trichomes: different regulatory networks lead to convergent structures

Trichomes: different regulatorynetworks lead to convergent structuresLaura Serna1 and Cathie Martin2

1Environmental Sciences Faculty, University of Castilla-La Mancha, 45071 Toledo, Spain2Department of Cell and Developmental Biology, John Innes Centre, Norwich, NR4 7UH, UK

Sometimes, proteins, biological structures or even

organisms have similar functions and appearances but

have evolved throughwidely divergent pathways. There

is experimental evidence to suggest that different

developmental pathways have converged to produce

similar outgrowths of the aerial plant epidermis,

referred to as trichomes. The emerging picture suggests

that trichomes in Arabidopsis thaliana and, perhaps, in

cotton develop through a transcriptional regulatory

network that differs from those regulating trichome

formation in Antirrhinum and Solanaceous species.

Several lines of evidence suggest that the duplication

of a gene controlling anthocyanin production and

subsequent divergence might be the major force driving

trichome formation in Arabidopsis, whereas the multi-

cellular trichomes of Antirrhinum and Solanaceous

species appear to have a different regulatory origin.

Trichome evolution

Trichomes are unicellular or multicellular appendagesthat originate from cells of the aerial epidermis [1]. Theyvary considerably in morphology, location, ability tosecrete and mode of secretion, and different types oftrichome can be produced by the same plant. Trichomeshave a range of functions: non-glandular trichomesfunction to reduce the heat load of plants, increasetolerance to freezing, seed dispersal, water absorptionand protection of plant tissues from UV light and bioticfactors such as insect herbivores [1–3], whereas glandulartrichomes offer chemical protection against herbivoresand pathogens, are associated with attracting animals oraccumulate salt [1]. Some types of non-glandular tri-chome, such as those called fibres from the seeds of cotton(Gossypium spp.), consist of extremely elongated singlecells, which are important in the textile industry [4].

Convergent evolution involves the emergence of bio-logical structures that show similar function and/orappearance but that evolved via different evolutionarypathways. The similarities that are shared are not theresult of derivation from a common ancestor but aretypically explained as the result of common adaptivesolutions to similar environmental pressures. Examples ofconvergent evolution include the evolution of functionallysimilar but distinct antifreeze proteins in divergentspecies of fish [5], or the multiple origins of compound

Corresponding author: Serna, L. ([email protected]).Available online 11 May 2006

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eyes of arthropods [6]. Are trichomes the result ofconvergent evolution? If so, when and how did lineagesradiate? Our understanding of how trichome formation iscontrolled is now becoming detailed enough to addressthese questions.

Trichomes in Arabidopsis and cotton

In Arabidopsis, trichomes are unicellular, non-glandular,and usually have three branches. Trichome production inthis model plant has been elucidated through thecharacterization of numerous genes. Mutations in theGLABROUS1 (GL1) gene result in plants with no or fewtrichomes, showing that GL1 promotes trichome forma-tion [7]. GL1 encodes an R2R3 MYB transcriptionalregulator with two MYB repeats that comprise its DNA-binding domain [8].

Some MYB proteins regulate transcription as part ofmulti-protein complexes. Four solvent-exposed amino acidresidues (Lx2Rx2[R/K]L) in the first helix of the R3 MYBrepeat of R2R3 MYB protein COLOURLESS1 (C1) providea surface for interaction with the basic-helix–loop–helix(bHLH) RED (R) protein in maize [9]. Such residues areboth necessary and sufficient to confer the ability tointeract with R and to induce transcription from specificpromoters [9]. Both C1 and R control anthocyaninproduction in maize [10]. More recently, the identificationof a related amino acid signature in a subset ofArabidopsis MYB proteins ([D/E]Lx2[R/K]x3Lx6Lx3R)that interacts with R-like bHLH proteins [11] (Figure 1)indicates that such an interaction domain is conservedamong higher plant species.

The six residues of the extended interaction domain arepresent in the GL1 [11] (Figure 1) and the AtMYB23proteins [11] (Figure 1). AtMYB23 is also an R2R3 MYBtranscriptional regulator that has partially overlappingfunctions with GL1 [12,13]. In yeast, AtMYB23 interactswith the bHLH protein ENHANCER OF GLABRA3(EGL3) [11], and the MYB domain of GL1 physicallyinteracts with the N terminal-domain of the bHLHproteins GLABRA3 (GL3) and EGL3 [14,15]. ThesebHLH proteins promote trichome cell fate determinationin a redundant fashion [15].

