consistent with the study of Helliwell et al. [35].Arabidopsis with no increase in antisense transcription during vernalization showed normal FLC repression and corresponding H3K27me3 changes [35], indicating that COOLAIR is probably not an indispensable part of vernalization-induced FLC repression.Moreover, before PRC2 adds H3K27me3 to FLC, PRC2 was revealed to play a potential role in the establishment of FLC repression [35].However, some studies in Arabidopsis argued that the mechanism of FLC silencing leading to f lowering is the synergistic effect of PRC2 and FLOWERING LOCUS D (FLD, the f loral accelerator via repressing FLC) complex by upregulation of H3K27me3 and downregulation of H3K4me2 [36,37] (Fig. 1a).In addition to the above FLC pathway, lysine methylation involved in vernalization-induced f lowering may also act in the following pathways.SDG8/EFS (SET Domain Group 8/Early Flowering in Short days), a histone lysine methyltransferase, is required for gene expression of FLC clade and functions in delaying vernalization-induced f lowering in Arabidopsis [38] (Table 1).Additionally, as an identified H3K27me3 reader, BAH domain-containing transcriptional regulator 1 [BDT1 (AT4G11560/AIPP3)] modulates Arabidopsis f lowering time [39] (Table 1).However, it remains to be seen whether BDT1 is a player in vernalization.The methylation of H3K4/36 activates FLC expression and suppresses vernalization-induced f lowering, while the methylation of H3K27/9 has the opposite effect, repressing FLC expression and promoting vernalization-induced f lowering (Fig. 1a).In winter wheat, the upregulated expression of vernalization-responsive genes VERNALIZATION 1 (TaVRN1) and FLOWERING LOCUS T-like 1 (TaFT1) in Triticum aestivum by vernalization is linked to the increased level of H3K4me3 [40], suggesting that the f loral transition is probably related to histone methylation.During vernalization, VRN1 is activated by a decrease in H3K27 methylation and by an increase in H3K4 methylation to repress VRN2, and thereby promote Agrostis stolonifera f lowering [41].In a collaborative study of vernalization and photoperiod, histone lysine (K)-mediated methylation changes in VRN genes probably bypassed the vernalization requirement for f lowering to boost f lowering, in which miR39 is involved [41].Transgenic plants overexpressing miR396 show advanced A. stolonifera f lowering via enhancing expressions of VRN1 and VRN3, which are accompanied by alteration of H3K4/27 methylation [41].The are two possibilities for the bypass: one is that histone methylation initiates pathways other than vernalization and induces f lowering, and the other is that histone methylation substitutes (partially) for vernalization to induce f lowering.
For arginine (Fig. 1b), the methylation of arginine plays an essential regulatory role in PTM [42].Arginine (R) is distributed in nuclear and cytoplasmic proteins, and controls the processes of chromatin remodeling, gene transcription, cell proliferation, and differentiation in animals and plants [43][44][45][46].Chromosome recombination and RNA transcription [47] in plants are regulated by the catalytic action of protein arginine methyltransferases (PRMTs).The key part is arginine methylation in the tail of core histones.A series of complex PTMs, including methylation, occur at the N-and C-termini of histones [46].A methyl group at S-adenosyl-l-methionine (AdoMet or SAM) is transferred to the end of guanidinium nitrogens of arginine residues via PRMTs.This removal process generates monomethyl-arginine, symmetric dimethyl-arginine, and asymmetric dimethyl-arginine [46].There have been preliminary studies of the roles of arginine in vernalization-induced f lowering.The pre-mRNAs of plant serine/arginine-rich (SR) protein is widely selectively spliced, which greatly increases the transcriptional complexity.SRs are essential for plant development.Overexpression of atSRp30 involves pre-mRNA splicing and growth in plants, leading to a late-f lowering phenotype after vernalization treatment [48].Histone arginine methylation is a requisite for vernalizationinduced epigenetic silencing of FLC [49].PRMT, an evolutionarily conserved enzyme family, has been illustrated in a previous review on the regulation of f lowering time, vegetative growth, the physiological cycle, and the response to high medium salinity and abscisic acid (ABA) [50].Mutation of AtPRMT5 in FLC chromatin leads to pleiotropic phenotypes, including low sensitivity to vernalization, delayed growth and postponed f lowering [51].Shk1 binding protein 1 (SKB1, also known as PRMT5) and AtPRMT10 inf luence Arabidopsis flowering [52,53] (Table 1).AtPRMT10 has self-methylation activity and its mutation represses FLC expression, thereby resulting in late f lowering [52].In contrast, SKB1 catalases the formation of histone H4R3 symmetric dimethylation (H4R3sme2) in FLC chromatin, and stimulates blossoming by competitive binding with the FLC promoter [53] (Fig. 1b).The late-f lowering phenotype of skb1-1f lc-3 is inhibited, verifying that the early f lowering function of SKB1 is realized by repressing FLC [53].Interestingly, the reduction of H4R3sme2 due to the mutation of SKB1 does not affect the asymmetric H4R3me2.The SKB1 mutation just upregulates FLC expression and triggers late f lowering under short days (SDs) or long days (LDs), whereas this late-f lowering effect may be reversed by vernalization.Fascinatingly, crosstalk between histone arginine and lysine methylation during vernalization-induced f lowering has been verified.During vernalization, atprmt5 mutant plants cannot acquire the FLC inhibitor H3K27me3 [54].Vernalizationmediated methylation of H3K27/9 at the FLC locus requires the methylation of H4R3 in Arabidopsis [49].Accordingly, SKB1/PRMT5mediated H4R3sme2 is a new histone marker that is required for repressive expression of FLC and regulation of f lowering time.Another study has further confirmed this.Single mutation of AtPRMT4a/AtPRMT4b does not lead to delayed f lowering, but double mutation causes late f lowering during vernalization due to hypomethylation of H3R17 and upregulation of FLC expression [55].Four PRMTs (AtPRMT5/10 and AtPRMT4a/4b) are involved in vernalization-induced f lowering based on the regulation of FLC expression (Fig. 1b).Moreover, vernalization is initiated by VIN3 and then maintained by VRN1, VRN2, LIKE-HETEROCHROMATIN PROTEIN1 (LHP1), and PRMT5.PRMTs seem to be vital markers involved in vernalization-induced f lowering.Arginine methylation at the FLC locus may be essential for the stability of the vernalized state.

DNA methylation changes before and after FLC reactivation, the substitutability of DNA methylation for vernalization, and the heritability of methylation memory
In Arabidopsis, it was reported that a transient and slight decrease in DNA methylation might be related to vernalization-induced f lowering [56].In plants, the main consequences of DNA methylation are CG, CHG, and CHH.Guo et al. classified the process of f loral transition into three phases through comparative transcriptome analysis between OB (Rosa chinensis 'Old Blush') and GIG (Rosa odorata var.gigantea): vegetative meristem (VM), pref loral meristem (TM), and f loral meristem (FM) [6].Then they found that some differentially expressed genes (DEGs) relevant to DNA methylation in VM-GIG and TM-GIG are primarily engaged in vernalization.Methyltransferase changes the level of DNA methylation and then affects f lowering.Some genes and proteins with catalytic activity may also affect plant f lowering during vernalization, such as DECREASED IN DNA METHYLATION 1 (DDM1) [57].The expression of FLC is suppressed by vernalization, and is suppressed in plants with DNA hypomethylation.FLC is associated with many f lowering-relevant signals in vernalization-induced f lowering.
