Advances in understanding epigenetic regulation of plant trichome development: a comprehensive review

Abstract Plant growth and development are controlled by a complex gene regulatory network, which is currently a focal point of research. It has been established that epigenetic factors play a crucial role in plant growth. Trichomes, specialized appendages that arise from epidermal cells, are of great significance in plant growth and development. As a model system for studying plant development, trichomes possess both commercial and research value. Epigenetic regulation has only recently been implicated in the development of trichomes in a limited number of studies, and microRNA-mediated post-transcriptional regulation appears to dominate in this context. In light of this, we have conducted a review that explores the interplay between epigenetic regulations and the formation of plant trichomes, building upon existing knowledge of hormones and transcription factors in trichome development. Through this review, we aim to deepen our understanding of the regulatory mechanisms underlying trichome formation and shed light on future avenues of research in the field of epigenetics as it pertains to epidermal hair growth.


Introduction
The plant trichome is a specialized accessory structure that covers aboveground organs and is differentiated from epidermal cells [1].Trichomes can be classified into unicellular or multicellular types based on their cell composition, and glandular or non-glandular types depending on their secretory function [2].The morphology and structure of trichomes exhibit significant diversity among different species, and interestingly, even within the same species, different types of trichomes can be found.The size and density of trichomes also vary depending on their location and developmental stage [3].These variations provide a theoretical foundation for studying the biological functional diversity of trichomes [4].
Arabidopsis thaliana and Gossypium hirsutum (cotton) are representative examples of unicellular trichome plants.Arabidopsis trichomes are three-branched, non-glandular and relatively simple in structure (Fig. 1A) [5].In contrast, cotton fibers are long and unbranched unicellular trichomes (Fig. 1B) [6].Cucumis sativus (cucumber) and Solanum lycopersicum (tomato) are multicellular trichome plants that exhibit a great diversity of trichome types [7,8].Cucumber trichomes can be categorized into eight types, all of which are multicellular.The majority are type I glandular trichomes and type II non-glandular trichomes [9].Type I trichomes have a rod-shaped stem of three or four cells and a gland head composed of four or five cells.Type II non-glandular trichomes consist of a multicellular base and a rod-like head composed of three to eight cells [9,10].The other types of cucumber trichomes are non-glandular, with the exception of type I and type VI (Fig. 1C) [9].Tomato epidermal hairs can also be classified into eight types according to their morphological structure.Among them, type II, III, V, and VIII trichomes lack secretory functions and are composed of base and neck cells.Type I, IV, VI, and VII epidermal hairs possess a glandular tip, distinguishing them from the other non-glandular epidermal hairs (Fig. 1E) [11].Trichomes have distinct morphologies compared with surrounding epidermal cells, making their structure easily observable.Thus, trichomes have become an ideal system for studying the regulation of fate identification, cell differentiation and polarity [12].
