An overview of the gene regulatory network controlling trichome development in the model plant, Arabidopsis

1Trichomes are epidermal protuberances that, depending on species, are located on the aerial parts of plants such as the leaves, stems, petioles, petals, and seed coat. They are generally classified into two types: simple, or non-glandular, and glandular secreting trichomes (GSTs; ). Trichomes play an important role in plant growth and development by protecting them from UV light, insect predation, and excess transpiration. The phytochemicals secreted by GSTs provide protection against pathogens and pests, and also attract pollinators. GSTs can also be considered “chemical factories” as they synthesize and secrete many economically important compounds. The unicellular non-glandular trichomes of Arabidopsis serve as an excellent experimental system to study molecular mechanism of cell differentiation and pattern formation in plants.

2The production and distribution of trichomes is spatially and temporally controlled. During the early vegetative phase, trichomes are present only on the adaxial side of the rosette leaves, whereas they are found on both adaxial and abaxial surfaces in the adult vegetative phase. During the reproductive stage, the trichome number gradually decreases on the main inflorescence stem and flowers. Trichomes originate from the protodermal cells of the developing leaf primordia. The protodermal cells destined to become trichomes cease to divide and enter an endoreduplication cycle in which DNA replication continues in the absence of nuclear and cellular division. The mature trichome has a stalk with two to three branches and an average DNA content of 32C, suggesting that trichome cells undergo at least four rounds of endoreduplication during development. Over 30 different genes are known to control the developmental processes. Extensive genetic and molecular analyses suggest that a network of transcription factors (TFs), belonging to three major groups: the R2R3 MYBs, the basic helix-loop-helix (bHLH) factors, and the WD40 repeat (WDR) protein, plays a crucial role in trichome development. These three groups of TFs form a trimeric activator complex, MYB-bHLH-WDR (MBW) that positively regulates the expression of downstream targets, which, in turn, induces trichome formation. The single repeat R3 MYBs act as negative regulators of trichome development. They compete with the R2R3 MYB to bind the bHLH factors and form a repressor complex. Additionally, a group of C2H2 zinc finger TFs has been implicated in trichome formation on inflorescence stems and flowers in Arabidopsis.

3Phytohormones, including gibberellins (GA), cytokinins(CK), and jasmonic acids (JA), are involved in numerous developmental processes in plants. These phytohormones also play a crucial role in trichome development in Arabidopsis. In Arabidopsis, microRNAs have been shown to be involved in trichome development by controlling the expression of key regulatory genes. Recently, a posttranslational control mechanism has been implicated in trichome development. Here, we focus on transcriptional regulatory network involved in the development of Arabidopsis trichomes. Furthermore, we discuss the influence of different phytohormones and their interactions on gene expression affecting trichome formation. The role of miRNA and 26S/ubiquitin proteasome system (UPS) in trichome development is also briefly discussed.

4In Arabidopsis, trichome development is regulated by a transcriptional network involving several groups of TFs, namely, the MYB, bHLH, WDR, and C2H2 zinc finger proteins. The R2R3 MYB family in Arabidopsis is comprised of 126 members. Based on the conservation of the DNA binding MYB domain and the amino acid motifs in C-terminal domain, the R2R3 MYBs are divided into 25 sub-groups. The R2R3 MYBs belonging to sub-group 15, MYB0/GLABROUS1 (GL1) and AtMYB23/MYB23, are involved in trichome development. These two proteins are functionally equivalent during trichome initiation but not during trichome branching. Recently, a newly characterized R2R3MYB, AtMYB82, that does not belong to sub-group 15, has also been shown to regulate trichome development. Expression of AtMYB82 under the control of the GL1 promoter complements the trichome defect of the gl1 mutant, suggesting that both GL1 and AtMYB2 are functionally equivalent.

