Integrating signals in stomatal development

Dominique C Bergmann
Stomata are specialized epidermal structures that control the exchange of water and carbon dioxide between the plant and the atmosphere. The classical developmental mechanisms that dene cell fate and tissue patterning cell lineage, cellcell interactions and signals from a distance are employed to make stomata and to dene their density and distribution within the epidermis. Recent work has shown that two genes that are involved in stomatal pattern may encode components of a classical cell-surface-receptor-mediated signaling cascade. Additional work has suggested that signals from the overlying cuticle and the underlying mesophyll also inuence stomatal pattern. These ndings highlight the need for models that explain how the signals that regulate stomatal development are integrated and how they act to regulate cell polarity, the cell cycle and, ultimately, cell fate.
Addresses Carnegie Institution, Department of Plant Biology, Stanford, California 94305, USA e-mail: bergmann@andrew2.stanford.edu

guard cells; however, optimal gas exchange appears to require regulation of the numbers and positions of stomata as well as the ability to open and close them [2]. In monocots such as maize, stomata are found in linear arrays parallel to the leaf veins [1]. In Arabidopsis and other dicots, there is no obvious arrangement of stomata on the leaf surface. Interestingly, however, stomata are almost never found in contact [2]. Stomata normally obey several patterning rules: rst, they are formed through a stereotyped lineage of asymmetric divisions; second, they are patterned locally so that two stomatal complexes are never adjacent to one another (the one-cell-spacing rule); and third, the overall numbers of stomatal complexes are controlled globally in response to environmental cues. The latter two rules imply that cell signaling is critical to the establishment of stomatal pattern. This review summarizes recent data on the nature and sources of such signals, and on the nascent receptor-mediated signaling cascade formed by the TOO MANY MOUTHS (TMM) and STOMATAL DENSITY AND DISTRIBUTION1 (SDD1) genes. I concentrate on stomatal pattern in Arabidopsis, but ndings from other plant species are included when particularly enlightening.

Current Opinion in Plant Biology 2004, 7:2632 This review comes from a themed issue on Growth and development Edited by Vivian Irish and Philip Benfey 1369-5266/$ see front matter 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/j.pbi.2003.10.001

Cell fate and cell signaling in the plane of the epidermis

The epidermis of a newly germinated Arabidopsis seedling shows no overt cellular differentiation. Within 24 hours, however, physically asymmetric divisions take place and these correspond to the creation of meristemoids, the rst precursor type in the guard cell lineage [2]. Meristemoids have a limited stem-cell character and can divide again asymmetrically up to three times, each time retaining the meristemoid fate in the smaller daughter cell from the division [3]. Eventually, the meristemoid differentiates into a guard mother cell (GMC) and becomes rounded due to the modication and reinforcement of its cell wall [4]. The GMC divides a single time, symmetrically, to form the two paired guard cells of the mature stoma ([4]; Figure 1). Loss-of-function mutations in FOUR LIPS (FLP) result in additional divisions of the GMCs, suggesting that this gene is involved in limiting the cell division competence of these cells [5]. In the hypocotyl, the same set of divisions takes place to create stomata but the divisions are restricted to cell les that are contiguous with the hair-producing les of the root ([6]; Figure 2b). Protodermal cells distributed throughout the young leaf epidermis enter into the lineage pathway that leads to the formation of stomata. The sister cells of the meristemoids
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Abbreviations C16 16-carbon cer eceriferum CLV2 CLAVATA2 FLP FOUR LIPS GA gibberellin GMC guard mother cell HIC HIGH CARBON DIOXIDE M meristemoid SDD1 STOMATAL DENSITY AND DISTRIBUTION1 TMM TOO MANY MOUTHS VLCFA very long-chain fatty acids Xcl1 extra cell layers1

Introduction
The epidermis is the interface between the plant and the world, and stomata are specialized structures within it that serve as the conduit for the exchange of water vapor and carbon dioxide. Minimally, a stoma consists of paired guard cells, which ank a pore in the epidermis, and an airspace in the underlying mesophyll [1]. To maximize photosynthetic efciency while minimizing water loss, pore size is modulated by the ion-driven swelling of the
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Integrating signals in stomatal development Bergmann 27