The TRANSPARENT TESTA GLABRA1 (TTG1) gene,which encodes a protein containing four conserved WDrepeats [16], is also required for trichome formation inArabidopsis [17]. TTG1 interacts with both GL3 andEGL3, but through a different domain in the bHLH

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AtCPC EEEEDLISRMYK LVGDRWELIAG RIPGRTPEEIERYWLMKH AtECT1 QEEEDLICRMYK LVGERWDLIAG RIPGRTAEEIERFWVMKN AtTRY EQEEDLIFRMYR LVGDRWDLIAG RVPGRQPEEIERYWIMRN AtGL1 EQEEDLIIRLHK LLGNRWSLIAK RVPGRTDNQVKNYWNTHL AtMYB23 DQEEDLIIRLHK LLGNRWSLIAK RVPGRTDNQVKNYWNTHL GaMYB2 DEEEDLIIRLHK LLGNRWSLIAG RLPGRTDNEIKNYWNSHL GhMYB109 EEEEDLVIRLHK LLGNRWSLIAK RVPGRTDNQVKNYWNSHL

AmMIXTA LQEEQTIIQLHA LLGNRWSAIAS HLPKRTDNEIKNYWNTHL AmMYBML1 LQEEQAIIQLHA FLGNRWSAIAT HLPKRTDNEIKNYWNTHL PhMYB1 LQEEQTIIQLHA LLGNRWSAIAT HLPKRTDNEIKNYWNTHL

AtMYB16 LQEEQTIIQLHA LLGNRWSAIAT HLPKRTDNEIKNYWNTHLAtMYB106 VQEEQTIIQLHA LLGNRWSAIAT HLPKRTDNEIKNYWNTHL AtMYB17 ADEEKLVIQLHA ILGNRWAAIAA QLPGRTDNEIKNLWNTHL

Physcomitrella patensPpMYB1 HEEDQMIVHLHA ILGNRWSAIAS HLPRRTDNEIKNYWNTHL

AtTT2 SDEEELIIRLHN LLGNRWSLIAG RLPGRTDNEIKNHWNSNL AtPAP1 SDEVDLLLRLHR LLGNRWSLIAG RLPGRTANDVKNYWNTHL AtPAP2 NDEVDLLLRLHK LLGNRWSLIAG RLPGRTANDVKNYWNTHL PhAN2 LDEVDLILRLHK LLGNRWSLIAG RLPGRTANDVKNYWNTHL PfMYBP1 VDEEELMIKLHA LLGNRWSLIAG RLPERTDNEVKNYWNSHM GhMYB10 EDEIDLIIRLHK LLGNRWSLIAG RIPGRTANDVKNWWNTHL ZmC1 YDEEDLIIRLHR LLGNRWSLIAG RLPGRTDNEIKNYWNSTL

Arabidopsis

Asterids

Rosids

Rosids, Asterids and maize

Trichome

MIXTA-like proteins Unknown functions

Shaping and growth of protonemal or chloronemal cells?

Anthocyanin

Figure 1. Sequence comparisons between the R3 domains ofMYB proteins regulating trichome initiation and anthocyanin production in different plant species. In Rosids, the

six solvent-exposed amino acid residues in the MYB R3 region that specify the interaction with the R-like bHLH proteins ([D/E]Lx2[R/K]x3Lx6Lx3R) are conserved in all the

proteins regulating trichome initiation (GL1, AtMYB23, CPC, ETC, TRY, GaMYB2, GhMYB109). In the AmMIXTA, AmMYBML1 and PhMYB1 proteins, which control trichome

initiation in Asterids, the amino acid signature is not conserved. Similarly, theMIXTA-like proteins ofArabidopsis do not contain the amino acid signature for interactionwith

the R-like bHLH proteins. All the MYB anthocyanin regulators from all plants characterized to date contain this amino acid signature. The sequence identifiers consist of

initials for genus and species, followed by their common names. Proteins were aligned using the ClustalW software.

Opinion TRENDS in Plant Science Vol.11 No.6 June 2006 275

proteins to that interacting with the MYB proteins[11,14,15]. Together, these data support the view that amultimeric complex involving GL1, GL3, EGL3 and TTG1triggers trichome cell fate specification in Arabidopsis(Figure 2).

Four single-repeat MYB proteins, CAPRICE (CPC),TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1(ETC1) and ETC2, counter the activity of the GL1 protein.They repress trichome determination in a redundantmanner [18–23]. CPC and TRY interact with the Nterminal-domain of the bHLH proteins GL3 and EGL3in yeast [15], and ETC1 interacts with the EGL3 protein[11]. A short region in CPC, TRY and ETC1 contains themotif for interaction with the R-like bHLH proteins, and issufficient to confer interaction with EGL3 in yeast [11](Figure 1). From their amino acid sequences, the single-repeat MYB proteins are not predicted to have DNAbinding activity. Gel mobility shift experiments and yeastone-hybrid assays have shown that CPC does not bind toDNA [24]. It is thought that TRY, CPC and ETC1 prevent(or reduce) the formation of the multimeric complex ofGL1, GL3, EGL3 and TTG1 by competing with GL1 forbHLH binding in the pavement epidermal cells thatneighbour trichomes [21,22,25,26] (Figure 2). Supporting

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this model, three-hybrid analysis in yeast has shown thatTRY physically interacts with GL3 and that thisinteraction prevents the physical interaction betweenGL3 and GL1 [27].

The homeodomain-leucine zipper protein GLABROUS2(GL2) might be the down-stream target of theGL1–GL3–EGL3–TTG1 complex [25,28,29] (Figure 2). A500-bp fragment of the GL2 promoter that contains aputative MYB binding site is required for GL2 expressionin the leaf epidermis [30]. Therefore, it is possible thatGL1, as part of the multi-protein complex, binds andactivates the GL2 promoter. The single-repeat MYBproteins probably repress GL2 expression by competingwith GL1 to bind with bHLH proteins.

