Research Focus

bHLH proteins know when to make a stoma

Update TRENDS in Plant Science Vol.12 No.11

Laura Serna

Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, E-45071 Toledo, Spain

In Arabidopsis thaliana, stomata develop through astereotypical pattern of cell divisions. Three recent pub-lications demonstrate that three bHLH proteins act suc-cessively in such lineages to drive the formation ofstomata. SPEECHLES drives the division that initiatesthe stomatal-cell lineage. Then MUTE induces the for-mation of the immediate stomatal precursor cell. Finally,FAMA causes the stomatal precursor cell to divide intothe two guard cells that surround each stomatal pore.

How to know how often to divide?Stomata are by far the most influential components ingas exchange. They consist of two guard cells thatchange shape as a result of turgor pressure; this shapechange leads to the opening and closing of the stomatalpore, which, as a result, regulates gas exchange. InArabidopsis, stomatal development starts with an asym-metric division from an epidermal cell called a meriste-moid mother cell [1] (Figure 1). This division yields atriangular meristemoid and a larger cell. Meristemoidscan divide asymmetrically up to three more times, andalways produce a larger cell and a smaller meristemoidthat maintains its stem cell activity. After these asym-metric divisions, the meristemoid loses its stem cellactivity and forms a guard mother cell (GMC). TheGMC undergoes a symmetric cell division that producesthe paired guard cells. The larger cells that result fromthe asymmetric divisions can divide or become pavementcells. That being said, how does a stomatal precursor cellknow how often it should divide before making a stoma?The recent isolation and functional characterization ofthree members of the basic helix-loop-helix (bHLH)family of transcriptional factors have deepened the mys-tery [2–4]. Two recent papers have highlighted thesefindings* [5,6].

Several signalling genes, including STOMATALDENSITY AND DISTRIBUTION1 (SDD1), TOO MANYMOUTHS (TMM), members of the ERECTA family recep-tor-like kinases [ERECTA, ERECTA LIKE1 (ERL1) andERL2], YODA (YDA) and four genes of aMITOGEN-ACTI-VATED PROTEIN KINASE (MAPK) signalling module,negatively regulate the development of supernumerary sto-mata [7–11]. By contrast, the three members of the bHLHfamily of transcriptional factors promote stomatal develop-ment [2–4]. Because these regulators control, in an oppositemanner, the same functions (the entrance into the stomatallineage and/or the stem cell activity of the meristemoids), a

Corresponding author: Serna, L. (laura.serna@uclm.es).

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dynamic balance between themmust be crucial for stomataldevelopment.

SPEECHLESS and MUTE: driving the first and the lastasymmetric cell divisionsMacAlister et al. found that plants homozygous for thespeechless-3 (spch-3) or spch-4 alleles, which lack detect-able transcripts, do not develop stomata, and all theirepidermal cells consist of jigsaw-puzzle-shaped pavementcells (Figure 2b) [3]. The mutation in spch-1, which elim-inates the last seven amino acids of the protein, alsoinduces an identical phenotype. The absence of stomatain these mutants suggests that SPCH drives the divisionthat initiates stomatal development [3]. Supporting such arole, the overexpression of SPCH increases the number ofcells entering into the stomatal pathway [3,4].

The missense mutation spch-2 affects the carboxyterminus of the protein, and it reduces the number ofstomata [3]. This finding supports the proposed role forSPCH in the regulation for stomatal development andsuggests some residual activity of the protein in thespch-2 mutant. In addition, the ability of the spch-2mutant to develop stomata provides an opportunity touncover additional functions regulated by the SPCHgene. Based on studies of static pictures of the pedicelepidermis in spch-2, MacAlister et al. concluded thatSPCH also maintains the stem cell activity of the mer-istemoids [3].

The SPCH gene encodes a protein that belongs to thebHLH family of transcription factors [3]. It is expressed inundifferentiated cells and persists in stomatal lineagecells, including guard cells [3]. However, the protein isrestricted to early stages of the stomatal lineage,suggesting that the SPCH proteinmight be downregulatedpost-transcriptionally [3]. These expression patterns areconsistent with a role for SPCH in determination ofentrance into the stomatal lineage and, perhaps, a laterrole in the maintenance of the stem cell activity of themeristemoids, as suggested by the phenotype of spch-2 inthe pedicel epidermis.