Apart from perennials, vernalization every generation is necessary for blossoming, so the cell memory of the vernalization experience should be reprogrammed when the life cycle is approaching These genes have been submitted to the DDBJ/EMBL/GenBank/TAIR databases see in corresponding literatures.the terminal stage [57].This means that in the memory system, plants need to reactivate the cleared FLC expression before the developmental completion of seeds or upon fertilization, and then guarantee and maintain appropriate f loral behaviors in each round of f lowering [58] (Fig. 2).Notably, a pivotal example of plant epigenetic (methylation) reprogramming is the resetting of FLC expression in Arabidopsis [59].A study has shown that methylation with vernalization memory may not be effectively erased due to the mutation of the jumonji-domain-containing protein ELF6, so part of the FLC expression may be retained and unite the methylation memory to be inherited by the next generations (with early f lowering phonotype) [59].However, Tao et al. demonstrated that the vernalization state/memory of Arabidopsis parents in FLC is mainly reset by a factor other than ELF6, namely the seed-specific transcription factor LEAFY COTYLEDON1 (LEC1), the main regulator of embryogenesis [60] (Fig. 2).LEC1 boosts the initial establishment of the FLC chromatin state and activates FLC re-expression in the pro-embryo, thus reversing the epigenetic silencing state (marked by H3K27me3) inherited from gametes [60].A review from Niu and He shows that three LEC genes function in resetting the vernalization memory of parents during early embryogenesis in Arabidopsis [61].Shortly after fertilization, LEC1 reactivates FLC as a pioneer, and completely activates FLC with the cooperation of LEC2 and FUSCA3 (FUS3).Meanwhile, LEC2 and FUS3 [recruiting FRIGIDA (FRI), a complex required for FLC reactivation] gradually increase in the cold memory element (CME) region, while the level of homologous B3 protein VP1/ABI3like (VAL) protein [recruiting Polycomb-group (PcG) proteins, like PRC2, a H3K27 methyltransferase complex] gradually decreases.This leads to the destruction of the original epigenetic silencing state of embryonic FLC, an adaptive means for plants to prevent precocious f lowering before cold exposure in winter.Yeast RNA polymerase II-associated factor 1 (Paf1) is a necessity for high expression and reprogramming of FLC [62].EARLY FLOWERING 7 (ELF7) and VERNALIZATION INDEPENDENCE4 (VIP4), Arabidopsis homologous genes of members of the Paf1 complex, are involved in vernalization.Mutation of the two genes causes severe defects in FLC reactivation [58] (Fig. 2), which means they are essential to the reactivation.Histone methylation of FLC chromatin is mediated by the Paf1 complex and interacts with the vernalizationresponsive process [38,62,63].Unlike histone methylation, effectors referred to the DNA methylation pathway are seemingly not involved in FLC regulation during reproduction.Thus, we will focus on the role of DNA methylation in FLC reprogramming.
DNA methylation has no effect on gametogenesis-specific FLC repression, and METHYLTRANSFERASE1 (MET1) is not involved in FLC reactivation [58].On the contrary, the low CG DNA methylation in the sporophyte generates FLC inhibition, but the DNA methylation at the FLC site does not change.In addition, FLC activation requires the FRI family, which is a determinant of the vernalization-requiring and winter-annual habit in Arabidopsis [64].FRI, as the upstream activator of FLC, is indispensable to the complete reactivation of high-level FLC during embryogenesis (Fig. 2).However, no matter how active the FRI complex is, DNA methylation still does not participate in the FLC reactivation in the reproductive stage.Finnegan et al. also showed that DNA methylation does not seem to be involved in the resetting of FLC expression before vernalization in Arabidopsis [65].All the above suggests that DNA methylation may really have no effect on FLC reactivation.The specific molecular process remains to be explored.Nevertheless, when FLC is reactivated, DNA methylation plays an important role in vernalization-induced f lowering (Fig. 3).In Arabidopsis, a study has shown that FLC is downregulated by DNA demethylation, which may occur through a separate vernalization-independent pathway [65].However, some opposite arguments are that FLC or FLC regulators are actually controlled by DNA methylation during vernalization [5] and that the FLC locus is epigenetically silenced by vernalization [59].In addition, FLOWERING LOCUS F (FLF, a repressor of f lowering), the homologous gene of FLC, is suppressed by vernalization and by a decrease in genomic DNA methylation in Arabidopsis [66].Another study showed that a reduced level of DNA methylation represses FLC expression and then facilitates f lowering in Perilla frutescens (a vernalization-requiring short-day plant) and Silene armeria (a non-vernalization-requiring long-day plant) under a non-inductive photoperiod [67], suggesting an efficient effect of the reduced DNA methylation on FLC repression during vernalization.Therefore, more experiments are needed to determine whether DNA (de)methylation is involved in the process called vernalization silencing FLC after FLC reactivation.Actually, there are increasing clues to the involvement of DNA (de)methylation in vernalization-induced f lowering.A recent study supports this argument [68].In the early vernalization stage, an increase in genomic DNA methylation was observed in orchardgrass; mCHH occurring in the promoter region of vernalization-related genes contributes to the upregulation of these genes in this stage; both exogenous application of the DNA methylation accelerator methyl trif late (MTFMS) and overexpression of the RNA-directed DNA methylation (RdDM)-related gene NUCLEAR POLY (A) POLYMERASE (DgPAPS4) elevate the DNA methylation level, thus ultimately advancing vernalization-induced f lowering [68].These results indicate the important role of DNA hypermethylation in vernalization-induced f lowering.DNA hypermethylation induced by vernalization is one of the inducements for the initiation and development of the f loral primordium during vernalization.
FT, a vital mobile f lorigen, can take advantage of vernalization to break FLC entrapment and facilitate f lowering.Before vernalization, FT is restrained by interaction with FLC and the FT promoter.When vernalization is initiated, vernalization begins to relieve the inhibited transcription of f lowering genes.For example, FT shows to almost completely overcome for the f lowering delay effect of FLC via some signals acting in phloem, and subsequently promotes early f lowering in Arabidopsis [69][70][71].Some researchers thought that FT and TWIN SISTER OF FT (TSF) bypasses the f lowering barrier produced by FLC by activating SOC1 expression in Arabidopsis [72].FTa1 is identified as an important f lowering time gene in Medicago truncatula and the only FT gene upregulated by long days and vernalization simultaneously [73].The synergistic effect of vernalization and long-day signal in M. truncatula indicates that FTa1 mutation delays f lowering, while overexpression of FTa1 accelerates f lowering violently [73,74] (Fig. 3).However, during vernalization, FT may not regulate f lowering through changes in DNA methylation.Medicago truncatula spring2 and 3 mutants overexpressing FTa1 f lower early [75] (Fig. 3).Tnt1 retroelement marking at the FTa1 locus eliminates the vernalization requirement.The retroelement is usually correlated with block of elevated DNA methylation.Analysis using mock digestions (using McrBC, a methylation-dependent restriction enzyme) shows that no changes in DNA methylation occur at the FTa1 locus in spring mutants [75] (Fig. 3).This means that DNA methylation does not seem to be directly involved in vernalization-induced f lowering promoted by FT.However, a recent study in orchardgrass (Dactylis glomerata) indicates that FT may be associated with the increase in DNA methylation involved in vernalization pathways [68].It was shown that the DNA methylation accelerator MTFMS can promote the expression of vernalization-related genes, including FT, CURLY LEAF (CLF), FERTILIZATION INDEPENDENT ENDOSPERM (FIE), MULTICOPY SUPPRESSOR OF IRA (MSI), VIN3, VIN3 LIKE 1 (VIL1), SWINGER (SWN), VRN1, and AGL20 [68].However, many details about the involvement of FT in the regulation of DNA methylationmediated f lowering during vernalization are still unknown.