In cotton (Gossypium arboretum), fibre cell determina-tion also seems to be controlled by an R2R3 MYB gene,GaMYB2 [31]. This gene is highly expressed in the earlystages of fibre cell development, and its expression inArabidopsis, under the control of the GL1 promoter,rescues trichome formation in a gl1 mutant [31]. Theoverexpression of this gene in Arabidopsis under thecontrol of the Cauliflower Mosaic Virus 35S (35S)promoter induces the formation of seed-trichomes [31].GaMYB2 contains the conserved amino acid signature for


R2R3 MYB: GL1

Anthocyaninproduction

Anthocyaninproduction

R2R3 MYBs: PAP1, PAP2

Hypothetical single-repeat MYB

TTG1

bHLHs: GL3, EGL3

GL2 promoter Single-repeat MYBs:CPC, TRY, ETC1

Promoter of pigmentation genes synthesis

(a) (b)

Figure 2. Working model for trichome and anthocyanin pattern specification. (a) In Arabidopsis, a multimeric complex comprised of TTG1 (WD40), GL3 and/or EGL3 (bHLH)

and GL1 (R2R3-MYB) triggers trichome cell fate specification by inducingGL2 expression. CPC, TRY and ETC1 (single-repeatMYBs) compete with the R2R3-MYB regulator for

bHLH binding, producing an inactive complex (WD40–bHLH–single-repeat MYB) that inhibits trichome initiation by repressing GL2 expression. These complexes might not

operate to control trichome formation outside the Rosid division. (Modified from Figure 1 in Ref. [29].) (b) In a wide range of species, anthocyanin production is positively

regulated by the WD40–bHLH–R2R3 MYB complex by inducing the expression of biosynthetic genes. PAP1 and PAP2 are R2R3 MYB proteins controlling anthocyanin

production inArabidopsis through their interactionwith TTG1 and EGL3 and/or GL3. In Petunia hybrida, a single-MYB repeat protein, MYBx, has been suggested to sequester

the bHLH protein AN1 into inactive complexes, repressing pigment biosynthesis presumably by a mechanism similar to the action of CPC, TRY and ETC1 in trichome

regulation in Arabidopsis [55].


interaction with bHLH proteins (Figure 1), suggestingthat it regulates fibre cell fate determination through itsphysical interaction with these proteins. In upland cotton(Gossypium hirsutum), an R2R3 MYB gene, GhMYB109,is also expressed in developing fibre cells [32]. GhMYB109has a motif for interaction with the R-like bHLH proteins(Figure 1). In G. hirsutum, two genes encoding WD-repeatproteins have been found that restore trichome formationin the ttg1 mutant of Arabidopsis [33]. So, it seemspossible that similar multimeric complexes between MYB,bHLH and WD repeat proteins trigger trichome formationin Arabidopsis and cotton. These species belong to theRosids, one of the main divisions of the dicots. Although nobHLH protein has yet been identified as being involved infibre formation, it is possible that homologues of GL3 orEGL3 control such processes in cotton and perhaps inother species of the Rosid division.

Trichomes in Antirrhinum and solanaceous species

The role of MYB transcriptional regulators in trichomeformation extends beyond Arabidopsis and cotton. TheR2R3 MYB-related transcriptional factor MIXTAregulates the formation of conical shape in petalepidermal cells of snapdragon (Antirrhinum majus)[34–36]. Because the differentiation of conical cells isreminiscent of the early steps of trichome initiation, andconical cells are referred to as ‘trichomes’ by some plantanatomists [37], it is possible that MIXTA-like proteinscontrol the expression of genes required for the productionof trichome cell type. In support of this idea, over-expression of MIXTA under the control of the 35Spromoter triggers the formation of ectopic multicellulartrichomes (and conical cells) in the leaves of this species,showing that this gene has the potential to controlmulticellular trichome production in Antirrhinum [36].


The ability of MIXTA to trigger multicellular trichomedevelopment is linked to the competence of expressingcells for further rounds of division [35,36].

The possible role of MIXTA-like genes in snapdragontrichome formation probably extends to several otherplant species. The overexpression of MIXTA in Nicotianatabacum induces the formation of multicellular trichomesas well as conical cells on all aerial epidermal surfaces,suggesting that unidentified MIXTA-like proteins controlconical cell and multicellular trichome formation intobacco [35,38]. Supporting evidence for such a role ofMIXTA-like proteins in Solanaceous species includes over-expression of MIXTA in woody nightshade (Solanumdulcamara), which results in the development of ectopictrichomes on the anthers [39]. In Petunia hybrida, conicalcell formation in the petals also requires a MYB-relatedtranscription factor named PhMYB1, which is closelyrelated, structurally, to MIXTA [40,41].

The MYB MIXTA LIKE 1 (AmMYBML1) gene fromA. majus encodes an R2R3 MYB-related transcriptionalregulator that has a DNA-binding domain almost identicalto that of MIXTA [35]. It also promotes trichome andconical cell formation on floral tissues when it is over-expressed under the control of the 35S promoter in tobacco[36,42]. It is expressed in the developing ventral petalwhile epidermal cells are still competent for furtherrounds of cell division, and particularly in the trichomesof the corolla tube, suggesting that it controls both conicalcell and trichome formation in A. majus [35,36,42].