The developmental processes of trichomes vary across species due to differences in their morphology.In Arabidopsis and cotton, trichome development is divided into four stages.In Arabidopsis, trichome growth involves pattern formation, endoreduplication, branch formation, and directional growth (Fig. 1A) [13,14].On the other hand, cotton fiber development consists of four sequential and continuous stages, namely initiation, elongation, secondary cell wall (SCW) deposition, and maturity (Fig. 1B) [6].The development processes of multicellular trichomes are more complex, and as a result studies on these processes have only been reported recently.Cucumber trichomes have five development stages: (i) initiation, (ii) first division, (iii) tip head formation/glandular head transition, (iv) elongation/glandular head formation, and (v) base formation/active metabolism.The third stage is where differentiation of glandular and non-glandular trichomes occurs (Fig. 1D) [15].However, there is still no clear stage division of tomato epidermal hairs.[5,14].(B) Morphology of cotton fibers at different stages of development [6].(C) Eight morphological classifications of cucumber trichomes [9].(D) Five developmental stages of cucumber trichomes [15].(E) Eight morphological classifications of tomato trichomes [11].
Plant trichomes hold significant biological importance and economic value, although they are not essential for the survival of plants.As a barrier between plants and the external environment, trichomes provide protection against various biotic and abiotic stresses [16].The physical structure of non-glandular trichomes and the chemicals secreted by the glandular trichomes play a role in defending against herbivorous insects [17].Glandular trichomes possess the ability to synthesize, store, and secrete a wide range of compounds, including proteins, polysaccharides, polyphenols, alkaloids, organic acids, and terpenes.These compounds find applications in the production of spices, essential oils, medical drugs, pesticides, and food additives [18].Moreover, some specific trichomes, such as cotton fibers, hold considerable economic value.In the case of cucumber fruits, the trichomes, known as fruit spines, significantly inf luence the market value of commercial fruits, thereby holding important economic significance.
Trichome initiation occurs prior to plant organogenesis, and the initiation and growth of trichomes are controlled through the coordinated actions of plant hormones.Salicylic acid and jasmonic acid (JA) can affect the establishment of trichomes [19].Overexpression of the Torenia fournieri gibberellin 20-oxidase gene (TfGA20OX2) increases trichome size and number in Artemisia annua [20].The exploration of key genes has contributed to a gradual enrichment of our understanding of the molecular mechanisms underlying trichome formation.However, there remains a significant research gap, particularly when it comes to comparing different species.Our current knowledge of the regulatory mechanism is more comprehensive for unicellular trichomes compared with multicellular trichomes.The regulatory model of multicellular trichome development involves only a limited number of transcription factors [21].Research efforts have mainly focused on hormonal and transcriptional regulation, leaving numerous gaps to be filled in our understanding of trichome formation.
In addition to hormones and transcriptional control, epigenetic modifications play a crucial role in plant growth and development [22].However, a limited number of epigenetic regulators have been reported to regulate the initiation and growth of trichomes.Most of the identified regulators have focused on RNA posttranscriptional modification and microRNA (miRNA)-mediated regulation.Therefore, building upon a brief summary of hormonal and transcriptional regulation, we present a review of the current research progress on epigenetic factors that regulate trichome development in some model plants.Furthermore, we outline potential future research directions in this area.This comprehensive investigation aims to contribute to a deeper understanding of the regulatory mechanisms underlying trichome formation.