Intriguingly, other studies indicate that vernalization-mediated f lowering promotion is mediated by DNA demethylation [56,76].Vernalization probably induces f lowering by DNA demethylation at specific sites in the genome.For example, a DNA-methylation inhibitor, 5-azacytidine (5-azaC), may regulate f loral regulatory networks to accelerate f lowering of multiple species.Some reports revealed the induction of f lowering by 5-azaC in flax (Linum usitatissimum) [77][78][79] and Pharbitis nil [80].DNA hypomethylation mediated by 5-azaC elevates the f lowering percentage on explants from young, vernalized roots of chicory (Cichorium intybus) [81].In late-f lowering ecotypes and mutants, vernalization-requiring Arabidopsis treated with 5-azaC can boost f lowering, which demonstrates that demethylation may mediate vernalization-induced f lowering through the control of one or more genes essential for f lowering induction [76].This control probably includes a step related to a GA biosynthetic enzyme gene with apex specificity (Fig. 3).GA is a phytohormone required for f loral transition, and kaurenoic acid hydroxylase (KAH) is the rate-limiting enzyme in its biosynthesis [82].Interestingly, vernalization leads to demethylation of the KAH gene and subsequent transcriptional activation, resulting in the increased production of GA essential for f loral transition of Arabidopsis [82] (Fig. 3).DNA demethylation also induces vernalization and f lowering of Arabidopsis via the repression of FLC, the central repressor of the f lowering response [65].Moreover, the vernalization-mediated genes BrCKA2 (casein kinase II αsubunit) and BrCKB4 (casein kinase II β-subunit) show increased expression and DNA demethylation in vernalized Brassica rapa that is deficient in DNA methylation through silencing MET1 or applying 5-azaC [83].By silencing BrCKA2 and BrCKB4, the vernalized B. rapa delays f lowering, suggesting that BrCKA2 and BrCKB4 may be positive regulators of f lowering, and there is a potential positive relationship between DNA demethylation and f lowering during vernalization [83] (Fig. 3).However, in Arabidopsis, although the new function of the casein kinase 2 (CK2) α subunit in f lowering has been validated, it does not seem to be involved in vernalization-mediated f lowering [84][85][86].The questions of whether CK2 is involved in vernalization-induced f lowering and whether the corresponding DNA methylation occurs still need additional exploration.DNA demethylation by 5-azaC or methyltransferase mutation promotes vernalization-induced f lowering, which means that maybe we can replace the effect of cold (vernalization) on f lowering via demethylating.We propose a hypothesis that DNA demethylation mediated by externally ingested 5-azaC may replace vernalization by inducing early f lowering in vernalization-requiring lines or otherwise provide an avenue to manipulate f lowering time.This hypothesis has been partly confirmed.In vernalized winter wheat, reagent 5-azaC and γ rays can partially substitute for vernalization to boost f lowering [87].In vernalized Arabidopsis and C. intybus, 5-azaC and antisense RNA can partially substitute for the vernalization requirement by repressing methylase activity [88].A recent study has also shown the possible substitution of 5-azaC for vernalization to accelerate f lowering in Iranian Anemone accessions [89].They explored the possibility of pretreatment with GA3 and 5-azaC substituting for cold storage (vernalization) in Iranian Anemone accessions and found that both 5-azaC and GA3 treatments advance A. bif lora f lowering under non-vernalized conditions [89].These results suggest that the substitutability of DNA methylation for the cold effect (vernalization) exists.However, it is also controversial that 5-azaC, as a demethylating reagent, is also a universal inhibitor of transcription, and thereby the promotion of f lowering by 5-azaC might be caused by effects other than DNA demethylation.Because of this controversy, some studies used Arabidopsis plants in which the DNA methylation level is decreased by antisense METI transgenes or DDM1 mutation [76,90,91].DNA-methylationdeficient plants bloom early without vernalization, suggesting that demethylation is enough to prompt early f lowering.Therefore, although it cannot be asserted, 5-azaC is likely to use the DNA demethylation function in promoting f lowering.Additionally, demethylation by antisense MET1 has been proved to have a superimposing effect on promotion of f lowering by low temperature in winter wheat or Arabidopsis [76].The above shows that downregulated DNA methylation (demethylation) is a potential substitute for vernalization to promote f lowering.
However, upregulated DNA methylation levels may also contribute to vernalization.Methyl-CpG-binding domain (MBD) protein, a vital trans-acting factor, functions in specially recognizing/interpreting methylated DNA [92].MBD interacts with chromatin remodeling protein to silence genes.In Arabidopsis, by mutating AtMBD8 of C24 (vernalization-responsive accession), atmbd8-1 displays late f lowering under long days/short days, downregulated expression of FT1 and SOC1, and the same response to vernalization as the control [93].These results suggest that AtMBD8 is a novel f lowering promoter, but may not be involved in vernalization-induced f lowering.However, the mutation of AtMBD9 induces DNA methylation at the FLC locus in the vernalization pathway and leads to Arabidopsis early f lowering [94].MBD protein has been proved to be associated with active DNA methylation (Fig. 3).For example, AtMBD7 is necessary for active demethylation and anti-silencing of highdensity DNA methylation [95].Hence, MBD is likely to serve as a reminder of changes in DNA methylation during vernalization.MBD has exhibited similar functions in other species.In wheat with vernalization treatment, TaMBD6 homologs are differentially expressed in developing wheat [96].Among them, the expression of TaMBD6_B and TaMBD6_D is upregulated, while the expression of TaMBD6_A is very weak, indicating that TaMBD6 is induced by prolonged chilling and may be involved in the vernalizationinduced transition to f lowering [96].A recent study in Chrysanthemum lavandulifolium found that MBD protein may recognize f lowering control genes regulated by DNA methylation in the vernalization pathway [97].Of the 89 candidate genes identified by MBD sequencing, 49 genes exhibited changes in DNA methylation status during f lowering induction [97].Among them, VIP2 in the vernalization pathway, FCA and REF6 in the autonomic pathway, ZTL in the photoperiod pathway, and GAI in the GA pathway were identified as f lowering control genes highly likely to be regulated by DNA methylation [97].Moreover, the expressions of two DNAmethylated genes of lily, LoCMT (CMT-type DNA methyltransferase genes) and LoHDAC, are promoted by vernalization [98] (Fig. 3).The gene expression of LoCMT and LoHDAC increases with increasing vernalization time [98].In other words, increased DNA methylation may also have a positive regulatory effect on vernalization.Above all, DNA methylation and demethylation may all have a positive regulatory effect on vernalization-induced f lowering.The reason for this contradiction may relate to the fact that f lowering behavior is a combined action of various signals in and out of plants, or the different methylation status of different f lowering-related genes.In addition, long days are accepted as the most important stimulus correlated to f lowering, so interaction of long days with vernalization and methylation and other factors cannot be ignored in the process of f lowering induction.Furthermore, the functional scope of 5-azaC as a general transcriptional inhibitor is uncertain, which may require more rigorous verification.After all, little is known about which (de)methylation pathway is specifically affected by 5-azaC in plants.Further studies, which take into account the contradiction mentioned above, will need to be undertaken.