Figure 1

SDD1 TMM
SDD1 TMM

FLP

SDD1 TMM

MMC

GMC

GC GC

Meristemoid mother cell

Meristemoid formed

Meristemoid converted to a guard mother cell

Immature guard cells formed

Mature stoma

Subsidiary meristemoid

Continued division of meristemoid

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Lineage pathway for guard-cell formation in Arabidopsis. A meristemoid (M; blue) is the smaller daughter of the asymmetric division of the meristemoid mother cell (MMC). The meristemoid may divide again or may convert directly into a guard mother cell (GMC). The GMC divides only once to produce the two guard cells (GCs). New meristemoids may be produced from the neighboring cells, but at positions distal from the mature stoma. FLP prevents multiple divisions of the GMCs (red T-lines). SDD1 and TMM promote oriented divisions of neighbor cells (green arrows), but prevent the conversion of meristemoids into GMCs and the division of cells that contact multiple stomata or precursors (red T-lines). SDD1 and TMM also promote cell divisions in subsidiary meristemoids (not shown).

can also enter this pathway and generate a temporally distinct, but otherwise functionally equivalent, set of subsidiary meristemoids [3,7]. Lineage alone is not sufcient to ensure adherence to the one-cell-spacing rule, so signals that ensure that stomata are not formed in contact with each other are superimposed on the lineage [3]. The major factors in determining the pattern of stomata appear to be signals from mature guard cells (or their
Figure 2

precursors, GMCs or meristemoids) to their neighboring cells. Two messages are relayed. Cells that are in contact with a single stoma or precursor are instructed to orient their future division planes such that asymmetric divisions place the smaller cell (and potential stoma) distal to the pre-existing stoma [3]. Cells that are in contact with two or more stomata are instructed not to divide ([3]; Figure 2a).

(a)

(b)

(c)

(d)

Current Opinion in Plant Biology

Development of stomata in the Arabidopsis epidermis. (a) Confocal image of a 3-day-old Arabidopsis cotyledon. Meristemoids are false-colored in yellow, GMCs in pink and guard cells in blue. The asymmetric division of the meristemoid mother cell to create the meristemoid is indicated by . Signals from mature stomata orient the divisions of neighbor cells (arrow) or prevent their division (T-line). (b) Bright-field image of a 12-day-old Arabidopsis hypocotyl showing the alternation of cell files and a single mature stoma (indicated in blue). (c) Hypocotyl of a 12-day-old EF2-expressing Arabidopsis plant showing extra cell divisions in alternate cell files but no extra stomata. (d) Hypocotyl of 3-week-old E2FaDpa-expressing plant showing the massive overproliferation of cells in all cell files but still no ectopic stomata. (bd) Reprinted, with permission, from [14]. www.sciencedirect.com Current Opinion in Plant Biology 2004, 7:2632

28 Growth and development

Figure 3

Influences on the developing Arabidopsis epidermis (a) High [CO2], low humidity (b) (via HIC) Epidermis Cuticle CER6 Stomata CER1 WAX2 HIC

Gibberellin (possibly via TMM)

? Mesophyll ?

(c) Promotes stomatal fate Inhibits stomatal fate TMM SDD1 ?

Current Opinion in Plant Biology

Sources of signals that direct stomatal pattern. (a) Plants receive long-range signals from external stimuli or via the action of plant hormones. (b) The epidermis is then subject to influences from the cuticle that lies above it and the mesophyll that lies below. (c) Within the epidermis, SDD1, TMM and FLP act to influence cell cycling and the polarity of cell divisions. ?, unknown factor.