A second bHLH protein, namedMUTE and very similarin sequence to SPCH, also controls stomatal development[4]. The loss-of-function mute mutant does not developstomata but forms meristemoids that abort after excessiveasymmetric cell divisions [3,4] (Figure 2c). Both MUTEpromoter activity and theMUTE protein are restricted to asubset ofmeristemoids, with residual activity inGMCs andimmature stomata [3,4]. It is therefore probable that, inthis subset of meristemoids, MUTE represses stem cellactivity and induces GMC formation. The fact that theoverexpression of MUTE converts all epidermal cells into

d. doi:10.1016/j.tplants.2007.08.016

Figure 1. SPEECHLESS (SPCH) starts stomatal development by inducing the first asymmetric division, which produces the first meristemoid. Two or three divisions after

SPEECHLESS, MUTE drives the last asymmetric cell division causing the formation of the guard mother cell. Then, FAMA drives the symmetric division that gives rise to the

two guard cells. The three genes encode bHLH proteins with very similar sequences. Adapted from Refs [3,4].

Figure 2. Epidermal phenotype of wild type (a) and mutants plants (b–d). (a) Wild-type plants develop stomata, (b) which fail to develop in spch mutants. (c) The mute

mutant does not develop stomata but forms meristemoids that abort after excessive asymmetric cell divisions. (d) fama lacks mature stomata and, instead, develops

clusters of guard mother cells or young guard cells. Adapted from Ref. [3].

484 Update TRENDS in Plant Science Vol.12 No.11

stomata is consistent with the role assigned to this gene[3,4].

FAMA and the symmetric division that produces thestomaThe FAMA gene encodes a third protein, which belongs tothe bHLH family of transcription factors and which islikely to act as a transcriptional activator [2]. Plantshomozygous for the insertion mutant fama-1, which haveno detectable FAMA transcripts, lackmature stomata and,instead, develop cluster of GMCs or incipient guard cells [2](Figure 2d). Kyoko Ohashi-Ito and Dominique Bergmannhave shown that the induction of the FAMA promoter isrestricted to GMCs and young guard cells, and that theFAMAprotein localizes to the nucleus of these cells [2]. It isthen likely that FAMA causes both the GMC to divide intotwo guard cells and that it promotes guard cell differen-tiation.

The overexpression of FAMA converts non-stomatalcells to guard cells [2]. These guard cells are not organizedinto pairs, and it seems that they develop from GMCs thatswitch to become guard cells without undergoing a sym-metrical division [2]. The simplest explanation for thesefindings is that the activity of FAMA is essential in theregulation of cell division, with high levels repressing celldivision and forcing GMCs to differentiate directly intoguard cells [2].

Playing downstream of the signalling genesSimilarly to the signalling genes (SDD1,TMM, members ofthe ER-family and YDA), SPCH controls both functions:entrance into the stomatal-cell lineage and the balancebetween meristemoid renewal and stomatal differen-tiation, with spch-1 being epistatic to tmm, er, erl1, erl2and yda mutants [3]. This suggests that SPCH operatesdownstream of theses genes [3]. In agreement withthis view, the overexpression of SPCH driven by its own

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promoter promotes the development of stomata in thehypocotyl of tmm [3], which usually has no stomata.

MUTE and the signalling genes control the stemcell fate of the meristemoids, however, they do so in anoppositemanner,withMUTE repressing such cell fate andthe signalling components inducing it. Like mute, thethree mutants mute tmm, mute erecta erl1 erl2 and mutesdd1 do not develop stomata, but the arrangement ofthe meristemoids is similar to the arrangement of thestomata induced by the mutation(s) in the signallinggene(s) in each case [4]. This suggests that all these genesinteract additively [4]. In addition, the absence of stomatain the double or quadruple mutants suggests that MUTEmight act downstream of these genes triggering GMCformation [4].

The absence of stomata in mutant combinationsbetween fama and the mutants of the signalling genes( fama sdd1, fama tmm, fama er erl1 erl2 and fama yda) [2]also places FAMA downstream of such genes with respectto the regulation of guard cell differentiation. Moreover,the arrangement of the GMCs or immature guard cellsreflects the phenotype of mutations in the signalling com-ponents [2], which suggests that they interact additivelywith FAMA.