Latest progress of RNA methylation in the vernalization pathway
Vernalization induces f lowering mainly through DNA demethylation and histone modification, which are the results of epigenetic regulation [99].Vernalization-mediated silencing/promotion of f loral regulators probably occurs through histone modifications and DNA methylation.RNA methylation (e.g.m 5 C in RNA) was discovered a long time ago, but its mechanism in some biological processes (e.g.f lowering) is still equivocal [100].Perhaps its role might lie in the effectiveness of mRNA translation and stability.Very few studies have been devoted to the role of RNA methylation in vernalization-induced f lowering.In the study of vernalized sugar beet (Beta vulgaris altissima, a long-day plant) [101], like phenolic compounds, RNA methylation also plays a role in f loral transition.In identified differentially methylated regions (DMRs), two RNA METHYLCYTOSINE TRANSFERASE (RNMT, DMRs related to bolting) sequences, BvRNMTa and BvRNMTb, were identified during cold exposure (vernalization) and/or between genotypes [101].These genotypes are mainly divided into two categories: a sensitive early bolting genotype (S) and a resistant late bolting genotype (R).It is generally acknowledged that sugar beet can bolt without f lowering, but seldom f lowers without bolting [102], indicating a tight link between bolting and f lowering.Contrary to one of the RNMT sequences, the other exhibits hypermethylation of the gene body and its expression is activated by cold exposure (Fig. 3).The mRNA methylation of f loral suppressor FLOWERING LOCUS 1 gene including BvFL1 occurs in the bolting-resistant genotype under cold exposure [101] (Fig. 3).BvFL1, an FLC-like and vernalized gene, functions as a f lowering repressor like FLC and is downregulated by vernalization [103].Methyl RNA immunoprecipitation (MERIP) ensured BvFL1 could be involved in the RNA methylation pathway; BvRNMTa and BvRNMTb show CG, CHG, and CHH methylation under cold exposure; the former gene shows hypomethylation and expression silencing, while the latter shows hypermethylation and expression activating (Fig. 3); the S genotype has a higher total RNA methylation level than the R genotype [101].These results suggest that RNMT (RNA methylation) plays a role in bolting and f lowering under cold treatment.Consistent with the study on the Arabidopsis rnmt mutant, the RNA methylation level decreases in rnmt vernalized sugar beet.Surprisingly, under short days cold treatment can still promote bolting and f lowering in the rnmt mutant.However, an Arabidopsis rnmt mutant experiment demonstrated that RNMT does not seem to be necessary for stem bolting and f lower formation [101].Overall, rnmt mutants show the phenomenon of early bolting/f lowering, suggesting that vernalization can accelerate the bolting and f lowering process through RNMT.RNA methylation may promote or delay bolting and f lowering by regulating vernalization at the RNA level.But more information is still required about RNA methylation for vernalization; this may be a more open question than histone and DNA methylation.
Generally, methylation interacts with temperature (vernalization) to regulate blossoming through a variety of regulatory factors.Among the three major methylation modes, vernalizationdependent plants may regulate f lowering behaviors mainly through histone modification and DNA methylation.In plants, histone and DNA methylation may inf luence vernalizationinduced f lowering mainly around FLC, followed by other f lowering-related genes, sRNAs, phytohormones, and the MBD.Of course, a broader and more complex mechanism of RNA methylation may be involved in vernalization-induced f lowering, and this needs careful exploration.

Methylation is involved in photoperiod-regulated flowering
Each plant has evolved a complex mechanism of f lowering transition.Photoperiod, light quality, and light intensity, like temperature (vernalization), deeply affect the timing of f lowering in plants [76].A study has found that light quality has a significant effect on plant f lowering [104].In Arabidopsis, farred light and blue light can foster f lowering but, inversely, red light may postpone f lowering [104].CO, FT, CRYPTOCHROME 2 (CRY2), FHA, GIGANTEA, and FWA are the main regulators in the f lowering pathway, and are induced by photoperiod [105][106][107].In Arabidopsis, light (photoperiod, light quality) and temperature (vernalization) are the external conditions inf luencing the pivotal f loral transition, and they play a role through the CO-FT and FLC pathways, respectively [4,108].Light signals measure the time of f lowering, and CO is a key link protein used by light signals to regulate f lowering time [18,109].Thus, CO is an important protein that mediates plant circadian clocks and f lowering time.
FLC is a classical central f lowering suppressor.FLC can integrate vernalization, autonomy, and other pathways to regulate f lowering in a dosage-dependent manner [110].FT, as a component of the photoperiod-induced pathway, orchestrates signals from photoperiod, vernalization, and autonomy to stimulate f lowering with increasing day length.FT, which is activated by CO, integrates photoperiod-dependent and FLC-dependent pathways to regulate f lowering time by controlling the expression of f lowering-specific identity genes [110].After the integration of f lowering signals in the photoperiod pathway and FLC signals, the expression of common downstream integrators of f lowering, such as SOC1 and FT, is controlled [18,104,111].These expressions are restrained by FLC, while the photoperiodic f loral regulatory signals mediated by CO antagonize FLC, and CO is responsible for activating the expression of these integrators [18,104,111].Thus, there is an antagonistic relationship between CO and FLC in the promotion of f lowering.Photoperiodic f lowering is probably affected by a variety of chromatin modifications in the plant, such as DNA methylation, H3K4 methylation, and RNA methylation, by controlling f loral regulatory behaviors of FLC and CO.

DNA methylation in photoperiodic flowering and its substitution for photoinduction and the heritability of methylation memory
DNA methylation regulates photoperiodic f lowering in plants.MBD is a protein that recognizes methylated DNA.The mutation of AtMBD8 delays photoperiod-induced Arabidopsis f lowering with the downregulation of FT and SOC1 expression in an FLCindependent and CO-independent manner [93] (Fig. 4a).AtMBD8, perhaps being a f lowering promoter, may regulate photoperiodinduced f lowering through DNA methylation [93].Consequently, MBD may be involved in photoperiod-induced f lowering via DNA methylation.In Arabidopsis, consistent with antisense METI, the mutation of DDM1 causes DNA demethylation [76,90] (Fig. 4a).By comparing the f lowering time of ddm1-homozygous mutants (self-pollination progeny), it was found that ddm1 mutants f lower later under long days [76,112].Also, the f lowering time in self-pollinated progenies became gradually later generation by generation [76,112], indicating that DNA demethylation mediated by DDM1 mutation promotes photoperiodic f lowering.This shows that Arabidopsis of the ecotype Colombia blossoms mainly through the photoperiod pathway rather than vernalization under long days.Under short days, the progenies of ddm1 blossom earlier generation by generation, and the effect of promoting f lowering is better in ddm1 with vernalization treatment [76], suggesting that the ecotype depends on vernalization to blossom under short days.This shows that DNA demethylation by DDM1 mutation inhibits photoperiodic f lowering, while it is conducive to promoting vernalization-induced f lowering.As one of the main pathways to induce f lowering, photoperiod is directly or indirectly related to vernalization.After vernalization is initiated at low temperature, it is not until several weeks or months that plants blossom, and thereby the specific conditions for photoperiod f lowering are gradually satisfied during this period [83].To study the interaction between photoperiod and vernalization in f lowering, Duan et al. constructed DNA methylation-deficient plants and viral silencing vectors [83].It was found that vernalization induces DNA demethylation of two subunits of CK2 (BrCKA2 and BrCKB4), which shortens the period of the clock gene BrCCA1 (an important gene in photoperiod perception) in vernalized B. rapa and consequently boosts f lowering under long days (Fig. 4).Studies have shown that casein kinases (such as CK1 and CK2) are involved in regulating the f lowering time of many plant species, such as Arabidopsis, rice, and B. rapa [113].As a consequence, photoperiod and vernalization interact directly through DNA demethylation and synergistically accelerate f lowering.In other words, DNA demethylation is able to participate simultaneously in both vernalization-and photoperiod-induced f lowering.So far, vernalization and photoperiod are the two pathways that simultaneously regulate f lowering time [114,115].However, in model plants such as Arabidopsis, it is unknown whether DNA methylation participates in both vernalization and photoperiod to regulate f lowering.In other species DNA methylation plays the same role in plant f lowering.5-azaC induces the f of the long-day plant S. armeria and the short-day plant P. frutescens var.crispa under a non-inducing photoperiod, indicating that f lowering gene expression is modulated by DNA methylation [67].However, the progeny of plants whose f lowering is induced by 5-azaC cannot f lower under a non-inducing photoperiod because de novo methylation occurs in the progeny [67] (Fig. 4a).Regarding heritability, the early f lowering phenotype produced by antisense METI-mediated demethylation can be passed on to the next generations.DNA demethylation probably regulates photoperiod f lowering, but this is independent of the stability of the photoperiod-induced f lowering state [116].Intriguingly, demethylation has also been proved to have a substituting role in photoperiodic f lowering.This is the first finding that photoinduction can be replaced by DNA demethylation in P. frutescens seeds via 5-azaC treatment [117].Perilla frutescens is an absolute short-day plant and does not require vernalization.Treatment with 5-azaC broke the suppression of P. frutescens f lowering by long days, and its f lowering promotion effect was slightly weaker than that of short-day treatment, which means that 5-azaC can at least partially replace the short-day requirement for f lowering in absolute short-day plants [117].This is similar to our hypothesis stated above in the section 'DNA methylation changes before and after FLC reactivation, the substitutability of DNA methylation for vernalization, and the heritability of methylation memory' that '5-azaC-mediated DNA demethylation may replace vernalization by inducing early f lowering in vernalization-requiring lines', and here DNA demethylation may (partially) replace photoinduction by inducing early f lowering in plants that need short days.Not requiring vernalization means that long-day/short-day plants bloom mainly through the photoperiod pathway.Spinach is a long-day plant and does not require vernalization.In another study, co-treatment of 5-azaC and photoinduction greatly promoted spinach f lowering compared with the control treatment of photoinduction or 5-azaC, implying that 5-azaC treatment can potentially partly replace photoinduction [118].These observations suggest that DNA demethylation (induced by 5-azaC) may substitute for photoinduction in photoperiodic f lowering.In addition to vernalization-and photoperiod-related genes, DNA demethylation (5-azaC) may also induce/trigger other f lowering-related genes during substitution.DNA demethylation (5-azaC) may provide an additional avenue to manipulate f lowering time.Therefore, we hypothesize that this is the real reason why DNA demethylation can replace or partially replace the cold effect (vernalization) and photoinduction (photoperiod) to promote plant f lowering.