These findings mirror the role of the R2R3 MYB geneGL1 in trichome formation in Arabidopsis suggesting, atfirst glance, that trichomes in the Schrophulariacae andSolanaceae (which belong to the Asterid division) developthrough a similar network of transcription factors to thosein the Rosids. However, overexpression of GL1 in tobacco


TRENDS in Plant Science

ZmC1

ZmP

AtMYB16

AtMYB106

PhMYB1

AmMIXTA

AmMYBML1

AtMYB17

PpMYB1

AtGL1

AtMYB23

GHMYB109

GaMYB2

ZmPl

AtTT2

AtGL1

AtMYB23

AmMIXTA

AmMYBML1

PhMYB1

PpMYB1

AtMYB34

Lamiales (Anthirrinum)

Solanales (tomato, petunia, tobacco)

Brassicales (Arabidopsis)

Malvales (cotton)

Monocots (maize)

Eudicots

Asterids

Rosids

[D/E]Lx2[R/K]x3Lx6Lx3R

(a)

(b)

(c)

Figure 3. Phylogeny of the MYB proteins and plant species discussed in this review.

(a)MYB proteins implicated in trichome formation. The GL1-like R2R3-MYB proteins

cluster in a clade distinct from the MIXTA-like proteins. (b) MYB proteins regulating

trichome formation and anthocyanin production. The grouping of both GL1 and

AtMYB23 in the anthocyanin biosynthesis branch suggests that they arose from the

duplication and subsequent diversification of a gene involved in anthocyanin

production. AtMYB34, which regulates indolic glucosinolate homeostasis [68], was

used as an outgroup. PpMYB1 clusters in the MIXTA-like protein clade, suggesting

that a trichome initiation pathway in Asterids might have originated from a pathway

controlling the growth of Physcomitrella patens protonema. Full-length proteins

were used to calculate the Neighbour-Joining Phylogenetic trees. (c) Simplified

phylogeny of the species used in this work. The arrow indicates the hypothetical

acquisition of GL1-like proteins involved in trichome initiation.


has no effect on trichome formation in this species [38].In addition, the MIXTA-like MYB proteins MIXTA,AmMYBML1 and PhMYB1 do not contain the aminoacid signature required for interaction with the R-likebHLH proteins, which is conserved in the GL1, AtMYB23,GaMYB2 and GhMYB109 proteins (Figure 1), suggestingthat MIXTA-like proteins do not physically interact withthe R-like bBHLH proteins. This is supported byco-expression of MIXTA and the R-like bHLH proteinDELILA in tobacco where the two proteins appear tofunction independently [35].

These different strands of evidence suggest that thetrichomes of Arabidopsis (and perhaps of cotton) and thoseof tobacco and A. majus might be analogous structuresrather than homologous ones, or at least the mechanismsinducing their formation are analogous rather thanhomologous. If so, R-like bHLH proteins play no role inmulticellular trichome (and conical cell) formation inAntirrhinum and Solanaceous species, and this could bea feature of the Asterid division, generally. Supportingthis view, the overexpression of an R-like bHLH factorfrom maize, which confers excess trichomes on leaves andstems of Arabidopsis [43], had no effect on trichomeformation in tobacco [38,43,44], petunia [45,46] or tomato[44]. R is also not associated with trichome formation inmaize. Moreover, in petunia, neither mutations norectopic expression of ANTHOCYANIN11 (AN11) (whichencodes a WD repeat protein) [47], cause any obvioustrichome phenotypes [48]. Because AN11 is a single genein petunia [47], the absence of a trichome phenotype inan11 mutants cannot be because of genetic redundancy.The pale aleurone colour (pac) mutant of maize also has notrichome phenotype [49]. PAC encodes a WD repeatprotein involved in regulating the anthocyanin pathway[49]. Interestingly, 35S::PAC in Arabidopsis complementsall ttg1 phenotypes, which argues against divergence ofthe WD repeat proteins in the different species havingresulted in the loss of their control of trichome cell fatespecification in maize [49].

The view that the induction mechanisms controllingtrichome formation in these different species are analo-gous rather than homologous is supported by thephylogenetic tree of GL1-like MYB proteins and MIXTA-like proteins, which shows that they fall into differentevolutionary lineages (clades) (Figure 3a). One cladeincludes GL1, AtMYB23, GhMYB109 and GaMYB2proteins, all from the Rosids. The other one consists ofthe MIXTA, AmMYBML1 and PhMYB1 proteins and theArabidopsis MIXTA-like proteins (AtMYB16, AtMYB17and AtMYB106). Because neither R-like bHLH nor WDrepeat proteins seem to play a role in trichome formationin maize, it is possible that GL1-like genes arose after theAsterid–Rosid division (Figure 3c). However, more data onthe genetic control of trichome production in other plantspecies, in particular in monocots, are required to confirmthis idea.

Do both networks function in either Arabidopsis or

Antirrhinum and solanaceous species?

