The establishment of new cell lineages during development often requires a symmetry-breaking event. An asymmetric division in the epidermis of plants initiates a lineage that ultimately produces stomatal guard cells. Stomata are pores in the epidermis that serve as the main conduits for gas exchange between plants and the atmosphere; they are critical for photosynthesis and exert a major influence on global carbon and water cycles. Recent studies implicated intercellular signalling in preventing the inappropriate production of stomatal complexes. Genes required to make stomata, however, remained elusive. Here we report the identification of a gene, SPEECHLESS (SPCH), encoding a basic helix-loop-helix (bHLH) transcription factor that is necessary and sufficient for the asymmetric divisions that establish the stomatal lineage in Arabidopsis thaliana. We demonstrate that SPCH and two paralogues are successively required for the initiation, proliferation and terminal differentiation of cells in the stomatal lineage. The stomatal bHLHs define a molecular pathway sufficient to create one of the key cell types in plants. Similar molecules and regulatory mechanisms are used during muscle and neural development, highlighting a conserved use of closely related bHLHs for cell fate specification and differentiation.

Coordination between cell proliferation and differentiation is essential to create organized and functional tissues. Arabidopsis thaliana stomata are created through a stereotyped series of symmetric and asymmetric cell divisions whose frequency and orientation are informed by cell-cell interactions. Receptor-like proteins and a mitogen-activated protein kinase kinase kinase were previously identified as negative regulators of stomatal development; here, we present the characterization of a bona fide positive regulator. FAMA is a putative basic helix-loop-helix transcription factor whose activity is required to promote differentiation of stomatal guard cells and to halt proliferative divisions in their immediate precursors. Ectopic FAMA expression is also sufficient to confer stomatal character. Physical and genetic interaction studies combined with functional characterization of FAMA domains suggest that stomatal development relies on regulatory complexes distinct from those used to specify other plant epidermal cells. FAMA behavior provides insights into the control of differentiation in cells produced through the activity of self-renewing populations.

Stomata are innovations of land plants that allow regulated gas exchange. Stomatal precursor cells are produced by asymmetric cell division, and once formed, signal their neighbors to inhibit the formation of stomatal precursors in direct contact. We report a gene of Arabidopsis thaliana, EPIDERMAL PATTERNING FACTOR 1 (EPF1) that encodes a small secretory peptide expressed in stomatal cells and precursors and that controls stomatal patterning through regulation of asymmetric cell division. EPF1 activity is dependent on the TOO MANY MOUTHS receptor-like protein and ERECTA family receptor kinases, suggesting that EPF1 may provide a positional cue interpreted by these receptors. Although multicellularity evolved independently in animals and plants (Baldauf 2003), both utilize asymmetric cell division for creating new cell lineages during development (Scheres and Benfey 1999;Betschinger and Knoblich 2004). In plants, stomatal development offers an excellent, tractable system to study asymmetric cell division. Stomata consist of two guard cells and a pore between them for gas exchange. The guard cells are produced through a series of asymmetric and symmetric cell divisions that not only produce the differentiated cells, but control the overall density and pattern of stomata on the organ surface (Bergmann 2003). In most dicot leaves, stomata follow a "one-cell-spacing" rule in which two stomata are separated by at least one intervening nonstomatal epidermal cell (Sachs 1991). This spacing is hypothesized to be important for efficient gas exchange (Nadeau and Sack 2002b). The first morphologically discernible stomatal precursor cell is the meristemoid, which is the smaller, triangular-shaped daughter cell produced by asymmetric cell division. The larger daughter cell may differentiate into a nonstomatal epidermal cell (pavement cell) or it may undergo its own asymmetric cell division, creating a satellite meristemoid. A meristemoid has a self-renewing capability and can continue asymmetric divisions, but eventually converts into a guard mother cell (GMC), which then divides symmetrically to form two guard cells that constitute a stoma. The major enforcer of the one-cell-spacing pattern appears to be a signal sent from stomata and precursors (guard cells, GMCs, and mature meristemoids) to undifferentiated neighbor cells that influences the plane of cell division (Geisler et al. 2000).A number of key regulators of the asymmetric cell divisions that ensure the one-cell-spacing rule and control sto- . Because TMM mutations appear to affect only stomatal development, whereas the ER family is important for a broader range of biological processes (Shpak et al. 2004), it may be that a presumed TMM/ER family complex would rely on TMM for specificity of the positional information and the kinasecontaining ER family proteins to transmit signals to downstream elements. One potential downstream target is YDA, a mitogen-activated protein kinase kinase kinase (MAPKKK) that has been shown to act as a switch between stomatal and pavemen...