DNA methylation is involved in the f lowering of grain crop rice mainly through a mediator: sRNA, such as longday-specific male-fertility-associated RNA (LDMAR, a kind of lncRNA) and osasmR5864m [119,120] (Fig. 4a).LDMAR, which controls photoperiod-sensitive male sterility (PSMS) hybrid rice, is necessary for male fertility in rice under long days [121].The Psi-LDMAR in the LDMAR promoter region probably originates from the AK111270 gene, whose overexpression induces RdDM in the LDMAR promoter and inhibits the expression of LDMAR [119,122,123] (Fig. 4a).In other words, the increased methylation of the LDMAR promoter in PSMS rice leads to decreased expression of LDMAR under long days.The decreased LDMAR expression causes male sterility and delays fertility restoration under long or short days.The f lowering habits of PSMS rice are affected by RdDM in LDMAR.The male sterility caused by the high methylation may eventually weaken the f lowering peak; the average f lowering frequency may decline more seriously; and the pre-afternoon f lowering ratio may significantly decline.In short, RdDM can lead to f lowering obstacles in PSMS rice to some extent.In the photoperiod-thermosensitive male sterile (PTGMS) line PA64S, DNA methylation may also participate in the transformation of sterility and fertility [124].This transformation is regulated by photoperiod and temperature.Sterility of PA64S can be transformed into fertility under short days and low temperature [125].Similar to LDMAR in PSMS rice, osasmR5864m, a non-coding and mutant sRNA, is responsible for the sterilityfertility transformation in PA64S, which is closely related to DNA methylation [125] (Fig. 4a).BLAST analysis shows that DNA fragments occur in differential methylations and these differential fragments correlate to photosynthesis, signal transduction, metabolism, and transposon activation [124].DNA fragments relevant to photosystem signals show hypermethylation.PA64S (S) shows more methylated fragments than PA64S (F) [124].This means that male sterility may be induced by elevated methylation levels in PA64S rice, which causes poor f lowering habit.PSMS and PA64S rice may show male sterility resulting from increased RdDM, which then affects f lowering behaviors.In general, DNA hypomethylation seems to be a promoter of f lowering and may be a substitute for photoinduction in photoperiodic f lowering.

Histone and RNA methylation in photoperiodic flowering
In contrast to DNA methylation, existing evidence suggests that histone methylation acts on photoperiodic f lowering more as a demethylase modifier to regulate f lowering (Fig. 4b).Studies show that the photoperiod-mediated CO/FT module can activate f loral transition in chrysanthemum and poplar [6,126].CO, as a prime f lowering regulator, controls the expression of FT mRNA through the photoperiod pathway to induce f lowering [114].The expression of the photoperiodic switch gene CO is strictly restricted by light and clock genes such as CCA1 [74].FT, encoding a RAFkinase inhibitor-like protein, is a f lowering activator regulated by vernalization and photoperiod in Arabidopsis [114,115].FT is activated by CO in the photoperiod pathway and restrained by FLC before vernalization (Fig. 4b).FT acts as a component in the photoperiod pathway as well as a f loral integrator that integrates the perception of inductive photoperiods and the FLC-mediated ) and histone demethylase (JmjC protein) in the photoperiod pathway: a series of SDG-mediated histone lysine methylations participates in photoperiodic f lowering by affecting f lowering-related regulators FLC, SOC1, OsLF (suppressors of Hd1), Ehd1, Hd3a, and RFT1.PRMT6 not only mediates the interaction between the H3R2me2a system and the NF-YC-CO module to dynamically regulate the expression of FT, but also cooperates with its homologous protein AtPRMT4a/4b to repress FLC, an inhibitor of FT, thus promoting photoperiodic f lowering.A series of H3K9/4 demethylases such as JmjC domain protein 27 (JMJ27)-JMJ14 play a role in the FT-FLC-CO module and act on upstream and downstream regulatory factors APETALA 1 (AP1), SOC1, and LEAFY (LFY).The long-day (LD) signal and the occurrence of methylation in the photoperiod pathway are coordinated by these JmjC proteins.c RNA methylation in photoperiodic f lowering: FIONA1 (mediating m 6 A methylation) and METTL4 (with N 6 -methylation activity) play respective roles in photoperiodic f lowering.FIONA1 participates in the photoperiod pathway by regulating FLC methylation and the expression of f lowering genes SOC1, SHORT VEGETATIVE PHASE (SVP), CO, and FT, as well as clock genes CCA1 and LATE ELONGATED HYPOCOTYL (LHY).Additionally, the mutation/deletion of METTL4 leads to early f lowering under long days.f loral repression signal.JmjC DOMAIN-CONTAINING PROTEIN 27 (JMJ27), one of the H3K9 histone demethylases, was found to be involved in this activation and restraint of FT [127] (Fig. 4b).Plants usually respond to environmental stress by regulating the time of f lowering, and it has been proved that plant defense signals do exist in the f lowering regulation pathway [128][129][130].JMJ27 is a protein factor that can regulate both plant defense and f lowering time.In f lowering, JMJ27 shows positive regulation of the f loral repressor FLC and negative regulation of the f loral activator CO [127] (Table 1).The absence of JMJ27 causes early f lowering in Arabidopsis infected with Pseudomonas syringae [127] and thereby JMJ27 may be engaged in photoperiodic f lowering.JMJ18 shows demethylase activity of H3K4me2/3 on the induction of photoperiodic f lowering in Arabidopsis [110] (Table 1).JMJ18 inhibits FLC expression by reducing the methylation level of H3K4 in FLC chromatin, thus promoting FT expression and then stimulating f lowering [110] (Fig. 4b).Contrary to JMJ27, JMJ18, which relies on the high expression of functional FT, leads to early f lowering, while its mutation leads to late f lowering.JMJ14, an H3K4 demethylase, is vital for preventing early f lowering of Arabidopsis in the vegetative stage via the repression of the f loral activator FT and the f loral integrators AP1, SOC1, and LFY in an FLCindependent manner under long days [131,132] (Fig. 4b, Table 1).In Arabidopsis, a previous report highlights that JMJ14 functions at the H3K4me3 level in the FT transcription initiation region to modulate photoperiodic f lowering [133,134], while others reveal that JMJ14 does not cause any changes in histone methylation levels of the FT or FLC locus [131,134].JMJ30 and JMJ32 directly bind and demethylate H3K27me3 at the FLC locus in vitro/in vivo and modulate Arabidopsis f lowering under long days [135] (Table 1).Other JmjC group proteins, JMJ11 (ELF6) and JMJ12 (REF6), have a delayed and accelerated effect on photoperiodic f lowering in Arabidopsis, respectively [136] (Table 1).In addition to these JmjC proteins, histone methyltransferases (HMTases) also function in photoperiodic f lowering, such as PRMTs and SDGs.PRMT6 mediates the interaction between the H3R2me2a system and the NF-CO module to dynamically modulate FT expression.Moreover, AtPRMT6 cooperates with its homologous protein AtPRMT4a/4b to repress FLC, an inhibitor of FT, thus facilitating photoperiodic f lowering [137].Furthermore, AtPRMT6 physically interacts with the positive f lowering regulators nuclear factors Y (NF-YC3/9 and NF-YB3) to regulate photoperiodic f lowering [137] (Table 1).Collectively, AtPRMT6, NF-YCs, and PRMT4a/4b synergistically regulate photoperiodic f lowering via the CO-FT module or the FLC-dependent pathway.SDGs, as the encoders of histone lysine HMTases, may also act in the photoperiodic f lowering of multiple species, especially