As discussed above, neither an R-like gene nor the PAC1gene seems to regulate trichome initiation in maize. This


finding, together with the phylogenetic tree that placesthe GL1 and AtMYB23 proteins of Arabidopsis andGhMYB109 and GaMYB2 sequences of cotton on a



different branch to those of MIXTA, AmMYBML1 andPhMYB1 of Antirrhinum and Solanaceous species,suggest that the mechanism specifying trichome cell fatein Arabidopsis (and perhaps cotton) might have arisenafter the Asterid–Rosid split. However, Arabidopsis doeshave MIXTA-like genes (subgroup 9, Figure 1) [50]. Dothey (AtMYB16, AtMYB17 and AtMYB106) play anyrole in trichome initiation in this species? Although thefunction of these genes has not yet been defined,the ectopic expression of MIXTA had no effect on trichomeformation in Arabidopsis [35,38], which suggests that theMIXTA-like network is not controlling trichome initiationin Arabidopsis. In addition, the overexpression of MIXTAdoes not complement the Arabidopsis gl1-1 null mutation[38], which argues against the possibility that competitionfor common target motifs between GL1 and MIXTA-likeproteins prevents MIXTA-like action in Arabidopsis.

In tobacco, the overexpression of GL1 (or C1) [43] doesnot alter trichome initiation [38]. One interpretation isthat the GL1 network arose only in members of the Rosiddivision. Although a GL1-type network seems to play norole in trichome initiation in tobacco, convergent develop-mental pathways might trigger the initiation of differenttypes of trichome in this species. Tobacco develops fivedistinct types of trichome [51] and genetic data supportthe idea that they develop through distinct developmentalpathways [35,38]. This idea can extend to other plantspecies that develop more than one trichome type such astomato or maize.

Relationship between trichome formation

and anthocyanin production

The relationship between trichome production and antho-cyanin biosynthesis was first suggested by the isolation ofthe Arabidopsis ttg1 mutant, which is glabrous and hasdefective anthocyanin production [17]. The gl3 egl3 doublemutant, like ttg1, also impairs both processes [15].Although the Arabidopsis MYB proteins that controltrichome production (GL1, CPC, TRY and ETC1) seem tobe distinct from the flavonoid and anthocyanin-specificR2R3 MYB regulators [TRANSPARENT TESTA (TT) 2,PAP1 and PAP2], genetic and biochemical evidencesupports the idea that comparable multimeric complexesof WD40, bHLH and MYB proteins regulate bothprocesses (Figure 2). In yeast, both PAP1 and PAP2interact with GL3, EGL3 and the bHLH flavonoidregulator TT8 [11,15]. EGL3 (and probably GL3) andTT8 physically interact with TTG1 [11]. In addition,co-expression of GL3 or EGL3 and PAP1 causes a severephenotype and strong activation of the DFR promoter,which indicates that these regulatory proteins interactin vivo [11,15]. Genetic interactions between TTG1 andPAP1 have also been demonstrated [52]. The combinationof TT8, TTG1 and TT2 has been shown to control bothflavonoids and proanthocyanin biosynthesis in Arabidop-sis seed [53,54]. In yeast, TT2 physically interacts with thebHLH proteins EGL3 and TT8, and TT2 also activatesexpression of the DFR promoter when it is co-expressedwith either GL3, EGL3 or TT8 in plant cells [11]. Together,these findings support the view that a WD40–bHLH–MYBmultimeric complex controls anthocyanin production and


a similar complex controls proanthocyanidin productionin Arabidopsis.

The requirement for a WD40–bHLH–MYB transcrip-tion complex for the control of anthocyanin biosynthesishas been established in a wide range of species[25,29,55,56]. For example, in maize, the regulation ofanthocyanin genes by the R2R3 MYB proteins C1 and Plrequires the involvement of the bHLH proteins from the Rand B gene family [57] and the WD repeat protein PAC1 isalso required for high levels of anthocyanin production inaleurone [49]. In Petunia hybrida, the R2R3 MYB proteinAN2, the bHLH protein AN1 and the WD repeatprotein AN11 are required for anthocyanin accumulationin flowers [46,47,58]. In Perilla frutescens, the anthocyaninproduction regulators MYB-P1 and MYC-RP (bHLH)physically interact [59,60] and a WD-repeat proteinPFWD, which controls anthocyanin production, interactswith MYC-RP in yeast [61]. Anthocyanin regulatorsGMYB10 (R2R3MYB) and GMMYC1 (bHLH) are partof the same complex in Gerbera hybrida [62,63].These findings indicate that the multimeric complex(WD40–bHLH–MYB) that specifies trichome cell fate inArabidopsis is compositionally similar to those thatregulate anthocyanin synthesis in a wide variety ofplant species.

Phylogenetic reconstructions place the MYB proteinsthat regulate trichome formation in Arabidopsis (GL1and AtMYB23) on the same branch as those involved inanthocyanin and flavonoid biosynthesis, distinct from theAmMIXTA, AmMYBML1 and PhMYB1 regulatorsbranch [50] (Figure 3b). This shows that GL1-like genesand MIXTA-like genes are not orthologous, and thatGL1-like genes arose from the duplication and sub-sequent functional diversification (neo-functionalization)of a gene involved in regulating anthocyanin andflavonoid production. So, the mechanism regulating theformation of trichomes in Arabidopsis might haveoriginated from the duplication and divergence of aMYB gene encoding a protein that already interactedwith the protein regulators TTG1, GL3 and EGL3 in thecontrol of anthocyanin biosynthesis. This interpretationreinforces the idea that gene duplication and divergenceare the main sources in genomic and organismalevolution [64,65], and suggests that trichomes mighthave evolved several times, highlighting the functionalityof their design.