5The bHLH TF family is one of the largest known groups of TFs in Arabidopsis with more than 160 members divided into 12 subgroups. GLABROUS3 (GL3) and ENHANCER OF GLABROUS3 (EGL3), members of subgroup IIIf, are involved in trichome development in a partially redundant manner. Mutation in the GL3 locus results in fewer trichomes and reduced branching. The nuclei in gl3-1 mutants undergo three, rather than four, rounds of endoreduplication, and this correlates with reduced trichome branching observed in this mutant. The EGL3 locus has a moderate effect on trichome number. However, gl3 egl3 double mutants have a glabrous phenotype. In addition to GL3 and EGL3, TRANSPARENT TESTA 8 (TT8) and AtMYC1, other members of the bHLH subgroup IIIf, have also been shown to affect trichome development. TT8 controls anthocyanin and proanthocyanidin (PA) biosynthesis in vegetative tissues and the seed coat. have demonstrated that TT8 also controls trichome development on leaf margins in Arabidopsis. AtMYC1 mutants have less trichomes, compared with the wild type, indicating AtMYC1 acts as a positive regulator of trichome initiation.

6WD40 repeat proteins contain highly conserved 40–43 amino acid tandem repeats usually ending with Trp-Asp (WD). They are involved in the regulation of a number of processes, including cell cycle, cell fate determination, and cell signaling. In Arabidopsis, the WDR gene, TRANSPARENT TESTA GLABRA 1 (TTG1), is a single copy gene. The ttg1 mutant has pleiotropic phenotype, which is glabrous and deficient in anthocyanin accumulation.

7In addition to the R2R3 MYBs, a group of seven R3 MYBs, that include TRIPTYCHON (TRY;; ), CAPRICE (CPC; ), ENHANCER OF TRY, and CPC 1 (ETC1, ETC2 and ETC3;,; ), and TRICHOMELESS 1 (TCL1 and TCL2;; ), are also involved in trichome development. Analyses of loss-of-function mutants reveal that these R3 MYBs act as negative regulators. Loss-of-function mutation in CPC causes increased trichome density whereas mutation in TRY results in a trichome clustering phenotype. Mutation in ETC1 does not dramatically affect the trichome phenotype whereas mutation in ETC2 or ETC3 results in increased trichome numbers. The higher order ETC mutants (etc1 etc3 and etc1 etc2 etc3) exhibit significantly higher numbers of trichome compared to the respective single or double mutants, suggesting a redundant function by these regulators in trichome development. Loss-of-function mutations in the TCL1 or TCL2 locus result in ectopic trichome formation on inflorescence stems and pedicels. The number of trichomes on inflorescence stems and pedicels increase significantly in the cpc tcl1 double mutant. The higher order cpc etc1 etc3 tcl1 quadruple mutant, exhibits more trichomes on internodes and pedicels, compared to the tcl1 or cpc tcl1 double mutant, suggesting a role of CPC, ETC1 and ETC3 in trichome formation on inflorescence stems and pedicels.

8The MYB (GL1)-bHLH (GL3/EGL3)-WDR (TTG1) proteins form a trimeric MBW complex that activates the expression of the homeodomain protein, GLABROUS2 (GL2; ), which, in turn, induces trichome formation. GL3 contains three different protein-protein interaction domains: the N-terminal MYB-interacting region (amino acid 1-97) that interacts with GL1/CPC/TRY, the middle portion that includes the transactivation domain (amino acid 212-401) interacting with TTG1, and the C-terminal bHLH and ACT-like domain (amino acid 400-637) that homo/heterodimerize. Recent studies demonstrate that the C-terminal domain of GL3/EGL3 also interacts with a number of factors involved in phytohormone signaling and protein degradation. The MYB TFs contain a conserved amino acid signature motif, [D/E]Lx2[R/K]x3Lx6Lx3R, that is crucial for interaction with the bHLH proteins. Physical interaction between GL1 and TTG1 has not been demonstrated. These findings suggest that the bHLH factors act as docking sites for a number of regulatory proteins, including the MYB and WDR proteins in the MBW complex. AtMYC1, like GL3/EGL3, has also been shown to interact with GL1 and TTG1, but does not dimerize. An arginine (Arg173) residue in AtMYC1 is found to be critical for its interaction with GL1. This Arg residue is conserved in GL3 and EGL3, and is essential for interaction with MYB partners. The R3 MYBs typically lack the activation domain and compete with the R2R3 MYB, GL1, to bind GL3/EGL3, and form a repressor complex, thereby affecting the expression of downstream targets. There is a marked difference in the ability of these single repeat MYBs to disrupt the GL1-GL3 interaction. Using yeast three hybrid assay, CPC has been demonstrated as the most potent inhibitor of this activation complex, followed by ETC1, TRY, ETC2, and ETC3. In a protoplast assay, TCL1 has been shown to be stronger than CPC in binding to GL3. Using YFP-tagged CPC and ETC3, it has been demonstrated that these R3MYBs move from cell-to-cell and the strong binding of CPC to GL3 affects the mobility of CPC. Although the cell-to-cell movement signature motif (WxM) is conserved in all R3MYBs, the movement of TCL1 has yet to be experimentally demonstrated. Whether the strong interaction of TCL1 with GL3 affects its movement and biological activity still remains to be elucidated.