In addition to the bHLH regulators of stomataldevelopment, two other proteins belonging to the MYBfamily of transcription factors have been implicated in thisprocess [12]. Both FOUR LIPS and MYB88 control theformation of the stoma from the GMC [12]. Some MYBproteins regulate transcription by physical interactionwith bHLH proteins [13]. However, FOUR LIPS, MYB88and FAMA do not contain the amino acid signaturesdefined as necessary for the interaction between MYBand bHLH proteins [6]. Certainly, although several tech-niques have been used, no interactions between FOURLIPS or MYB88 and FAMA have been detected [2]. More-over, neither protein is required for the transcriptional

Update TRENDS in Plant Science Vol.12 No.11 485

activation of the other [2]. Together, this suggests thatFAMA and FLP/MYB88 control the GMC–guard celltransition independently.

Stomatal density is regulated by environmentalconditions [14]. Environmental factors that increase sto-matal density might be working through the positive reg-ulators (SPCH,MUTE and FAMA). By contrast, those thatdecrease the number of stomata might be working throughthe negative ones (SDD1, TMM, ER-family, YDA and theMAPK module).

Concluding remarksThese results bring us closer to an understanding of how astomatal precursor knows how often to divide. Among themany challenges that lie ahead is deciphering how SPCH,MUTE and FAMA interact with one another. It would alsobe interesting to determine not only which genes areregulated by these bHLH proteins, but also how theirproducts function to ensure that fully differentiated sto-mata develop in the right places.

*Note added in proofA new paper covering the same topic has been publishedduring the production of this issue: Pillitteri, L.J. and Torii,K.U (2007) Breaking the silence: three bHLH proteinsdirect cell-fate decisions during stomatal development.Bioessays 29, 861–870.

AcknowledgementsI thank C. Martin for critical reading of the manuscript. Work in mylaboratory is supported by grants from both the Communities Council of

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Rev. Plant Biol. 58, 163–1812 Ohashi-Ito, K. and Bergmann, D.C. (2006) Arabidopsis FAMA controls

the final proliferation/differentiation switch during stomataldevelopment. Plant Cell 18, 2493–2505

3 MacAlister, C.A. et al. (2007) Transcription factor control ofasymmetric cell divisions that establish the stomatal lineage.Nature 445, 537–540

4 Pillitteri, L.J. et al. (2007) Termination of asymmetric cell division anddifferentiation of stomata. Nature 445, 501–505

5 Gray, J.E. (2007) Plant development: three steps for stomata. Curr.Biol. 17, R213–R215

6 Barton, M.K. (2007) Making holes in leaves: promoting cell statetransitions in stomatal development. Plant Cell 19, 1140–1143

7 Berger, D. and Altmann, T. (2002) A subtilisin-like protease involved inthe regulation of stomatal density and distribution in Arabidopsisthaliana. Genes Dev. 14, 1119–1131

8 Nadeau, J.A. and Sack, F.D. (2002) Control of stomatal distribution onthe Arabidopsis leaf surface. Science 296, 1697–1700

9 Shpak, E.D. et al. (2005) Stomatal patterning and differentiation bysynergistic interactions of receptor kinases. Science 309, 290–293

10 Bergmann, D.C. et al. (2004) Stomatal development and patterncontrolled by a MAPKK kinase. Science 304, 1494–1497

11 Wang, H. et al. (2007) Stomatal development and patterning areregulated by environmentally responsive mitogen-activated proteinkinases in Arabidopsis. Plant Cell 19, 63–73

12 Lai, L.B. et al. (2005) TheArabidopsisR2R3MYB proteins FOURLIPSand MYB88 restrict divisions late in the stomatal lineage. Plant Cell17, 2754–2767

13 Serna, L. and Martin, C. (2006) Trichomes: different regulatorynetworks lead to convergent structures. Trends Plant Sci. 11, 274–280

14 Hetherington, A.M. and Woodward, F.I. (2003) The role of stomata insensing and driving environmental change. Nature 424, 901–908

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