A possible origin of one trichome initiation

pathway in Asterids?

A MIXTA-like gene, PpMYB1, has been identified in thegenome of the moss Physcomitrella patens suggesting thatthe biological function of MIXTA-like MYB genes(directing cell shaping) is an ancient one in plants.Given that P. patens does not produce flowers, let alonepetals or trichomes, PpMYB1 must have a differentdevelopmental function to MIXTA. Antisense suppressionof PpMYB1 was lethal to moss colony growth suggestingthat it might play a role in shaping and growth ofchloronemal or protonemal cells [66]. This idea hasexperimental support because mutants blocked in proto-nemal development have drastically reduced expression of


(a) (b)

Figure 4. Phenocopy of Physcomitrella patens protonemal growth by high-level

expression of MIXTA in Antirrhinum majus. (a) Protonema of P. patens showing

outgrowth (arrowed) as the first stage of branch initiation. (b) Effect of strong

ectopic expression of MIXTA in A. majus giving rise to protonemal-like, branched

growth of cotyledonary epidermal cells. Scale barsZ100 mm.


PpMYB1 [66]. The gene is also highly expressed duringthe phase of rapid branched protonemal growth. Further-more, some cellular changes associated with conical cellformation that are promoted by MIXTA (includingmicrotubular re-orientation) are remarkably similar tothe changes observed during outgrowth and branchformation in protonema (Figure 4) [67] (K. Baumannand C. Martin, unpublished). Therefore the pathwayleading to multicellular trichomes controlled by MIXTA-like proteins in plants might be a relatively ancient onehaving originated from the developmental pathwaycontrolling the branching growth of files of cells. Thisview is supported by phylogenetic analysis that placesPpMYB1 in the MIXTA-like protein clade (Figure 3b).

Concluding remarks

Together, these findings support the idea that trichomesin Arabidopsis (and perhaps cotton) develop through atranscriptional regulatory network that differs from thoseregulating trichome formation in Antirrhinum andSolanaceous species. GL1 and AtMYB23 proteins belongto the anthocyanin biosynthetic clade of R2R3MYBproteins (subgroup 6) [50], a clade distinct from that ofMIXTA, AmMYBML1 and PHMYB1, which stronglysuggests that duplication of a MYB gene controllinganthocyanin and flavonoid production and subsequentdivergence might have been a major step in the evolutionof trichome formation in the progenitor of Arabidopsis. Inthe Asterid division, the proteins directing formation of atleast one type of trichome appear to have a distinctevolutionary origin. The knowledge derived from studyingtrichome formation in diverse plant species and compari-son with those mechanisms understood in detail inArabidopsis should help us to fill in some of the manygaps that remain in our understanding of the evolutionof trichomes.

AcknowledgementsThis work was supported by both grants from the JCCM (PAI-05-058) andthe MEC (BIO2005-04493) to L.S., and by the Core Strategic Grant fromBBSRC to JIC to support C.M.

References

1 Werker, E. (2000) Trichome diversity and development. Adv. Bot. Res.31, 1–35

2 Johnson, H.B. (1975) Plant pubescence: an ecological perspective. Bot.Rev. 41, 233–258


3 Mauricio, R. and Rausher, M.D. (1997) Experimental manipulation ofputative selective agents provides evidence for the role of naturalenemies in the evolution of plant defense. Evolution Int. J. Org.Evolution 51, 1435–1444

4 Wilkins, T.A. et al. (2000) Cotton biotechnology. Crit. Rev. Plant Sci.19, 511–550

5 Chen, L. et al. (1997) Convergent evolution of antifreeze glycoproteinsin Antarctic notothenioid fish and Arctic cod. Proc. Natl. Acad. Sci. U.

S. A. 94, 3817–38226 Oakley, T.H. and Cunningham, C.W. (2002) Molecular phyl-

ogenetic evidence for the independent evolutionary origin of anarthropod compound eye. Proc. Natl. Acad. Sci. U. S. A. 99,1426–1430

7 Marks, M.D. and Feldmann, K.A. (1989) Trichome development inArabidopsis thaliana. I. T-DNA tagging of the GLABROUS1 gene.Plant Cell 1, 1043–1050

8 Oppenheimer, D.G. et al. (1991) A myb gene required for leaf trichomedifferentiation in Arabidopsis is expressed in stipules. Cell 67,483–493

9 Grotewold, E. et al. (2000) Identification of the residues in the Mybdomain of maize C1 that specify the interaction with the bHLHcofactor R. Proc. Natl. Acad. Sci. U. S. A. 25, 13579–13584

10 Winkel-Shirley, B. (2001) Flavonoid biosyntheis. A colour model forgenetics, biochemistry, cell biology, and biotechnology. Plant Physiol.126, 485–493

11 Zimmermann, I.M. et al. (2004) Comprehensive identification ofArabidopsis thaliana MYB transcription factors interacting withR/B-like BHLH proteins. Plant J. 40, 22–34

12 Kirik, V. et al. (2001) Ectopic expression of the Arabidopsis AtMYB23gene induces differentiation of trichome cells. Development 235,366–377