9TRICHOMELESS 1 binds the GL1 promoter in a chromatin immunoprecipitation (ChIP) assay and negatively regulates GL1 expression. TCL1 probably acts as a negative regulator of trichome development by affecting the expression of GL1, as well as competing with GL1 for binding to GL3. Although the expression of most single repeat MYBs appears to be regulated by the MBW complex, it is unclear whether MBW also controls TCL1.

10The C2H2 zinc finger proteins constitute one of the largest families of regulatory proteins, with 176 members in Arabidopsis, and are involved in numerous developmental and physiological processes in plants. GLABROUS INFLORESCENCE STEMS (GIS), GIS2, ZINC FINGER PROTEIN 8 (ZFP8;, ), ZFP5, and ZFP6, which encode C2H2 zinc finger TFs, are involved in trichome formation in inflorescence stems and floral organs. Gene expression analyses of knockout mutants reveal a transcriptional hierarchy with ZFP6 acting upstream of ZFP5 which regulates the expression of GIS, GIS2, and ZFP8. GL1 and GL3, key TFs in the MBW complex, function further downstream of GIS2 and ZFP8.

11Collectively, these findings suggest that a regulatory loop involving a group of activators and repressors fine tunes the expression of downstream gene targets and ultimately trichome formation. The activator complex (GL3/EGL3-GL1-TTG1) induces the expression of genes encoding the repressors (TRY/CPC) which can move into neighboring cells to form a repressor complex (GL3/EGL3-CPC/TRY-TTG1) and inhibit function of the activators. In addition to an activator–repressor mechanism, a depletion mechanism has also been proposed to operate, in parallel, during trichome development. In the depletion process, GL3 traps TTG1, resulting in depletion of TTG1 protein in neighboring cells. Consequently, cells with high levels of GL3 and TTG1 proteins are developed into trichomes. The depletion model is derived from the following findings: (a) TTG1 protein moves between cells, (b) TTG1 protein is preferentially accumulated in trichome initials and depleted in surrounding cells, and (c) depletion of TTG1 protein in neighboring cells, and its accumulation in trichome initials, is lost in the gl3 mutant. Supporting this model, show that GL3 controls TTG1 movement, and interaction between GL3 and TTG1 is necessary for intracellular movement and epidermal distribution.

12Phytohormones, including GA, JA, and CK, play pivotal roles in controlling a wide array of biological processes in plants. Accumulating evidences suggest that these phytohormones are also crucial in trichome development. Here, we discuss the influence of different phytohormones and underlying molecular mechanisms, which control trichome formation in Arabidopsis.