Two gene products required to regulate stomatal patterning are the leucine-rich repeat (LRR)-receptor-like protein encoded by TMM [8] and the putative serine protease encoded by SDD1 [7]. Mutations in either gene lead to an increase in stomatal index (i.e. the percentage of epidermal cells that are guard cells) and a breakdown of the one-cell-spacing rule. Transcripts for both TMM and SDD1 are expressed in the stomatal precursors, but SDD1 is additionally expressed at low levels in the mesophyll [8,9]. If TMM and SDD1 are involved in cellcell signaling, as suggested by their molecular identity, then the simplest model to explain their roles is that TMM serves as a receptor for a signal generated by SDD1. Evidence in support of this model comes from the nding that constitutive SDD1 expression reduces the number of stomata in the epidermis of wildtype plants (and of sdd1 and p1 mutants) but it is unable to suppress the tmm-1 extra stomata phenotype. This suggests that tmm acts downstream of sdd1 [9]. For details of the model for TMM action and the relationship between TMM and SDD1, readers are directed to an insightful recent review ([10]; Figure 3).

precursors remain diploid [11]. This has prompted some groups to propose that cell fate is a consequence of cellcycle regulation. If this model holds true, then experimental manipulation of ploidy levels would lead to changes in cell fate. Constitutive expression of the cell-cycle inhibitor KIPRELATED PROTEIN2 (KRP2) leads to a reduction of both endoreduplication and cell division rate, and gives rise to plants that have fewer cells that are larger than normal [12]. However, the stomatal index of the mature leaves of these plants is similar to that in wildtype plants [12]. Conversely, when the cyclin CYCD3:1 is overexpressed, plants have increased endoreduplication levels and produce many more small cells in the epidermis than is normal [13]. Again, neither stomatal index nor stomatal patterning is profoundly affected in these plants. Downstream of the D cyclins, E2FaDPa heterodimeric transcription factors promote the entry into S-phase in mammalian cells. In Arabidopsis, the 35S-driven expression of E2Fa (or E2Fa and DPa together) has effects similar to those caused by CYCD3:1 overexpression, but the phenotype is somewhat stronger [14]. In hypocotyls, the overexpression of E2Fa leads to a massive overproliferation of small cells (Figure 2c). These cells appear in the cell les that normally make stomata. Later in development, plants
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Cell cycles and differentiation

Cell identity in the epidermis is correlated with nuclear DNA content, as trichomes and pavement cells undergo rounds of endoreduplication whereas guard cells and their
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Integrating signals in stomatal development Bergmann 29

expressing E2FaDPa lose the cell-le restriction and divisions can seen in all hypocotyl cell les. Signicantly, the increase in the number of small cells does not lead to a concomitant increase in the number of stomata in the hypocotyls ([14]; Figure 2). Taken together, these results suggest that a pre-pattern is established in the hypocotyl cell les that inuences their ability to respond to mitogenic cues. Furthermore, cell division in both leaves and hypocotyls is only permissive for stomatal fate; additional competence factors are required to make cells mature into guard cells.

sence of a mesophyll airspace. Both stomata and meristemoids can form directly over mesophyll cells in pea [18]; and in the Arabidopsis fatty-acid biosynthesis2 (fab2) mutant, in which no airspaces are formed between the tightly appressed mesophyll cells, the epidermally derived parts of the stomata form normally [19]. Additional hints of signaling from mesophyll to the epidermis come from work in which the contacts between mesophyll and epidermal cells were genetically or physically altered and the effect on stomatal pattern observed. Arabidopsis double mutants for the protodermal factor2 (pdf2) and A. thaliana meristem layer1 (atml1) homeodomain transcription factors produce no morphologically distinguishable epidermis [20]. Scanning electron micrographs of the pdf2;atml1 leaf surfaces reveal a naked mesophyll layer, except for occasional patches of clustered stomata [20]. The preponderance of stomata in these patches may be due either to increased exposure to a signal from the mesophyll that promotes stomatal cell fate or to decreased exposure to an inhibitory signal that would normally be provided by the surrounding epidermal cells. Because the clonal origin of these islands of cells is unclear, it is also formally possible that these cells transdifferentiated from mesophyll and may not express the full complement of epidermal patterning genes (such as TMM). In the dominant maize mutant Xcl1 (for extra cell layers1), extra epidermal cell layers are found in the leaves [21]. Cells within these layers are derived from excess divisions of the L1, display epidermal cell morphology and express epidermal marker genes [21]. In the outermost epidermal layer of Xcl1 plants, stomata are underrepresented on the bottom leaf surface and nearly absent from the top. Xcl1 appears to be a hypermorphic allele and, using dosage studies, Sinha and coworkers [21] found that reduced xcl1 dosage (i.e. a single copy of the wildtype allele) gives rise to plants with a single epidermal layer that contains an increased density of stomata. The theme seems to be that proximity to mesophyll promotes stomatal formation, and additional epidermal cells either block the signal from the mesophyll or send a counteracting repressive signal to maintain the normal pattern of stomata in the epidermis.