rice and Arabidopsis.In short-day rice (Oryza sativa), SDG723 (OsTrx1) and SDG724/725 function in photoperiodic f lowering as H3K4 and H3K27 HMTase, respectively [138][139][140][141] (Table 1).SDG718 (OsiEZ1) and SDG711 (OsCLF), as H3K27 HMTases, function in photoperiodic f lowering by repressing OsLF (suppressor of Hd1) and silencing polycomb [142] (Table 1).Moreover, SDG708/724/725, encoding H3K36-specific HMTase, accelerates rice f lowering by stimulating the expression of the Ehd1, Hd3a, and RFT1 genes under short/long days [143] (Table 1).Furthermore, SDG712, encoding H3K9-specific HMTase, negatively regulates Hd1 expression.Subsequently, SDG712 inhibits the expression of f lorigen genes Hd3a and RFT1 by mediating the H3K9me2 of the two f lorigen genes, which eventually causes late f lowering in rice under short/long days [143].The sdg712 mutant f lowers early while the SDG712-OX line f lowers late, which further confirmed the function of SDG712 in photoperiodic f lowering [143].In Arabidopsis, AtSDG25 and AtSDG26 encode H3K36 and H3K4/36specific HMTase, respectively.They participate simultaneously in photoperiodic and vernalization-induced f lowering [144,145] (Table 1).AtSDG25 postpones f lowering by activating FLC expression, while AtSDG26 advances f lowering by binding to the SOC1 locus [144,145].Excitingly, a new study from Guo et al., through the isolation and identification of Arabidopsis H3K4/K36me2/3 reader MRG1/2, revealed a new mechanism for histone methylation to precisely regulate photoperiodic f lowering [146].They reported that MRG1/2 and histone deacetylase HD2C jointly repress FT to set an appropriate f lowering time so as to regulate photoperiodic f lowering.MRG and CO form a protein complex and collaboratively promote the expression of FT.Additionally, MRG1/2 can recruit HD2C to the FT gene under long days and catalyze the deacetylation of H3K9/23/27ac in the FT promoter to inhibit FT and thereby delay f lowering [146].These results suggest that histone methylation probably regulates photoperiodic f lowering by altering expression of f lowering genes such as FLC, CO, and FT or polycomb silencing.
As for RNA, especially non-coding sRNA affects f lowering time by directing the methylation status of DNA and histone [147].The circadian clock, a central player in f lowering/meristem transition in bulbous plants, serves as an integrator of lowtemperature signals and vernalization-/photoperiod-/meristem transition-related gene expression.In the study of Ben Michael et al., long dark cold exposure (photoperiod and vernalization) in Allium sativum bulbs induced f lowering and bulbing [148].They shed light on the result that RNA methylation functions in metabolic processes to play general developmental roles in meristem identity affected by vernalization.They identified a series of DEGs related to hormone transport, regulation of gene expression, DNA modification and methylation, cytokinin response, auxin inf lux, defense response, photoperiod, photosynthesis, and f lavonoid metabolism.Overall, RNA methylation or other potential methylation is probably involved in A. sativum f lowering under co-treatment of vernalization and photoperiod.RNA methylation may be involved simultaneously in both photoperiod-and vernalization-induced f lowering.In addition, circadian regulations are required to enable organisms to synchronize physiology with photoperiod.m 6 A is established by 'writer' and 'eraser' proteins, and its RNA methylation extensively and actively contributes to circadian regulation in seagrasses (Cymodocea nodosa and Zostera marina) [149].When the circadian rhythm mechanism senses suitable f lowering conditions, the plant will blossom and bear fruit.Seagrasses are a unique group of f lowering plants, and whether RNA methylation accelerates f lowering by regulating the circadian rhythm in seagrasses remains to be studied'.More regulatory details on whether and how RNA self-methylation directly/indirectly affects photoperiodic f lowering in seagrasses are still required.Notably, in Arabidopsis, several putative m 6 A erasers belong to the ALKBH family, such as ALKBH9B and ALKBH10B, both of which are functionally characterized in f lowering due to their m 6 A demethylase activity [150] (Fig. 4).Fascinatingly, FIONA1mediated m 6 A methylation may be involved in photoperiodic f lowering.FIONA1, a U6 m 6 A methyltransferase from Arabidopsis, has been identified as a genetic regulator of the circadian clock.FIONA1 affects day-length-dependent f lowering so that fio1-1 f lowers early in a photoperiod-dependent manner [151].A previous study also revealed that Arabidopsis fio1-1 mutants show an early-f lowering phenotype under long days/short days [152].However, Wang et al. reported that the m 6 A methylation activity of FIONA1 is necessary for phytochrome signalingdependent photomorphogenesis and photoperiod-independent f lowering [151] (Table 1).It is noteworthy that after absorbing different wavelengths of light, photoreceptors can induce plant morphogenesis and have a significant effect on some physiological processes.Phytochrome is a photoreceptor that mediates the reaction of red light and far-red light.FIONA1 is a positive regulator of photomorphology, especially in phytochrome A (Phy A) and Phy B signal transduction in Arabidopsis [151].Therefore, FIONA1-mediated m 6 A methylation might have an effect on f loral transition.This remains to be explored.Intriguingly, FIONA1mediated FLC 3 UTR methylation, which regulates Arabidopsis f lowering, is partially independent of the photoperiodic pathway [153] (Table 1).FIONA1, as an m 6 A writer and f loral repressor, stabilizes FLC expression by methylating the 3 end of FLC transcripts [153].The expression of FT and CO is elevated in fio1-1 and fio1-5, while fio1 ft and fio1 co f lower early under long days/short days [153].Surprisingly, Xu et al. demonstrated that the Arabidopsis f lowering regulated by FIONA1-mediated m 6 A methylation is closely related to SOC1, a key f lowering integrator [154] (Table 1).They indicated that FIONA1-mediated m 6 A methylation directly reduces the transcript abundance of SOC1 and indirectly inhibits the expression of its upstream regulators SHORT VEGETATIVE PHASE (SVP, a direct repressor of SOC1), CO, and FT to further suppress SOC1 expression, thus repressing photoperiodic f lowering.Mutant fio1 exhibited global m 6 A mRNA demethylation and an early-f lowering phenotype [154].In addition, the circadianregulated genes CCA1 and LATE ELONGATED HYPOCOTYL (LHY) in fio1-2 were identified as m 6 A-hypomethylated genes [154].These two clock genes, whose mRNAs are methylated directly by FIONA1, elevate the expression of CO and FT, and thereby indirectly participate in SOC1 suppression [154].Besides FIONA1, the MT-A70 family methyltransferase METTL4 may also be involved in photoperiodic f lowering.Intriguing discoveries by Luo et al. revealed that METTL4 acts as the U2 snRNA MTase for N 6 -2 -O-dimethyladenosine (m 6 Am) in vivo and specifically catalyzes the N 6 -methylation of Am in ssRNA in vitro [155].The deletion/mutation of METTL4 can promote Arabidopsis f lowering under long days [155] (Table 1).Notably, in their GO analysis data, a group of DEGs related to photosynthesis and response to low temperature is associated with the regulation of f lowering time [155], suggesting that METTL4 as an MTase might involve both photoperiod and vernalization pathways.In general, therefore, it seems that DNA methylation and histone methylation are two main potential methylation types involved in photoperiodic f lowering.More detailed information about the involvement of RNA methylation in photoperiodic f lowering is needed.

