Introduction to stomatal development
During postembryonic development, the plant epidermis generates several distinct cell types, including stomatal guard cells. Stomata act as valves through which atmospheric CO2 can enter the plant and O2 and water vapor can escape, and each consists of paired guard cells surrounding a central pore. Although the overall pattern of stomata on the leaf surface is variable among species, stomata are almost universally patterned according to a one-cell spacing rule, such that at least one intervening epidermal cell separates nearby stomata from one another. Both the patterned distribution of stomatal complexes and the differentiation of the guard cells themselves are associated with asymmetric, oriented divisions.

Arabidopsis stomatal development requires asymmetric entry divisions of MMCs to create meristemoids (M), which may self-renew via amplifying divisions or differentiate into GMCs  that divide symmetrically to form guard cells (GCs). Spacing divisions of MMCs next to stomatal precursors are oriented as well as asymmetric. 

Arabidopsis stomatal development
Arabidopsis stomatal precursors arise from asymmetric divisions of an apparently random subset of cells in the immature leaf epidermis. As of yet, no morphological or gene expression patterns have unambiguously marked this cell population, so these cells (meristemoid mother cells, MMCs) are defined retrospectively. An asymmetric entry division of the MMC creates a meristemoid and a stomatal lineage ground cell (SLGC) as its smaller and larger daughters, respectively.  The meristemoid has limited self-renewing capacity and may continue to undergo asymmetric amplifying divisions, with the smaller daughter of each division round retaining meristemoid identity and the larger becoming an SLGC. Eventually, the meristemoid will differentiate into a guard mother cell (GMC) that undergoes symmetric division to produce the paired guard cells of the stoma. The SLGCs produced at various stages of lineage progression may differentiate into large, lobed, pavement cells, or may also become MMCs, dividing asymmetrically to create secondary meristemoids.  These secondary entry divisions, called spacing divisions, lead to a sort of “fill in” pattern where new meristemoids arise among mature precursors and stomata. To maintain the one-cell spacing pattern, the spacing divisions creating secondary meristemoids are not only asymmetric, but are oriented such that the new meristemoid forms distal to the existing stoma/precursor. 

Signaling during stomatal development
Cell divisions that generate secondary meristemoids are oriented relative suggesting that external signals might play a key role in stomatal development, and indeed, several receptors and receptor like-kinases are required for the maintenance of one-cell spacing. The LRR-receptor like protein TOO MANY MOUTHS (TMM) was the first component of this network to be identified (Nadeau and Sack, 2002) and has subsequently been joined by potential LRR-RLK signaling partners ERECTA, ERECTA-LIKE1 and ERECTA-LIKE2 (collectively referred to as the ERECTA family, or ERf) (Shpak et al., 2005). Loss of TMM or ERf function results in the production of excess stomata arranged in clusters, and these factors appear both to orient asymmetric division and repress stomatal fate at various stages of lineage progression.

Loss-of-function mutations in two related genes encoding putative ligands, EPIDERMAL PATTERNING FACTOR 1 (EPF1, Hara et al., 2007) and EPF2 (Hunt and Gray, 2009), also confer defects in stomatal patterning. EPF2, which is expressed in early stomatal lineage cells, appears to limit the number of cells that undergo lineage entry. EPF1, on the other hand, is expressed in relatively late stomatal precursors and primarily regulates orientation of spacing divisions. Interestingly, epistasis analyses indicate that EPF1 activity depends on both TMM and the ERf but that EPF2 possesses some TMM-independent functions, potentially reflecting specificity in ligand-receptor interactions (Hunt and Gray, 2009).

Scheme of major signaling and transcriptional inputs into stomatal development; arrows and T-bars indicate positive and negative effects, respectively, and are directed to the stage in which they have been demonstrated to act.

Transcriptional regulation of stomatal development
Transcription factors also play a critical role in asymmetric division and cell fate establishment in the stomatal lineage (Kanaoka et al., 2008; Kutter et al., 2007; Lai et al., 2005; MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007). One set of these transcription factors belongs to the conserved bHLH family, which is found in animal as well as plant systems. Based upon loss and gain of function phenotypes, five bHLH transcription factors serve as major cell fate regulators in the stomatal lineage (Kanaoka et al., 2008; MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007), and three of these five (SPEECHLESS (SPCH), MUTE, and FAMA) display restricted expression patterns that correlate with specific stages of lineage progression (MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007).

SPCH and MUTE are responsible for the initiation and termination, respectively, of asymmetric divisions in the stomatal lineage. SPCH is expressed transiently in a subset of epidermal cells, many of which undergo entry (and later, spacing) divisions to produce meristemoids, and strong spch mutations block stomatal lineage initiation (MacAlister et al., 2007; Pillitteri et al., 2007). SPCH is a direct target of MAP kinase phosphorylation, and although overexpression of wild-type SPCH confers little phenotype, expression of SPCH variants lacking phosphorylation sites induces excess asymmetric divisions and meristemoid overproduction (Lampard et al., 2008). MUTE, which acts in late meristemoids to terminate amplifying division, is not subject to similar MAPK control and can, upon overexpression, generate an epidermis composed almost entirely of stomata by inducing all epidermal cell types to become GMCs, which then divide to become pairs of  guard cells (MacAlister et al., 2007; Pillitteri et al., 2007).