Hormonal control of stomata

Some of the competence factors that are required for the development of mature guard cells may be plant hormones. Using the Arabidopsis hypocotyl as a system to study the interactions between hormones in growth control, Saibo et al. [15] found that the application of gibberellins (GAs), especially in combination with ethylene or auxin, resulted in an overproduction of stomata. Moreover, the inhibition of gibberellin signaling, with paclobutrazol or in a GA-biosynthetic mutant, eliminated stomata from the hypocotyl but did not affect stomatal production in the leaves [15]. GA-induced production of stomata was conned to the cell les in which stomata are normally found, suggesting that gibberellins act in a normal signaling pathway to promote cell cycling within the hypocotyl [15]. That gibberellin affects stomatal production in the hypocotyl but not the leaves points to how these organs might differentially regulate cell identity. Interestingly, the organ-specic pattern of stomata produced in response to GA is similar to the tmm phenotype. tmm-1 mutants, although they make excess stomata on leaves and cotyledons, make no stomata on the hypocotyl, and this phenotype is epistatic to the overproliferation of hypocotyl stomata in other mutant backgrounds ([16]; DC Bergmann, unpublished). Perhaps TMM normally positively regulates a GA-dependent pathway to promote cell division in the hypocotyl.

Signals from below

Interactions between the mesophyll and epidermis are inferred from the correlation of stomata with internal features. In maize, stomata are formed in linear chains, and these chains are aligned such that they do not overlay veins [1,2]. In Arabidopsis, stomata are also restricted from forming above the midvein [2]. To form a functional stoma, the guard cells and pore must lie directly above an airspace in the mesophyll. Air spaces are formed by the degradation of the matrix between adjacent cell walls, thus guard cells should lie over the edges of mesophyll cells. Serna and Fenoll [17] found that, in the adaxial leaf surface, meristemoids and stomata were found over junctions in the underlying mesophyll cells more often than expected by chance. However, two other studies found that guard cell formation was not dependent on the prewww.sciencedirect.com

Signals from above

A waxy cuticle covers the external face of plant epidermal cells, and consists of two morphologically and chemically distinct layers. The inner cuticle layer is made up of cutin, a polyester of 16-carbon (C16) and 18-carbon (C18) fatty acids linked together to form an open meshwork that may serve as a scaffold for the overlying wax layer [22]. The wax layer is composed of very long-chain fatty acids (VLCFAs) and gives the cuticle its hydrophobic properties [23]. Plants grown in dry or open-air conditions have an increased wax load [24] and lower stomatal density than plants grown at high humidity, as would be expected if water loss is a constraint. Paradoxically, mutations that
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30 Growth and development