Conclusions and future outlook
Histone, DNA, and RNA methylation may regulate vernalization and photoperiod-induced f lowering through crosstalk with f lowering-related regulatory factors (FLC, FT, CO etc.), sRNAs [lncRNA (COOLAIR, LDMAR, and osasmr5864m)] and phytohormones.Methylation probably regulates vernalizationand photoperiod-induced f lowering simultaneously, and subsequently accelerates plant f lowering in coordination.Histone methylation acts on the vernalization and photoperiod pathways via the histone methyltransferase or demethylase and lncRNAs in the lysine and arginine pathways.DNA methylation regulates f lowering through the RdDM pathway and plant hormone pathway, and the methylation state carrying vernalization/photoperiod memory may be inherited by subsequent generations, especially plant endogenous methylase mutations (such as METI).In addition, based on the evidence that DNA methylation can replace prolonged cold (vernalization) and the photoinduced effect, DNA methylation may replace vernalization and photoperiod as f lowering regulators in the future.RNA methylation may regulate vernalization-induced bolting and f lowering via RNA methylcytosine transferase.It also participates in photoperiodic f lowering by regulating FIONA1 and METTL4 with N 6 -methylation activity.However, the exact mechanism by which RNA methylation regulates vernalization-and photoperiodregulated f lowering is still an open question and needs to be further studied.A large number of studies have shown that vernalization-and photoperiod-induced f lowering are often accompanied by significant methylation changes.More studies have confirmed that methylation is likely to replace vernalization and photoinduction to promote f lowering.Therefore, we propose the feasibility of methylation as a new f lowering regulator in the future.Notably, most model plants comply with the above methylation mechanisms of f lowering regulation, but there are unequal differences among different species.Methylation reagents may become new f lowering accelerators or inhibitors to selectively replace f lowering conditions such as low temperature and photoinduction.Methylation-regulated f lowering might develop into a novel f lowering pathway dependent/independent of other f lowering pathways in the future.This methylation regulatory mechanism may lead to the cultivation of plants more adaptable to changing environment.
Flowering is a crucial step in developmental transitions.Methylation in plants may be changed by factors/conditions in vitro or in vivo, such as methyltransferase/demethylase (Table 1), prolonged cold (vernalization), light quality or intensity, and long/short days.5-azaC may replace prolonged cold (vernalization) to induce f lowering, and the substitution of 5-azaC for the photoinduced f lowering effect is not specific to P. frutescens [116].However, 5-azaC cannot replace the photoinduced effect, but only slightly increase the f lowering percentage in induced Chenopodium rubrum [156].This means that the substitution of 5-azaC is not suitable for each plant.But it would be viable that the methylation level is changed to regulate f lowering time more conveniently and accurately by using these factors/conditions.It will be intriguing to further study the substitutability of f lowering conditions by methylation.This may represent the first step in the evolution of methylation treatments as a new practical means of regulating f lowering.In human and animal research, huge efforts have been made to develop methylation reagents as clinical drug candidates, such as the development of the DNA methyltransferase inhibitor PRMT5 (one of the most promising anticancer targets).Recent discoveries show that the druggability of histone lysine methylation or its reader is feasible, which means the development of MBD antagonists is feasible [157,158].The DNA methyltransferase inhibitor 5-azaC immunoregulates common epithelial cancers [159].Attenuated fungus treated with 5-azaC increases camptothecine production in host plants [160].A recent study has proposed the use of demethylation reagents to avoid the decline in the production of secondary metabolites during the maintenance of plant cell cultures in vitro [161].The function of the 5-azaC reagent as a f lowering accelerator has been gradually recognized.Methylation/demethylation reagents may show promising strategies for more convenient and finer f lowering regulation at molecular level in different species.
However, abnormal changes in DNA methylation may cause developmental abnormalities in plants.For example, a study on the somaclonal mantled African oil palm Elaeis guineensis showed that the hypomethylation of Karma LINE retrotransposons of the EgDEF1 gene results in abnormal fruit with low oil yield [162].In seed development, the enzymes/proteins involved in active DNA demethylation, including Repressor of silencing 1 (ROS1), Demeter (DME), and downstream proteins of DME, including ZDP, APE1L, and DNA ligase I, were reported to be essential for normal seed development [163].The mutation of maternal DME leads to failure of maternally expressed genes (MEGs, maternally imprinted genes) activation, and early seed abortion with cessation of embryo growth [164].In their progeny, homozygous DME-null mutants cannot survive, while 50% of the seeds of heterozygous DME/dme-1 and DME/dme-2 plants are aborted.Kim et al. indicated that a Arabidopsis dme-2-homozygous mutant with a seed abortion rate of 97.1% still produces some seeds and shows early f lowering [165].Single mutants of APE1L and ZDP show no abnormality, but their double mutants display an embryonic lethal phenotype and produce ∼50% abortive seeds after pollination [166].The expression of imprinted genes in the endosperm of these abortive seeds is downregulated and DNA hypermethylation occurs, such as DNA hypermethylation at selected MEG (FWA and FIS2) promoters.The cytosine methylation in antisense METI Arabidopsis decreases, causing many abnormal developmental phenotypes, including reduced apical dominance, smaller plant size, reduced fertility, and changes in leaf size and shape and f lowering time [90].Separating/removing the antisense structure in sexual hybridization cannot completely restore the methylation pattern of the progenies.Similarly, Lang et al. found that SlDML2 mutation leads to genomewide DNA hypermethylation and fruit ripening inhibition [167].Moreover, the number of seeds produced in the fruit of the Sldml2 mutant decreases, indicating that the abnormal increase in DNA methylation may cause the decline in seed yield [167].Furthermore, exogenous low (30 μM) and high (≥100 μM) concentrations of 5-azaC show extremely different effects on the growth and development of spinach seeds, including seed germination rate, root length, plant height, and f lowering time [118].This shows that 5-azaC-mediated demethylation must be within a certain limit to play a positive regulatory role in plants.Therefore, it is worth considering that the f lowering promotion effect mediated by DNA (de)methylation may make it a f lowering regulator to some extent, but there is still a big gap in research on whether the progeny will produce abnormal phenotypes, such as abnormalities in f lowering, fruit ripening, and seed development.Interestingly, in breeding, the paternal epigenome is the key determinant of the triploid blocking response (failure of endosperm development, arrest in embryogenesis, seed collapse).It was recently discovered that 5-azaC can help Arabidopsis bypass triploid seed collapse, and shows stable low-methylation intergenerational inheritance in strong suppressor line (Aza1, 14, 18 and 25; 'suppressor' means the suppression of 5-azaC for triploid blocking reaction) upon CG context, as well as the normal expression of paternally expressed genes (PEGs, the genetic component of triploid seed death) in triploid seeds [168].Thus, there is some controversy about the effect of applying methylation reagents on growth and development in the progeny.It will be a critical point to explore when and where to use DNA methylation reagents in the future.Of course, even if the use of a methylation reagent results in abnormal seed, f lowering and, fruit development in the progenies in some species, it is still available in some suitable scenarios.For example, it is still applicable to some ornamental plants, such as foliage plants, stem plants, and f lower plants.Therefore, in the future it is essential to clearly understand and accurately define the use of DNA methylation in the right place at the right time and at the right concentration.