RNA post-transcriptional modification and miRNA regulation
Epigenetic regulations encompass a diverse range of factors, including non-coding RNAs, histone modifications, RNA/DNA methylation and chromatin remodeling, which collectively  6 A modification in plants involves three primary regulatory elements: the reader, writer, and eraser.These elements play pivotal roles in modulating the fate of RNA by introducing, eliminating, and binding m 6 A sites on RNA.Writers, including MTA, MTB, HAKAI, FIP37, and VIRILIZER, function as methyltransferases to add methyl groups to RNA.Erasers, such as ALKBH9B/10B, serve as demethylases for removing methyl groups.Readers, represented by CPSF30 and ECTs, act as recognition factors to identify methylation sites.contribute to the transcriptional/translational regulation of RNA, DNA, and chromatin [23].Research on plant trichomes has progressively delved into the realm of epigenetics, with particular emphasis on RNA methylation and non-coding RNAs.

RNA methylation
Epitranscriptomics studies have led to the discovery of >160 RNA modifications.N6-Adenylate methylation (m 6 A), N1-adenylate methylation (m 1 A) and cytosine hydroxylation (m 5 C) are three types of commonly studied modifications in plant research.m 6 A, the first identified modification, is also the most abundant internal modification of mRNAs [24].The level of m 6 A and its regulatory role depend on the dynamic interplay among its methyltransferases (writers), demethylases (erasers), and binding proteins (readers) (Fig. 2) [25].m 6 A methylation modifications play crucial roles in plant growth and stress responses by inf luencing the translation efficiency and stability of mRNAs.The components of the m 6 A methyltransferase complex, such as MTA, MTB, FIP37, VIRILIZER, and HAKAI, modulate vascular development in Arabidopsis [26].Knockout of FKBP12 INTERACTING PROTEIN 37 KD (FIP37) results in excessive proliferation of the shoot apical meristem in seedlings and delays leaf formation [27].Additionally, the fip37-4 mutant exhibits an embryonic lethal phenotype [28].An RNA demethylase called SlALKBH2 that can bind to SlDML2 transcripts is required for tomato fruit maturation.Mutation of SlALKBH2 delays fruit ripening [29].Deletion of the m 6 A demethylase ALKBH9B reduces the level of viral RNA, restricting the transfer of viral RNA between plant organs, and thereby reducing its systemic infection ability in plants [30].Additionally, drought stress induces the m 6 A demethylases ZmALKBH10a/b, which can improve the drought resistance of maize [31].
In addition to m 6 A, 5-methylcytosine (m 5 C) is also an important internal mRNA modification.Mutation of tRNA-specific methyltransferase 4B (TRM4B), an RNA m 5 C methyltransferase, leads to a decrease in m 5 C peaks.TRM4B-mediated m 5 C methylation increases the transcript levels of root-related genes, thereby positively regulating root development [32].In rice, the major m 5 C methyltransferase OsNSUN2 promotes selective translation of certain mRNAs, thereby maintaining chloroplast function and improving heat tolerance [33].Given its importance in plant growth and stresses response, m 5 C modification likely plays a role in the regulation of trichome development.

Non-coding RNAs
In eukaryotes, there are certain RNAs that do not undergo protein translation and are referred to as non-coding RNAs (ncRNAs).These ncRNAs play a role in regulating gene expression.Based on their average size, regulatory ncRNAs are classified into long ncRNAs (lncRNAs, >200 nt) and small ncRNAs (sncRNAs, <200 nt) [34].microRNA is the most prevalent class of endogenous sncR-NAs [34].miRNAs typically range in length from 16 to 29 nt, the majority falling between 20 and 23 nt [35].The biosynthesis of mature miRNAs is a complex process coordinated by multiple enzymes and auxiliary proteins.
Plant miRNAs control the expression of target genes during or after transcription.They function by either degrading the target mRNAs through complete complementarity or inhibiting mRNA translation through incomplete complementarity (Fig. 3) [36].miRNAs have significant impacts on plant growth, morphogenesis, and stress responses [35].Three members of the miR164 family regulate NAC transcription factors, playing crucial roles in boundary establishment and maintenance, lateral root emergence, vegetative growth, and f loral organ formation in Arabidopsis [37][38][39].Overexpression of sly-miR156 leads to a reduction in fruit quantity and size [40].In addition, overexpression of miR319b inhibits TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (OsTCP21), thus reducing the resistance to blast disease in rice [41,42].

Regulation of unicellular trichome development
Unicellular trichomes, such as those found on the rosette leaves of A. thaliana and the seed coats of G. hirsutum (cotton), have Figure 3. Biosynthesis process and functional mechanism of miRNA.The orange half-arc represents the nuclear membrane.On the left side of the arc, miRNA biosynthesis takes place within the nucleus.miRNA is processed into pri-miRNA by various enzymes in the nucleus, which is further converted into pre-miRNA.The dimer cleaved by DCL1 is processed into a mature miRNA-induced silencing complex (miRISC).Subsequently, miRISC regulates the expression of target genes through three distinct mechanisms.
been extensively studied, leading to a significant understanding of their developmental mechanism.Researchers have identified numerous transcription factors and plant hormone-related genes that play crucial roles in this process.Additionally, miRNAs have emerged as important participants by inhibiting the expression of these genes post-transcriptionally.