Stomata are epidermal structures that modulate gas exchange between a plant and its environment. During development, stomata are specified and positioned nonrandomly by the integration of asymmetric cell divisions and intercellular signaling. The Arabidopsis mitogen-activated protein kinase kinase kinase gene, YODA, acts as part of a molecular switch controlling cell identities in the epidermis. Null mutations in YODA lead to excess stomata, whereas constitutive activation of YODA eliminated stomata. Transcriptome analysis of seedlings with altered YODA activity was used to identify potential stomatal regulatory genes. A putative transcription factor from this set was shown to regulate the developmental behavior of stomatal precursors.

Plants must coordinately regulate biochemistry and anatomy to optimize photosynthesis and water use efficiency. The formation of stomata, epidermal pores facilitating gas exchange, is highly coordinated with other aspects of photosynthetic development. However, the signaling pathways controlling stomata development are not fully understood1,2, although Mitogen Activated Protein Kinase (MAPK) signaling is known to play key roles. Here we demonstrate that brassinosteroid (BR) regulates stomatal development by activating the MAPKKK, YODA. Genetic analyses indicate that receptor kinase-mediated BR signaling inhibits development through the GSK3-like kinase BIN2, and BIN2 acts upstream of YODA but downstream of the known ERECTA family of stomatal receptor kinases. Complementary in vitro and in vivo assays show that BIN2 phosphorylates YODA to inhibit YODA phosphorylation of its substrate MKK4, and activities of downstream MAPKs are reduced in BR-deficient mutants but increased by treatment with either BR or GSK3-kinase inhibitor. Our results indicate that BR inhibits stomatal development by alleviating GSK3-mediated inhibition of the MAPK module, providing two key links; that of a plant MAPKKK to its upstream regulators and BR to a specific developmental output.

Stomata, epidermal structures that modulate gas exchange between plants and the atmosphere, play critical roles in primary productivity and the global climate. Positively acting transcription factors and negatively acting mitogen-activated protein kinase (MAPK) signaling control stomatal development in Arabidopsis; however, it is not known how the opposing activities of these regulators are integrated. We found that a unique domain in a basic helix-loop-helix (bHLH) stomatal initiating factor, SPEECHLESS, renders it a MAPK phosphorylation target in vitro and modulates its function in vivo. MAPK cascades modulate a diverse set of activities including development, cell proliferation, and response to external stresses. The coupling of MAPK signaling to SPEECHLESS activity provides cell type specificity for MAPK output while allowing the integration of multiple developmental and environmental signals into the production and spacing of stomata.

SUMMARY Development in multicellular organisms requires the organized generation of differences. A universal mechanism for creating such differences is asymmetric cell division. In plants, as in animals, asymmetric divisions are correlated with the production of cellular diversity and pattern; however, structural constraints imposed by plant cell walls and the absence of homologs of known animal or fungal cell polarity regulators necessitates that plants utilize new molecules and mechanisms to create asymmetries. Here, we identify BASL, a novel regulator of asymmetric divisions in Arabidopsis. In asymmetrically dividing stomatal-lineage cells, BASL accumulates in a polarized crescent at the cell periphery before division, and then localizes differentially to the nucleus and a peripheral crescent in self-renewing cells and their sisters after division. BASL presence at the cell periphery is critical for its function, and we propose that BASL represents a plant-specific solution to the challenge of asymmetric cell division.

Stomata are cellular epidermal valves in plants central to gas exchange and biosphere productivity. The pathways controlling their formation are best understood for Arabidopsis thaliana where stomata are produced through a series of divisions in a dispersed stem cell compartment. The stomatal pathway is an accessible system for analyzing core developmental processes including position-dependent patterning via intercellular signaling and the regulation of the balance between proliferation and cell specification. This review synthesizes what is known about the mechanisms and genes underlying stomatal development. We contrast the functions of genes that act earlier in the pathway, including receptors, kinases, and proteases, with those that act later in the cell lineage. In addition, we discuss the relationships between environmental signals, stomatal development genes, and the capacity for controlling shoot gas exchange.