In addition, the following aspects may be worth exploration.The f lowering promotion of the autonomous pathway in Arabidopsis is unlikely to be due to DNA methylation [169].Nevertheless, Bäurle and Dean have shown in Arabidopsis that FPA (protein containing RRM-type RNA-binding domains) may promote f lowering by inhibiting FLC at least partly through histone demethylase FLD, and that autonomous pathway-mediated silencing may act through DNA methylation-dependent/independent effects [170].The age pathway regulated by the miR156-SPL (SQUAMOSA promoter binding protein-like) module ensures that plants bloom under non-induced conditions (non-vernalization, non-induced photoperiod) [171,172]-is methylation involved?Is methylation involved in the GA pathway, the endogenous f lowering pathway?What is the relationship among RNA methylation, sRNA, and RdDM in f lowering regulation?More molecular evidence of RNA methylation regulating f lowering is also one of the next challenges.These derived problems may be worthy of further studies on methylation-regulated f lowering.

Figure 1 .
Figure 1.Histone methylation in vernalization-induced f lowering.At the center is FLOWERING LOCUS C (FLC). a Lysine (K) and b arginine (R) histone methylation pathways regulate FLC expression through the FLC promoter, Polycomb Repressive Complex 2 (PRC2), long non-coding RNA (lncRNA), COOLAIR (an lncRNA), FLOWERING LOCUS D (FLD), Shk1 binding protein 1 (SKB1) [functioning as protein arginine methyltransferase 5 (PRMT5)] and PRMT10.a Methylation of H3K4/36 activates FLC expression, while the methylation of H3K27/9 has the opposite effect, repressing FLC expression and promoting vernalization--induced f lowering.There are two controversial pathways of COOLAIR: (i) COOLAIR directly represses FLC by the increase in H3K36me3 and decrease in H3K27me3 at the FLC locus or is induced by WRKY63 to indirectly repress FLC (H3K27me3 reduction occurs), so as to participate in vernalization-induced f lowering; (ii) CRT/DRE-binding factors (CBFs) bind to the CRT/DRE of FLC, resulting in an increase in COOLAIR; CBF mutation causes a serious defect in COOLAIR, but shows almost normal vernalization.There are two controversial pathways related to whether COOLAIR is a necessity for FLC repression during vernalization.SDG8/EFS, a histone lysine methyltransferase, functions in the expression of the FLC clade.b Both SKB1 (PRMT5) and PRMT10 have potential effects on vernalization.Symbol explanations: → indicates promotion; ↔ indicates interaction; indicates inhibition or silence.The followings of symbol explanations are the same.

Figure 2 .
Figure 2. Schematic overview of the effect of DNA methylation on FLC reactivation.Yeast RNA polymerase II-associated factor 1 (Paf1) (including VIP2/4) and the FRIGIDA (FRI) family are required for FLC reactivation.Apart from perennials, vernalization memory should be reprogrammed at the end of the life cycle, such as the reactivation of FLC expression.The vernalization state/memory of parents in FLC may be reset by ELF6 or LEAFY COTYLEDON 1 (LEC1), and LEC1 may be involved in the reactivation of FLC by FRI.DNA methylation may have no effect on FLC reactivation.

Figure 3 .
Figure 3. Effects of DNA and RNA methylation on vernalization-induced f lowering after FLC reactivation.Under the synergistic or independent action of vernalization signals and photoperiod signals, DNA methylation may directly/indirectly affect the expression of the central f lowering repressor FLC and its homologous gene FLF, vernalized genes (BrCKA2 and BrCKB4), mobile f lorigen FLOWERING LOCUS T (FT) and its homologous genes TWIN SISTER OF FT (TSF) and FTa1 (overexpressed in spring), f lowering integrator SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1) and clock gene BrCCA1.AtMBD7 is necessary for active demethylation.AtMBD9-mediated methylation at FLC promotes early f lowering.5-Azacytidine (5-azaC) promotes vernalization-induced f lowering, which may be related to the GA biosynthetic enzyme gene KAH with apex specificity.In addition, vernalization induces upregulated expression of methylated genes LoCMT and LoHDAC.RNA methylation may regulate f lowering through RNA methyltransferase (RNMT) and the FLC homologous gene FLOWERING LOCUS 1 (FL1) in vernalized sugar beet.Snowf lakes represent prolonged cold (vernalization) and the following snowf lakes represent the same.

Figure 4 .
Figure 4. Methylation in photoperiodic f lowering.a DNA methylation in photoperiod-induced f lowering: METHYLTRANSFERASE (METI), DECREASED IN DNA METHYLATION1 (DDM1), 5-azacytidine (5-azaC) and ncRNA [long-day-specific male-fertility-associated RNA (LDMAR) and osasmR5864m] mediate DNA methylation (RdDM) to affect f lowering; methyl-CpG-binding domain (MBD) protein may regulate f lowering through methylation; the effect of 5-azaC may be reset in the next generation.b Histone methylation in photoperiodic f lowering.Two pathways of histone methyltransferase (SDGs and PRMT6) and histone demethylase (JmjC protein) in the photoperiod pathway: a series of SDG-mediated histone lysine methylations participates in photoperiodic f lowering by affecting f lowering-related regulators FLC, SOC1, OsLF (suppressors of Hd1), Ehd1, Hd3a, and RFT1.PRMT6 not only mediates the interaction between the H3R2me2a system and the NF-YC-CO module to dynamically regulate the expression of FT, but also cooperates with its homologous protein AtPRMT4a/4b to repress FLC, an inhibitor of FT, thus promoting photoperiodic f lowering.A series of H3K9/4 demethylases such as JmjC domain protein 27 (JMJ27)-JMJ14 play a role in the FT-FLC-CO module and act on upstream and downstream regulatory factors APETALA 1 (AP1), SOC1, and LEAFY (LFY).The long-day (LD) signal and the occurrence of methylation in the photoperiod pathway are coordinated by these JmjC proteins.c RNA methylation in photoperiodic f lowering: FIONA1 (mediating m 6 A methylation) and METTL4 (with N 6 -methylation activity) play respective roles in photoperiodic f lowering.FIONA1 participates in the photoperiod pathway by regulating FLC methylation and the expression of f lowering genes SOC1, SHORT VEGETATIVE PHASE (SVP), CO, and FT, as well as clock genes CCA1 and LATE ELONGATED HYPOCOTYL (LHY).Additionally, the mutation/deletion of METTL4 leads to early f lowering under long days.