Trichome development in A. thaliana
Since the 1990s, more than 70 trichome mutants of A. thaliana have been discovered [43].Through the analysis of these mutants and genetic studies, numerous genes have been identified as coregulators of trichome development.The activation-inhibition model based on Arabidopsis trichome development is widely recognized.In this model, the trimeric complex activator MBW is composed of GL1 (the R2R3-MYB transcription factor), TTG1 (WD40 repeat protein), and GL3/EGL3 (bHLH transcription factor).AtGL1 was the first gene identified, promoting trichome differentiation and development [44].TRIPTYCHON (TRY), encoding a small single R3-MYB-repeat protein, was the first negative regulator of trichome development to be reported, lacking an obvious activation domain [45].The MBW complex facilitates the movement of negative regulators, such as TRY, to neighboring non-epidermal hair cells.In these cells, TRY hinders the formation of the MBW complex, thereby inhibiting the activation of epidermal hairs.The complex also modulates the downstream gene GL2/TTG2 to control trichome formation [46].Additionally, numerous other genes have been implicated in controlling trichome formation in A. thaliana (Fig. 4).
Plant hormones, such as auxin (IAA), gibberellin (GA), cytokinin (CK), and JA, play important roles in trichome development by regulating downstream genes.C2H2 transcription factors GIS2, GIS3, and ZINC FINGER PROTEIN8 (ZFP8) positively regulate trichome growth via the GA and CK pathways [47,48].TEM2 binds to the promoters of GL1, GL2, GIS2, and ZFP8 to negatively regulate trichome development, and also functions as a link between GA and CK signaling pathways [49].JASMONATE-ZIM-DOMAIN1 (JAZ1) functions as a repressor of MBW complex formation in the absence of JA by interacting with GL1 and GL3 (the components of the MBW complex).When JA induces JAZ degradation, it promotes the formation of MBW complexes and leads to the development of trichomes (Fig. 4) [50].
One of the most significant epigenetic regulations in plant growth is miRNA-mediated gene silencing, and it also has a very crucial regulatory role in the formation of trichomes.For instance, miR319 modulates trichome branching by inhibiting PROLIFERATING CELL FACTOR4 (TCP4), which promotes the expression of trichome branching inhibitor GIS (Fig. 4) [51].Another important miRNA involved in trichome development is miR156, which targets 10 SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL2/3/4/5/6/9/10/11/13/15) genes involved in the distribution of trichomes during f lowering.Among them, SPL9 negatively regulates trichome development independently of the GL1 pathway by directly binding to the promoters of TCL1 and TRY [52].MIR156 is expressed during the juvenile growth stage and promotes miR172b transcription via SPL9 (Fig. 4) [53].The transition from juvenile to adult stages in A. thaliana is marked by the appearance of abaxial trichomes on leaves, and it is inf luenced by two factors: spatial location and time.Spatially, the leaf polarity determinant KAN1 is expressed exclusively on the abaxial surface of leaves.Temporally, as plants grow, SPLs promote the production of miR172 by reducing the inhibitory effect of miR156 on SPLs.Consequently, miR172 targets and inhibits AP2-like genes.AP2-like is required to form a complex with KAN1 to inhibit GL1 gene expression in the distal leaf axis through chromatin loop-mediated histone deacetylation.As plants transition to adulthood, AP2-like gradually decreases, relieving this inhibitory effect [54].Both lateral roots and trichomes are lateral organs, and some transcription factors have opposite effects on their regulation.When the expression of MIR164 is reduced, the number of lateral roots is increased with the upregulation of AtNAC1 [39].miR169 regulates NF-YA transcription factors to affect root growth and branching [55].Based on the functions of miR164 and miR169 in root development, we speculate that they may also regulate trichome formation.