13Gibberellin is known to regulate a number of developmental processes in plants including seed germination, hypocotyl elongation, flowering, and trichome development. Evidence for the involvement of GA in trichome development comes from the analyses of several mutants in GA biosynthesis and signaling pathways in Arabidopsis. The GA biosynthesis mutant ga1-3 has completely glabrous leaves, and application of exogenous GA to these plants restores trichome development. Additionally, GA stimulates the expression of GL1, and relative to wild type plants, the ga1-3 mutant contains less GL1 transcripts. SPINDLY (SPY) is a repressor of GA signaling in Arabidopsis. Mutation in the SPY locus results in increased trichome formation. The Arabidopsis DELLA proteins are inhibitors of GA signaling and encoded by a family of five genes: GIBBERELLIC ACID INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA), and three RGA-LIKE genes (RGL1, RGL2, and RGL3). Among the five DELLA proteins, RGA, and GAI play significant roles in trichome formation. Mutations in RGA and GAI restore trichome initiation in the ga1-3 mutant. Consistent with this, the expressions of several trichome regulators, including GL1 and GL3, are up-regulated in DELLA-defective ga1-3 mutants, whereas conditional over-expression of RGA-GR (RGA fused to rat glucocorticoid receptor) in these mutants reduces the expression of these trichome regulators. have demonstrated that GA stimulates trichome formation through up-regulation of key TF genes. Expression of GL1, MYB23, GL3 and EGL3 are induced, whereas expression of TRY, ETC1, and ETC2 are reduced, in response to GA treatment. TTG1 expression is not significantly affected following GA treatment. Taken together, these findings suggest that GA regulates trichome formation by modulating the expression of key regulatory genes. These conclusions are further substantiated by the elegant demonstrations that individual TFs in the MBW complex are direct targets of DELLA proteins. RGA or RGL2 physically interact with GL3, EGL3, and GL1 to repress the transcriptional function of the MBW complex. Furthermore, analysis of the DELLA mutant, penta (gai-t6 rga-t2 rgl1-1 rgl2-1 wild type for RGL3), gl3 egl3 double mutant, and penta gl3 egl3 mutants, suggest that the MBW complex acts downstream of DELLA proteins. Based on these findings, it is proposed that upon perception of the GA signal, protein–protein interactions between DELLA-GL1/GL3/EGL3 are disrupted as DELLA proteins are recruited to the SCFSLY complex, and subsequently degraded by the 26S proteasome system. GL3, EGL3, and GL1 are consequently released to form a complex with TTG1 and activate the expression of GL2, which, in turn, mediates trichome formation.

14Recent studies suggest that TFs other than MYB and bHLH proteins, also operate in the GA signaling pathway to regulate trichome development in inflorescence stems and flowers. The expression of C2H2 zinc finger TF genes, GIS, GIS2, ZFP8, ZFP5, and ZFP6, is stimulated in response to exogenous application of GA.

15Jasmonic acid and its derivatives, collectively known as jasmonates (JAs), act as key signaling molecules that regulate numerous developmental processes in plants. JA biosynthesis is triggered in response to a variety of signals including wounding. have shown that mechanical wounding and JA significantly induce trichome development in plants. The Arabidopsis aos mutant, deficient in JA biosynthesis due to a knock-out mutation in the ALLENE OXIDE SYNTHASE (AOS) gene, produces less trichomes compared to wild type plants and this defect can be rescued by JA treatment. have also demonstrated that JA stimulates trichome development by modulating the expression of several regulatory genes. In Arabidopsis, the F-box protein CORONATINE INSENTIVE1 (COI1), along with ASK1/ASK2, Cullin1, and Rbx1, form the SCFCOI complex that mediates JA signal transduction. JA induction of trichome formation is attenuated in the coi1-2 mutant, which is defective in JA signaling and produces less trichomes compared with wild type plants. Collectively, these findings show that JA plays an important role in trichome formation. Recent findings by elucidate the molecular mechanism underlying this process. They demonstrate that the MBW complex is involved in JA-induced trichome development in Arabidopsis. The expression of GL3 and GL1 is significantly induced in response to JA treatment in wild type plant, but severely weakened in the coi1-2 mutant. Moreover, GL3 and GL1 physically interact with the JA-ZIM domain (JAZ) proteins in yeast, as well as plant cells. The JAZ proteins are known negative regulators of JA signaling. They are recruited to the SCFCOI complex upon perception of the JA signal and are subsequently degraded by the 26S proteasome system. These observations suggest that, in the absence of JA, the JAZ proteins bind to GL3, EGL3, and GL1 and inhibit the formation of the MBW complex. Upon perception of the JA signal, the JAZ proteins are degraded by the 26S proteasome system, thereby releasing GL3, EGL3, and GL1 to form the complex with TTG1, and activate the downstream targets to promote trichome formation.

16Cytokinin (6-benzylaminopurine, BAP) acts as a positive regulator of trichome development in Arabidopsis. Plants treated with BAP produce more trichomes per leaf; however, the trichomes are shorter and nuclear DNA content is less than in untreated plants, indicating that BAP affects endoreduplication in trichomes. The expression of GL1, MYB23, GL3, and EGL3, is also stimulated following BAP treatment. The expression of GIS2, ZFP5, ZFP8, and ZFP6, that regulate trichome formation on inflorescence stems, is also influenced by cytokinins.