13 Kirik, V. et al. (2005) Functional diversification of MYB23 and GL1genes in trichome morphogenesis and initiation. Development 132,1477–1485

14 Payne, C.T. et al. (2000) GL3 encodes a bHLH protein that regulatestrichome development in Arabidopsis through interaction with GL1and TTG1. Genetics 156, 1349–1362

15 Zhang, F. et al. (2003) A network of redundant bHLH proteinsfunctions in all TTG1-dependent pathways of Arabidopsis. Develop-ment 130, 4859–4869

16 Walker, A.R. et al. (1999) The TRANSPARENT TESTA GLABRA1

locus, which regulates trichome differentiation and anthocyaninbiosynthesis in Arabidopsis, encodes a WD40 repeat protein. PlantCell 11, 1337–1349

17 Koornneef, M. (1981) The complex syndrome of ttg mutants.Arabidopsis Inf. Serv. 18, 45–51

18 Hulskamp, M. et al. (1994) Genetic dissection of trichome celldevelopment in Arabidopsis. Cell 76, 555–556

19 Schnittger, A. et al. (1998) Tissue layer and organ specificity oftrichome formation are regulated by GLABRA1 and TRIPTYCHON inArabidopsis. Development 125, 2283–2289

20 Schnittger, A. et al. (1999) Generation of a spacing pattern: the role ofTRIPTYCHON in trichome patterning in Arabidopsis. Plant Cell 11,1105–1116

21 Schellmann, S. et al. (2002) TRIPTYCHON and CAPRICE mediatelateral inhibition during trichome and root hair patterning inArabidopsis. EMBO J. 21, 5036–5046

22 Kirik, V. et al. (2004) The ENHANCER OF TRYAND CPC1 gene actsredundantly with TRIPTYCHON and CAPRICE in trichome and roothair cell patterning in Arabidopsis. Dev. Biol. 268, 506–513

23 Kirik, V. et al. (2004) ENHANCER of TRY and CPC2 (ETC2) revealsredundancy in the region-specific control of trichome development ofArabidopsis. Plant Mol. Biol. 55, 389–398

24 Koshino-Kimura, Y. et al. (2005) Regulation of CAPRICE transcrip-tion by MYB proteins for root epidermis differentiation in Arabidopsis.Plant Cell Physiol. 46, 817–826

25 Larkin, J.C. et al. (2003) How cells know what they want to be whenthey grow up? Lessons from epidermal patterning in Arabidopsis.Annu. Rev. Plant Biol. 54, 403–430

26 Schiefelbein, J. (2003) Cell-fate specification in the epidermis: acommon patterning mechanism in the root and shoot. Curr. Opin.Plant Biol. 6, 74–78



27 Esch, J.J. et al. (2003) A contradictory GLABRA3 allele helps definegene interactions controlling trichome development in Arabidopsis.Development 130, 5885–5894

28 Hulskamp, M. (2004) Plant trichomes: a model for cell differentiation.Nat. Rev. Mol. Cell Biol. 5, 471–480

29 Serna, L. (2004) A network of interacting factors triggering differentfates. Plant Cell 16, 2258–2263

30 Szymanski, D.B. et al. (1998) Control of GL2 expression inArabidopsis leaves and trichomes. Development 125, 1161–1171

31 Wang, S. et al. (2004) Control of plant trichome development by acotton fiber MYB gene. Plant Cell 16, 2323–2334

32 Suo, J. et al. (2003) Identification of GhMYB109 encoding a R2R3MYB transcriptional factor that expresses specifically in fiber initialsand elongating fibers of cotton (Gossypium hirsutum L.). Biochim.

Biophys. Acta 1630, 25–3433 Humphries, J.A. et al. (2005) Two WD-repeat genes from cotton are

functional homologues of the Arabidopsis thaliana TRANSPARENT

TESTA GLABRA1 (TTG1) gene. Plant Mol. Biol. 57, 67–8134 Noda, K-I. et al. (1994) Flower colour intensity depends on specialized

cell shape controlled by a Myb-related transcription factor. Nature

369, 661–66435 Glover, B.J. et al. (1998) Development of several epidermal cell types

can be specified by the same MYB-related plant transcriptional factor.Development 125, 3497–3508

36 Martin, C. et al. (2002) The mechanics of cell fate determination inpetals. Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 809–813

37 Martin, C. and Paz-Ares, J. (1997) MYB transcription factors inplants. Trends Genet. 13, 67–73

38 Payne, T. et al. (1999) Heterologous myb genes distinct from GL1

enhance trichome production when overexpressed in Nicotiana

tabacum. Development 126, 671–68239 Glover, B.J. et al. (2004) Convergent evolution within the genus

Solanum: the specialised anther cone develops through alternativepathways. Gene 331, 1–7

40 Avila, A. et al. (1993) Petunia hybrida genes related to the maizeregulatory C1 gene and to the animal myb proto-oncogenes. Plant J. 3,553–562