Development of cotton fibers
There are several similarities between the growth of cotton fibers and Arabidopsis trichomes.CPC interacts with MYC1 and TTG1 to negatively regulate cotton fiber initiation [61], while HOX1 and HOX3 promote cotton fiber initiation and elongation [62,64].MYB212 positively regulates SWEET12 to facilitate the transport of sucrose and glucose from the ovary to the fiber during cotton fiber elongation [64].Both MYB46 and FSN1 (a NAC transcription factor) are active during fiber SCW deposition [65], whereas the transcriptional repressor KNL1 negatively regulates cotton fiber development by reducing the transcription of SCW-related genes (Fig. 5) [66].
The formation of cotton fibers is significantly inf luenced by plant hormones like ethylene, JA, GA, and brassinosteroids (BRs) (Fig. 5).GA promotes the degradation of GhSLR1, thereby releasing GhHOX3 to form a transcriptional complex that promotes cotton fiber growth [63].Ethylene application to ovules in vitro enhances fiber elongation by activating genes crucial for cell wall production and loosening, as well as cytoskeleton organization [67].JAZ2, a repressor in the JA pathway, negatively regulates fiber initiation by inhibiting the downstream gene MYB25-like [68].Under the combined regulation of ethylene and reactive oxygen species (ROS), fibers progress from the initial stage to the elongation phase [13].BR stimulates the elongation of cotton fibers by promoting the interaction between Gh14-3-3 and BZR1 [69].Additionally, BR enhances secondary wall thickening and maturation of cotton fibers through BRI1 [70,71].
MYB2D is targeted by ghr-miR828 and ghr-miR858, which enhance fiber initiation (Fig. 5) [72].Under drought and salt stress, 163 miRNAs targeting 210 unique genes were identified to be involved in cotton fiber growth [73].ghr-miR156 may promote cotton fiber initiation by downregulating SPL9 expression, while ghr-miR36 modulates fiber growth by targeting the bZIP transcription factors (Supplementary Data Table S1) [74].Seven miRNAs related to fiber initiation were identified using high-throughput sequencing combined with bioinformatic analysis, and their target genes were predicted in the wild-type and fiberless mutants (Supplementary Data Table S1) [75].Several miRNAs exhibit differential expression patterns across the four developmental stages of fibers, indicating their involvement at different stages.During cotton fiber initiation, the expression level of 33 miRNAs

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differs between the fiberless type and wild type.In the wild type, 26 miRNAs regulate fiber initiation, and 48 miRNAs control secondary cell wall thickening potentially by targeting MYB, ARF, LRR, and 723 other genes [76].Deep sequencing identified 32 miRNAs with differential expression between cotton leaves and ovaries, and many fiber-related miRNAs have been validated by qRT-PCR.They are involved in initiation and elongation of fibers by targeting downstream genes (Supplementary Data Table S1) [77].Differential expression of 46 miRNAs was found during fiber elongation, and the target genes of eight miRNAs and a tasiRNA (trans-acting small interfering RNA) were experimentally verified to be associated with fiber elongation through different pathways (Supplementary Data Table S1) [78].Cellulose synthesis is an indispensable part of fiber maturation.At least three miRNA families (miR396/414/782) target cellulose synthesis-related genes, including fiber synthase, fiber protein Fb23, and fiber quinine oxidoreductase, which are important for the development of cotton fibers (Supplementary Data Table S1) [79].These miRNAs potentially control the development of cotton fibers (Fig. 5).
Apart from miRNAs, other epigenetic components are crucial for fiber formation as well (Fig. 5).For example, the histone deacetylase HDA5 is specifically expressed during cotton fiber initiation, and its silencing results in reductions of fiber initiation and lint yield [80].The bidirectional transcripts of MML3 A12 produce siRNAs that inhibit cotton fiber initiation by mediating selfcleavage of GhMML3 A12 mRNA in N 1 plants [81].Genome-wide analysis revealed that DNA methylation increased during fiber development and targeted genes involved in lipid biosynthesis and spatio-temporal modulation of ROS, thereby regulating fiber differentiation [82].HISTONE MONOUBIQUITINATION2 (HUB2), a histone H2B mononucleotide E3 ligase, is bound by the transcriptional repressor KNL1, which in turn regulates elongation and SCW deposition of fibers through the ubiquitin-26S proteasome pathway [83,84].