17Trichome formation is also affected by brassinosteroids (BR), ethylene (ET), and salicylic acid (SA). The Arabidopsis bls1 mutant, impaired in BR response, develops fewer trichomes on both abaxial and adaxial surfaces of the leaf, indicating a possible involvement of BR in trichome development. The ET receptor mutant etr2-3, has completely unbranched trichomes. Through epistatic and gene expression analysis, it has been shown that the ET signaling cascade involves CHROMATIN ASSEMBLY FACTOR 1 (CAF1) and TRY to control trichome branching, and is independent of the GL3, GL2 pathway. Application of SA significantly reduced the number and density of trichomes in different cultivars of Arabidopsis, indicating its negative regulatory role in trichome development.

18Phytohormones are known to act synergistically as well as antagonistically to regulate different developmental processes in plants. The antagonistic action of GA and JA regulate hypocotyl elongation, root growth, and flowering, whereas their synergistic action regulates stamen development and trichome formation. Trichome density and number are significantly higher in plants treated with a combination of GA and JA compared with plants treated with only JA. Consistent with this, the expression of GL2 and MYB23 are found to be significantly up-regulated by combined treatment of GA and JA. Moreover, the GA biosynthetic inhibitor, paclobutrazol, represses JA-induced trichome formation and expression of GL2 and MYB23. JA-induced trichome formation is also attenuated in the GA biosynthesis mutant ga1-3. Similarly, the coi1-1 mutant, defective in JA signaling, inhibits GA-induced trichome formation. Recent studies by reveal the molecular mechanism underlying the GA-JA synergy in trichome development. Both JAZ and RGA (DELLA protein) bind to trichome regulators, GL3, EGL3, and GL1. GA and/or JA signals control the level of these repressor proteins via 26S proteasome-dependent proteolysis and maintain the stable transcription of the activators that induce trichome formation.

19The positive regulatory role of GA and CK on trichome development is well documented. A recently identified C2H2 zinc finger TF, ZFP6, seems to function as an integrative hub of GA and CK signals in promoting trichome formation in Arabidopsis. ZFP6 expression is induced in response to GA and CK treatment. Moreover, GA- and CK-induced expression of the downstream targets of ZFP6, ZFP5, ZFP8, and GL1, is significantly affected in zfp6 mutant.

20The negative cross-talk between JA and SA-dependent pathways in Arabidopsis is well documented. These phytohormones act antagonistically to regulate trichome development. Arabidopsis plants treated with a combination of JA and SA produce lower numbers of trichomes compared with plants treated with JA alone.

21microRNAs (miRNAs) are small endogenous non-coding RNAs of 20–22 nt in length and present in plants, animals, and protozoa. miRNAs modulate the expression of their target genes at the posttranscriptional level and thus control many aspects of cellular functions. Recent studies indicate that miRNAs regulate trichome development by modulating the expression of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes. SPLs are a group of plant-specific TFs that share a highly conserved SBP DNA-binding domain first identified in a protein that binds to the promoter of the SQUAMOSA gene of Antirrhinum majus. SPLs regulate numerous fundamental aspects of plant growth and development, including phase transition, trichome distribution and flowering. In Arabidopsis, SPLs are negative regulators of trichome development in inflorescence stem and floral organs. The SPL gene family has 17 members, 10 of which are targeted by miRNA156. Expression of miRNA156 and SPLs (SPL3/SPL9) are temporally regulated. miRNA156 levels are highest in young plants and decline as the plant ages. On the contrary, expression of SPLs, targets of miRNA156, is low in young plants and increase gradually during the reproductive stage. The expression pattern fits well with the gradual loss of trichomes on stem and floral organs. Consistent with this, plants expressing a mimicry target of miR156 accumulate significant SPL transcripts and show a reduction in trichome density on stems. Similar results are found when miR156-resistant forms of SPL3, SPL10, SPL13, and SPL9 are over-expressed in plants. By comparison, transgenic Arabidopsis plants constitutively expressing miRNA156 produce ectopic trichomes on stem and floral organs. Moreover, SPL9 directly binds to the promoters of TCL1 and TRY, negative regulators of trichome development, and activates their expression in a GL1-independent manner, leading to reduced trichome formation. Together, these observations suggest that the temporal control of trichome development in Arabidopsis is regulated by the miR156-targeted SPL TFs.