41 van Houwelingen, A. et al. (1998) Analysis of flower pigmentationmutants generated by random transposon mutagenesis in Petunia

hybrida. Plant J. 13, 39–5042 Perez-Rodriguez, M. et al. (2005) Development of three different cell

types is associated with the activity of a specific MYB transcriptionfactor in the ventral petal of Anthirrhinum majus flowers. Develop-

ment 132, 359–37943 Lloyd, A.M. et al. (1992) Arabidopsis and Nicotiana anthocyanin

production activated by maize regulators R and C1. Science 258,1773–1775

44 Mooney, M. et al. (1995) Altered regulation of tomato and tobaccopigmentation genes caused by delila gene of Antirrhinum. Plant J. 7,333–339

45 Quattrocchio, F. et al. (1993) Regulatory genes controlling anthocya-nin pigmentation are functionally conserved among plant species andhave distinct sets of target genes. Plant Cell 5, 1497–1512

46 Spelt, C. et al. (2000) anthocyanin1 of petunia encodes a basic-helixloop helix protein that directly activates structural anthocyaningenes. Plant Cell 12, 1619–1631

47 de Vetten, N. et al. (1997) The an11 locus controlling flowerpigmentation in petunia encodes a novel WD-repeat protein conservedin yeast, plants, and animals. Genes Dev. 11, 1422–1434

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48 Spelt, C. et al. (2002) ANTHOCYANIN1 of petunia controls pigmentsynthesis, vacuolar pH, and seed coat development by geneticallydistinct mechanism. Plant Cell 14, 2121–2135

49 Carey, C.C. et al. (2004) Mutations in the pale aleurone color1regulatory gene of the Zea mays anthocyanin pathway have distinctphenotypes relative to the functionally similar TRANSPARENTTESTA GLABRA1 gene in Arabidopsis thaliana. Plant Cell 16,450–464

50 Stracke, R. et al. (2001) The R2R3-MYB gene family in Arabidopsisthaliana. Curr. Opin. Plant Biol. 4, 447–456

51 Goodspeed, T.H. (1954) The genus Nicotiana. In Chronica Botanica(Verdoorn, F., ed.), pp. 102–131, Chronica Botanica Co

52 Borevitz, J.O. et al. (2000) Activation tagging identifies a conservedMYB regulator of phenylpropanoid biosynthesis. Plant Cell 12,2383–2393

53 Nesi, N. et al. (2000) The TT8 gene encodes a basic helix–loop–helixdomain protein required for expression of DFR and BAN genes inArabidopsis siliques. Plant Cell 12, 1863–1878

54 Nesi, N. et al. (2001) The Arabidopsis TT2 gene encodes an R2R3 MYBdomain protein that acts as a key determinant for proanthocyanidinaccumulation in developing seed. Plant Cell 13, 2099–2114

55 Koes, R. et al. (2005) Flavonoids: a colorful model for the regulationand evolution of biochemical pathways. Trends Plant Sci. 10, 236–242

56 Ramsay, N.A. and Glover, B.J. (2005) MYB–bHLH–WD40 proteincomplex and the evolution of cellular diversity. Trends Plant Sci. 10,63–70

57 Mol, J. et al. (1998) How genes paint flowers and seeds. Trends PlantSci. 3, 212–217

58 Quattrocchio, F. et al. (1998) Analysis of bHLH and MYB-domainproteins: species specific regulatory differences are caused bydivergent evolution of target anthocyanin genes. Plant J. 13, 457–488

59 Gong, Z.Z. et al. (1999a) A constitutively expressed Myc-like geneinvolved in anthocyanin biosynthesis from Perilla frutescens: molecu-lar characterization, heterologous expression in transgenic plants andtransactivation in yeast cells. Plant Mol. Biol. 41, 33–44

60 Gong, Z.Z. et al. (1999a) A constitutively expressed Myc-like geneinvolved in anthocyanin biosynthesis from Perilla frutescens: molecu-lar characterization, heterologous expression in transgenic plants andtransactivation in yeast cells. Plant Mol. Biol. 41, 33–44

61 Sompornpailin, K. et al. (2002) A WD-repeat-containing putativeregulatory protein in anthocyanin biosynthesis in Perilla frutescens.Plant Mol. Biol. 50, 485–495

62 Elomaa, P. et al. (1998) A bHLH transcriptional factor mediates organ,region and flower type specific signals on dihydroflavonol-4-reductase(dfr) gene expression in the inflorescence of Gerbera hybrida(Asteraceae). Plant J. 16, 93–99

63 Elomaa, P. et al. (2003) Activation of anthocyanin biosynthesis inGerbera hybrida (Asteraceae) suggests conserved protein–protein andprotein–promoter interactions between the anciently diverged mono-cots and eudicots. Plant Physiol. 133, 1831–1842

64 Ohno, S. (1970) Evolution by Gene Duplication, Springer-Verlag65 Zhang, J. (2003) Evolution by gene duplication: an update. Trends

Ecol. Evol. 18, 292–29866 Leech, M.J. et al. (1993) Expression of myb-related genes in the moss

Physcomitrella patens. Plant J. 3, 51–6167 Cove, D.J. and Ashton, N.W. (1988) Growth regulation and develop-

ment in Physcomitrella patens: an insight into growth regulation anddevelopment of bryophytes. Bot. J. Linnean Soc. 98, 247–252

68 Celenza, J.L. et al. (2005) The Arabidopsis ATR1 Myb transcriptionfactor controls indolic glucosinolate homeostasis. Plant Physiol. 137,253–262

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