Regulation of multicellular trichome development
Although the regulatory mechanisms of uni-and multi-cellular trichomes have numerous similarities, there are also notable differences.It remains unclear whether there is a unified model that governs the regulation of multicellular trichomes, and further exploration is required to understand the underlying mechanisms.At present, the network of trichome growth has been extensively studied only in cucumber and tomato.A preliminary model for the regulation of multicellular trichome development was proposed by studying different mutants of cucumber and tomato, which provides a basis for understanding similar processes in other multicellular trichome-bearing plants.

Development of cucumber trichomes
Considerable research has been conducted on the regulatory mechanisms underlying the formation and growth of cucumber trichomes, particularly fruit spines.Studies have revealed that trichome initiation and development in cucumber are primarily inf luenced by specific genes and hormones, such as GA, IAA, and CK.Through the interplay of these genes and the crosstalk of plant hormones, a regulatory network of trichome development is proposed (Fig. 6).
The phenotype of a hairless mutant is attributed to the two alleles, CsGL3 and Tril, arising from different mutations of a HD-Zip IV transcription factor (Csa6M514870) that controls trichome initiation [85][86][87].Positive regulation of trichome development is achieved through the alleles CsGL1, Mict, and TBH, which result from various deletions of Csa3G748220.Mutant plants exhibit only cell protrusions on their surfaces, as the complete morphological construction of cucumber trichomes cannot form in csgl1, mict, and tbh mutants [9,88,89].CsMYB6 and CsTRY act as modules to inhibit cucumber trichome initiation [90].CsTTG1 positively regulates epidermal hair initiation and differentiation by interacting with CsGL1/TBH/Mict [91].CsGL1 epistatically acts on CsTu to control fruit tubercule formation [92,93].Notably, to modulate the density of fruit spines and warts, CsHEC2 directly interacts with CsGL3 and CsTu [94].
Studies have shown that plant hormones such as GA, IAA, and JA regulate the formation of multicellular trichomes.Transcriptome data analysis showed that the GA biosynthetic enzyme gene CsGA20ox1 was upregulated in csgl1.Overexpression of CsGA20ox1 resulted in shorter trichomes [95].The auxin transporter-like protein 3 is encoded by a highly conserved NS gene in plants, which controls the number of cucumber fruit spines [96].CsTu directly binds to the promoter of CsTS1 and synergistically regulates the size of fruit tubercules by affecting auxin content in fruit spines [97].The expression of CsNACs initially increases and then decreases in response to exogenous GA, IAA, and methyl jasmonate (MeJA).The conserved MeJA-responsive elements (CGTCA), GA-responsive elements (AAACAGA), IAA-responsive elements (AACGAC), and ethylene-responsive elements (ATTTCAAA) are present in the vast majority of CsNACs promoters, indicating a link between CsNACs, hormones, and trichome formation [98].
The comprehensive understanding of the impact of epigenetic regulation on cucumber trichomes is still lacking.Analysis of eight publicly available cucumber sRNA-seq databases, coupled with bioinformatic prediction of target genes, resulted in the identification of a limited number of miRNAs targeting several important genes involved in cucumber trichome development in a single database.For instance, csa-miR4249, csa-mir-21, and csa-mir-216 target CsGL1, while csa-mir-23, csa-mir-73, and csamir-153 target CsTu.Furthermore, csa-mir-153 also targets CsTRY (Fig. 6) [99].These findings suggest that miRNAs may play a role in the growth of cucumber trichomes, but their specific contributions have not been definitively established.Currently, there is a lack of molecular experimental evidence to substantiate the significance of miRNAs in cucumber trichome development.An miRNA can target multiple downstream genes, and conversely, a gene can be regulated by multiple miRNAs.Therefore, it requires further investigation whether these miRNAs affect the occurrence and growth of cucumber trichomes through regulating these key genes in the network, along with the underlying molecular mechanisms involved.