22The 26S proteasome is a multi-subunit ATP-dependent protease complex assembled from two particles: the 20S core particle (CP) and the 19S regulatory particle (RP; ). The UPS-dependent proteolysis is the most elaborate and complex regulatory mechanism controlling activities of short-lived proteins in eukaryotes. Over 1300 genes in the Arabidopsis genome are associated with the 26S proteasome pathway. Loss-of-function mutations in one of the RP components, RPT2a, result in several physiological abnormalities including aberrant trichome development. The rpt2a mutant has larger trichomes with increased branch number. Additionally, trichomes of rpt2a plants have larger nuclei compared with the wild type, suggesting RPT2a is involved in regulation of endoreduplication in trichomes.

2326S/ubiquitin proteasome system comprises E1, E2, and E3 enzymes that act coordinately to conjugate ubiquitin moieties to the target proteins and pave the way for subsequent degradation by the 26S proteasome. E3 enzymes determine substrate specificity by recognizing a single or small group of proteins and in plants, are divided into two subgroups, RING/U boxes and HECT ubiquitin ligases. In Arabidopsis, mutation in the UPL3/KAK locus, which encodes a HECT domain E3 ligase, results in trichomes with increased branch number and higher nuclear DNA content, suggesting that UPL3 regulates of ploidy level in trichomes by controlling the activities of proteins that normally promote the endoreduplication cycle. GL3 is a potential target of UPL3 because it is a positive regulator of endoreduplication in trichomes, and the supernumerary trichomes in upl3/kak seedlings is suppressed in kak gl3 double mutant background. Our recent demonstration, that GL3 and EGL3 are short-lived proteins, supports the hypothesis that GL3/EGL3 are targets of UPL3. UPL3 physically interacts with GL3 and EGL3, and mediates degradation via UPS. Additionally, we have also shown that TTG1 and TT8, the regulator of trichome formation on leaf margin, are targets of UPS. However, the specific E3 ligase that mediates this degradation remains to be identified. Endoreduplication cycles in Arabidopsis trichomes are also controlled by another class of ubiquitin ligases, the RBX1-containing Cullin-RING E3 ubiquitin ligases (CRLs). Of five known cullin genes, CUL1, CUL3A, and CUL4 are strongly expressed in young trichomes. CUL1 and CUL3A loss-of-function mutants are phenotypically indistinguishable from wild type. However, a knock-down line of CUL4 produces small trichomes with less nuclear DNA content. The CUL4-CRL complex modulates cyclin-dependent kinase (CDK) activity presumably by mediating the degradation of a class of CDK inhibitors during endoreduplication cycles.

24During the past decade, substantial and significant progress has been made in delineating the elaborate gene regulatory network that controls trichome development in Arabidopsis. Multiple lines of evidence suggest that a number of transcriptional activators and repressors act in concert to fine tune the spatial and temporal distribution of trichomes. Additionally, phytohormones such as JA, GA, and CK, act synergistically or antagonistically to modulate the expression of genes encoding these regulators. Recent findings, pertaining to miRNAs and 26S/UPS-dependent regulation of TFs in trichome development, highlight the complexity of the regulatory network. Whether other regulators such as GL1/MYB23 and R3 MYBs are targets of miRNA and/or 26S/UPS remains to be elucidated.

25Our current knowledge about the gene regulatory network is largely limited to the unicellular non-glandular trichomes in the model plant, Arabidopsis. Very little is known about the regulatory network that controls the development of glandular secretory trichomes. This type of trichomes is found in many plants, including tobacco, tomato, basil, and mint, and is thus an important source of phytochemicals. Over-expression of GL1 in tobacco does not augment trichome formation, suggesting that different regulatory mechanisms control trichome development in Arabidopsis and tobacco. Recently, an integrated genomic database, TrichOME (www.planttrichome.org), has been developed. The database hosts more than a million EST sequences from both trichome and corresponding non-trichome tissues from 13 species, including tobacco, basil, and mint, and provides a potential source for genes involved in development of glandular and non-glandular trichomes. Understanding the regulatory network that controls the development of multicellular trichomes will aid our efforts to engineer trichomes to produce commercially important phytochemicals.

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.