reduce wax (e.g. eceriferum [cer]) can result in increased stomatal density and the appearance of clustered stomata in open-air-grown plants. Not all waxless mutants have this phenotype, suggesting that it is not the quantity but the quality of the lipids and waxes in the cuticle that inuences stomatal pattern. For example, only the eceriferum-g locus of barley produces stomatal defects (mainly double and triple stomatal complexes), whereas alleles at 44 other wax-decient loci do not [25]. In Arabidopsis, mutations in the genes that encode the VLCFA-producing enzymes CER6, CER1 and HIGH CARBON DIOXIDE (HIC) increase the stomatal index [26], but mutations in WAX2, which is required for the integrity of the internal cuticle layer, result in reduced stomatal index [27]. The extent of saturation of shorter-chain fatty acids also appears to play a role in determining stomatal density and stomatal responses to the environment. FATTY-ACID DEASATURASE4 (FAD4) is required to desaturate palmitic acid (16:0) [28], and fad4 mutants are unable to change their stomatal index in response to elevated CO2 [29]. Metabolic proling of sdd1 plants, which have stomatal densities at ambient CO2 that are 34-fold higher than those of wildtype plants, showed a ve-fold reduction of unsaturated C16 fatty acids compared to wildtype plants and a concomitant rise in 16:0 species [30]. The ultimate fate of these C16 fatty acids is not known; one possibility is that they are incorporated into the cutin layer and that the changes in desaturation affect the extent of crosslinking in the meshwork. As the interface between the environment and the plant, as well as a contiguous layer that connects epidermal cells, the cuticle is an excellent medium for the perception and distribution of signals that regulate stomatal density, distribution or function [31]. The mutations that affect stomatal pattern and cuticle structure would be expected to alter the accessibility or diffusion of signaling molecules, or could affect the creation of lipid-based signals. To date, the molecular nature of the signals that transit through the cuticle eludes us.

these putative ligands are expressed in the developing leaves, although further studies are needed to dene the cellular localization of these ligands [32]. If these ligands behave like CLV3, then they travel only a short distance, and a ligand for TMM would thus be expected to be synthesized in the meristemoids, GMCs or guard cells. A signaling cascade that incorporates both SDD1 and TMM cannot be the only one that directs stomatal pattern. Mature guard cells do not express SDD1, yet they are capable of signaling to their neighbors. It is possible that a component that is embedded in the mature wall of guard cells can act as a signal; such a tethered molecule could provide not only a signal but also a direction along which the neighbor cell may orient its division axis. tmm mutants have defects in both the signal that tells neighbor cells to cease dividing and the signal that polarizes cell division, suggesting that these signals might differ only quantitatively. A hypothesis to explain how the same signal could specify two outcomes is that a cell receiving signals from two different sources attempts to align its future division plane relative to two conicting axes. When the cell fails to align correctly relative to the two axes, it arrests its division process. In the future, new approaches to complement ongoing forward genetic screens might increase the number of identied players in the stomatal development pathway. Both TMM and SDD1 are expressed in stomatal precursor cells, and HIC is expressed in guard cells. It is reasonable to expect that other regulators of guard-cell identity or pattern may also be expressed in these cell types. Transcriptome analyses of precursor cells that have been isolated by laser-capture microdissection (LCM) [34,35], selected by cell sorting, or enriched in certain mutant backgrounds might reveal those regulators. The available genome-wide Arabidopsis gene knockouts could be used to conrm a role for these genes in stomatal pattern [36]. In the emerging eld of chemical genetics, the discovery and recent characterization of specic chemical inhibitors of VLCFA synthesis could lead to useful conditional tools with which to assay the effect of altering the cuticle on aspects of stomatal biology [37].

Conclusions and prospects

The data reviewed here suggest that many tissues in the plant have the potential to inuence the nal arrangement and numbers of stomata on the leaf surface (Figure 3). The challenges that remain are to identify these signals at the molecular level, and to understand how external signals impinge on cell polarity and the cell cycle. The cloning of SDD1 and TMM provides a starting place. TMM and SDD1 might act together in a common signaling pathway, but if so, the upstream signal and downstream targets have yet to be identied. On the basis of the similarity of TMM to CLAVATA2 (CLV2), it is likely that a small peptide that is similar CLV3 serves as a ligand in this signaling pathway. Recently, a family of CLV3-like proteins was described [32,33]. At least ten of
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Acknowledgements
I thank P Jenik, J McConnell, S Mohr, TK Raab and members of the Somerville laboratory for discussions and helpful comments on the manuscript. I also thank Chris Somerville for guidance and for sharing his extensive knowledge of plant lipid metabolism. I have been supported by a National Research Service Award (NRSA) fellowship (1F32GM64273-01).