Development of tomato trichomes
The regulatory network governing tomato trichomes is relatively straightforward, involving several important transcription factors (Fig. 7).SlMYC1 was reported to be specifically involved in glandular trichome formation.Knockout of SlMYC1 led to the disappearance of type VI trichomes [100].WOOLLY is the first gene identified during the development of tomato trichomes and is homologous to AtGL2.In the woolly mutant, the numbers of both type III and type V trichomes increase, while the number of type IV glandular trichomes decreases.The quantity of type I glandular trichomes increases in SlWo-overexpressing plants [101][102][103].Overexpression of SlZFP8-like (SlZFP8L, a close homolog of Hair) increases the length and density of epidermal hairs by interacting with WOOLLY [104].When overexpressing SlCycB2, almost all glandular trichomes and non-glandular trichomes (such as types III and V) disappear.Knockdown of SlCycB2 might stimulate the growth of type III and type V trichomes [105].SlMIXTA1 (an MYB transcription factor) promotes the production of tomato trichomes [106].Transcription factors still play a role in regulating the morphology of tomato trichomes.For instance, SlHDZIV8 governs Hairless-2 expression, which in turn inf luences trichome shape [107].
The growth of tomato trichomes is controlled by three main hormones: JA, GA, and IAA (Fig. 7).JA regulates the expression of downstream genes SlCycB2 and WOOLLY through JAZ2.Overexpression of JAZ2 reduces the density of trichomes [108].H and HL genes, which are part of the JA signaling pathway, directly repress the expression of the downstream gene THM1, which inhibits the development of trichomes [109,110].COI1, a critical component of the JA receptor, promotes the occurrence of trichomes [111].The SlHD8-SlJAZ4 complex plays a significant role in JAinduced elongation of epidermal hairs [112].bHLH95 regulates GA synthesis by inhibiting GA20ox2 and KS5, thereby regulating the formation of trichomes [113].The number of tomato type I, V, and VI trichomes decreases in response to downregulation of SlIAA15 (an auxin family gene) and SlARF3 (an auxin-responsive factor) [114,115].By inhibiting the expression of THM1 and MYB52, ARF4 promotes type II, V, and VI tomato trichome growth [116].SlMYB75 negatively regulates type II, V, and VI trichomes by affecting several transcriptional pathways [117].
The study of the regulatory network underlying tomato trichome development has primarily focused on transcriptional and hormone regulation, while the role of epigenetic factors in this process has been rarely investigated.Only a few epigenetic regulatory factors have been identified thus far in relation to tomato trichome formation.For instance, overexpression of lncRNA000170 in mature transgenic strains prevents type I trichome production in the lower stem [118].In addition to slow growth, severe leaf curling, and deepening of leaf fissures, miR319a-overexpressing plants exhibit increased trichome density (Fig. 7).However, which target genes of miR319a contribute to the development of trichomes require further investigation [119].

Summary and outlook
Trichomes play a crucial role in plants, enhancing their ability to withstand diverse stresses and providing significant economic value.For example, the presence of trichomes on cucumber fruit is an important characteristic that inf luences consumer preferences.Glandular trichomes, found in many plants, produce secondary metabolites with substantial utility in various industries, including production and medicinal applications.
The regulatory functions of several well-known transcription factors involved in the initiation and morphogenesis of plant trichomes have been partially elucidated.Transcription factor families such as MYB, bHLH, WD40, WRKY, HD-ZIP, NAC, and others, along with hormone-related genes, form the core model for understanding the regulation of plant trichome development.However, the complete and detailed regulatory network is still unknown.The formation of trichomes in plants is modulated by various transcription factors and may be inf luenced by additional types of transcription factors.Notably, research on multicellular trichomes has progressed at a slower pace compared with that on unicellular trichomes, and many aspects of the regulatory network governing trichome development remain unknown.Therefore, there is ample opportunity to further explore and identify potential regulatory transcription factors for trichome development by leveraging the existing network composed of known genes.
Advancements in molecular biotechnology have greatly contributed to the understanding of epigenetic regulation in various fields, including medicine, microbial metabolism, and plant growth.Epigenetic regulators govern gene expression through multiple pathways and inf luence nearly all aspects of plant development.In recent years, systematic studies have shed light on the impact of epigenetic modifications on f lowering, stress responses, and senescence in different model plants, such as A. thaliana and Oryza sativa.
As research on plant epigenetic modifications continues to surge, scientists have started to shift their focus towards understanding how epigenetic modifications regulate the development of plant trichomes beyond the scope of transcriptional and hormone regulation.Some epigenetic regulators, such as ETC2, CAF1, GCN5, UPL3, and miR319, were identified as important players in epidermal hair formation in A. thaliana.However, only a limited number of miRNAs have been found to be involved in this process.Overall, it remains poorly understood how epigenetic modifications regulate the development of trichomes in plants.
MicroRNAs, although confirmed in a limited number of plant species, have only been studied to a preliminary extent in the context of trichome development.However, the molecular mechanism and regulatory network of these specific miRNAs involved in trichome development are still obscure.Recent advancements in sequencing technologies have rapidly evolved.Small RNA sequencing technology enables in-depth exploration of miRNAs, while degradome sequencing technology efficiently detects the target genes of miRNAs.Differentially expressed miRNAs were identified by analyzing and comparing sRNAseq databases from wild-type and trichome-defective mutants.Combined with degradome data, the downstream target genes of these miRNAs were further studied to elucidate the molecular mechanism of trichome development.
In addition to interacting with their target genes, miRNAs can also establish connections with other regulators, contributing to a more comprehensive understanding of trichome development.
Here are three potential approaches to exploring these interactions.(i) Deciphering the transcriptional regulation of miRNAs by analyzing cis-regulatory elements on MIRNA promoters.By identifying the upstream transcription factors of miRNAs that participate in epidermal hair growth, we can expand the TF-miRNA network.(ii) Exploring the role of lncRNAs as endogenous competitive RNAs that indirectly regulate biological processes posttranscriptionally by miRNAs.Incorporating lncRNAs into the network helps establish a large lncRNA-miRNA-gene regulatory network, thereby enriching the regulatory mechanism of trichome formation.(iii) Considering the inf luence of DNA methylation, which regulates genes involved in lipid biosynthesis and ROS metabolism, thereby inf luencing cotton fiber development.Given that miRNAs can mediate DNA methylation [120,121], investigating the regulation of miRNA-regulated DNA methylation in plant trichome formation holds great scientific significance and potential.In conclusion, starting from miRNAs as a focal point will facilitate further breakthroughs in unraveling the mechanisms through which epigenetic factors regulate plant trichome development.

Figure 2 .
Figure 2. m6 A modification in plants involves three primary regulatory elements: the reader, writer, and eraser.These elements play pivotal roles in modulating the fate of RNA by introducing, eliminating, and binding m 6 A sites on RNA.Writers, including MTA, MTB, HAKAI, FIP37, and VIRILIZER, function as methyltransferases to add methyl groups to RNA.Erasers, such as ALKBH9B/10B, serve as demethylases for removing methyl groups.Readers, represented by CPSF30 and ECTs, act as recognition factors to identify methylation sites.

Figure 4 .
Figure 4.The regulatory network of trichome growth in A. thaliana.The rectangular box in the center represents the various stages of trichome formation in A. thaliana.The four ovals above the rectangle correspond to every stage of trichome development and contain the genes involved in each stage.The regulatory network for trichome development is shown below the rectangle.The dashed box represents the MYB-bHLH-WD40 (MBW) complex.

Figure 5 .
Figure 5.The regulatory network for cotton fiber development.The four dashed rectangles from top to bottom in the middle indicate the four developmental stages of cotton fibers.

Figure 6 .
Figure 6.The regulatory network for cucumber trichome development.The miRNAs in the dashed gray diamonds may regulate trichome development by acting on downstream genes.All dashed lines without arrows in the diagram represent only potential regulation.

Figure 7 .
Figure 7.The regulatory network for tomato trichome development.Several important transcription factors and hormones participate in tomato trichome growth.