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1. Esau K: The Anatomy of Seed Plants, edn 2. New York: John Wiley and Sons; 1977. www.sciencedirect.com

Integrating signals in stomatal development Bergmann 31

2.

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8. Nadeau JA, Sack FD: Control of stomatal distribution on the  Arabidopsis leaf surface. Science 2002, 296:1697-1700. The authors of this paper describe the cloning and expression pattern of the TMM gene. On the basis of the similarity of TMM to CLV2 and the expression of TMM in stomatal precursors and their neighbors, the authors suggest that TMM is required to receive an orienting signal. They propose a model in which TMM expression denes a stem-cell compartment that is analogous to that at the shoot meristem. Von Groll U, Berger D, Altmann T: The subtilisin-like serine protease SDD1 mediates cell-to-cell signaling during Arabidopsis stomatal development. Plant Cell 2002, 14:1527-1539. The localization of SDD1 is explored using RNA in-situ and reporter constructs. Constitutive expression of SDD1 leads to a phenotype that is opposite to that of the sdd1 loss-of-function mutant. Plants that express SDD1 constitutively have leaves that have a lowered stomatal index and many arrested precursors. The authors use lines that constitutively express SDD1 in epistasis tests with other stomatal mutants to conclude that TMM and SDD1 may work in the same signaling pathway. 10. Nadeau JA, Sack FD: Stomatal development: cross talk puts mouths in place. Trends Plant Sci 2003, 8:294-299. 11. Larkin JC, Brown ML, Schiefelbein J: How do cells know what they want to be when they grow up? Annu Rev Plant Biol 2003, 54:403-430. 12. De Veylder L, Beeckman T, Beemster GT, Krols L, Terras F, Landrieu I, van der Schueren E, Maes S, Naudts M, Inze D: Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell 2001, 13:1653-1668. 13. Dewitte W, Riou-Khamlichi C, Scoeld S, Healy JM, Jacqmard A, Kilby NJ, Murray JA: Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3. Plant Cell 2003, 15:79-92. 14. De Veylder L, Beeckman T, Beemster GT, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A, Engler G et al.: Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2FaDPa transcription factor. EMBO J 2002, 21:1360-1368. 15. Saibo NJ, Vriezen WH, Beemster GT, Van Der Straeten D: Growth  and stomatal development of Arabidopsis hypocotyls are controlled by gibberellins and modulated by ethylene and auxins. Plant J 2003, 33:989-1000. The combinatorial effects of gibberellin, auxin and ethylene are explored using the growth of the Arabidopsis hypocotyl as a model. Gibberellin promotes the formation of stomata in a tissue-dependent manner. Elevated GA levels lead to extra stomata, but only within the cell les that normally make them. Reduced GA signaling leads to a reduced number of hypocotyl stomata. 16. Geisler M, Yang M, Sack FD: Divergent regulation of stomatal initiation and patterning in organ and suborgan regions of the Arabidopsis mutants too many mouths and four lips. Planta 1998, 205:522-530. www.sciencedirect.com 9. 

32 Growth and development

expressed differentially in epidermal cells or vascular tissues of maize. Plant Cell 2003, 15:583-596. This paper and [34] describe a technique that is based on the laser dissection of plant tissues. The cells that are captured are suitable for RNA extraction and RNA amplication, and may be used for microarrays to identify genes that are specically upregulated in specialized cell types, such as meristemoids or guard cells. The Nakazono paper contains an extensive list of genes that are specic to the maize epidermis. A subset of these is likely to be required for stomatal identity.

36. Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al.: Genomewide insertional mutagenesis of Arabidopsis thaliana. Science 2003, 301:653-657. 37. Lechelt-Kunze C, Meissner RC, Drewes M, Tietjen K: Flufenacet herbicide treatment phenocopies the ddlehead mutant in Arabidopsis thaliana. Pest Manag Sci 2003, 59:847-856.

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