After the initial decision: how to execute an asymmetric division
The identification of signaling components and transcription factors (at least one of which is a direct target of the signaling pathways) as key regulators of asymmetric division in the Arabidopsis stomatal lineage fits well with what is observed in later rounds of meristemoid production, when new precursors must be intercalated with existing stomata in a patterned fashion. Identification of these factors, however, does not explain how the relatively high-level ‘decision’ process is translated into asymmetry at the cell biological level. BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), an unequally segregated protein, may offer insight into the problem of executing an asymmetric division (Dong et al., 2009).

Polarized expression pattern of BASL at cell periphery and in nuclei of asymmetrically dividing stomatal lineage cells

In the absence of BASL, stomatal lineage divisions show reduced physical and fate asymmetry, generating daughter cells inappropriately similar in size, marker expression, and ultimate identity (Dong et al., 2009). BASL encodes a novel, plant-specific protein possessing no recognizable functional domains. BASL is expressed primarily in asymmetrically dividing stomatal lineage cells, and it is the dynamic behavior of BASL protein within these cells that is most informative.  Prior to a typical asymmetric division, BASL is found in the nucleus, but begins to accumulate in a cortical crescent; this crescent is always positioned so that it is inherited by the larger daughter. After division, the smaller daughter has BASL in the nucleus, while the larger has BASL both in the nucleus and at the cortex (and Dong et al., 2009). Time-lapse experiments tracing BASL dynamics in single cells revealed two possible developmental trajectories for each daughter. The smaller (meristemoid) can become a GMC, losing nuclear BASL in the process, or it can divide again asymmetrically after first establishing a new cortical crescent. The larger (SLGC) can differentiate into a non-stomatal epidermal cell, losing nuclear BASL (but sometimes retaining the cortical crescent), or it can divide asymmetrically to form a secondary meristemoid—a fate that correlated with retention of both nuclear and cortical BASL. Production of a secondary meristemoid requires that the SLGC reorient its axis of polarity in order to maintain one-cell spacing. This reorientation is reflected in the cortical BASL crescent’s relocation to the opposite side of the cell, ensuring that it is distal to the newly forming meristemoid.

Scheme of BASL localization and its correlation with cell fate in the stomatal lineage

Outstanding questions
In the past decade work from many groups has generated a developmental framework for Arabidopsis stomatal development and identified many of the crucial genes.  There are undoubtedly more regulators to be found; these may include new components of signaling pathways that modulate spacing and stomatal density, other transcriptional regulators that promote specific cell identities and certainly more components that generate the physical asymmetries of stomatal lineage divisions.  Once we have populated the lists of “stomatal genes”, however, it is critical to begin building connections among these genes.  Our group is taking a variety of experimental approaches to expand the repertoire of stomatal regulators and understand how they work together. Vignettes about some of our favorite current projects can be accessed by clicking the icons on the lab research page.

Relevant publications (Bergmann lab publications in bold)

Bergmann, D. C., Lukowitz, W., Somerville, C. R. (2004) Stomatal development and pattern controlled by a MAPKK kinase. Science. 304(5676), 1494-7.

Bergmann, D. C. and Sack, F. D. (2007). Stomatal development. Annu. Rev. Plant. Biol. 58,163-81.

Dong J, Macalister CA, Bergmann DC (2009) BASL Controls Asymmetric Cell Division in Arabidopsis. Cell. 2009 Jun 10. PMID: 19523675

Hara, K., Kajita, R., Torii, K. U., Bergmann, D. C., Kakimoto, T. (2007). The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev. 21(14), 1720-1725.

Hunt, L  and  J. Gray. (2009) Signaling Peptide EPF2 Controls Asymmetric Cell Divisions During Stomatal Development. Curr Biol

Kanaoka, M. M., Pillitteri, L. J., Fujii, H., Yoshida, Y., Bogenschutz, N. L., Takabayashi, J., Zhu, J. K., Torii, K. U. (2008). SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to arabidopsis stomatal differentiation. Plant Cell 20(7), 1775-1785.

Kutter, C., Schob, H., Stadler, M., Meins, F.,Jr, Si-Ammour, A. (2007). MicroRNA-mediated regulation of stomatal development in arabidopsis. Plant Cell 19(8), 2417-2429.

Lai, L. B., Nadeau, J. A., Lucas, J., Lee, E. K., Nakagawa, T., Zhao, L., Geisler, M., Sack, F. D. (2005). The arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell .

Lampard, G. R., Macalister, C. A., Bergmann, D. C. (2008). Arabidopsis stomatal  initiation is controlled by MAPK-mediated regulation of the bHLH, SPEECHLESS. Science 2008 Nov 14;322(5904):1113-6. PMID: 19008449

MacAlister, C. A., Ohashi-Ito, K., Bergmann, D. C. (2007). Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445(7127), 537-540.

Nadeau, J. A. and Sack, F. D. (2002). Control of stomatal distribution on the Arabidopsis leaf surface. Science 296(5573), 1697-1700.

Ohashi-Ito, K. and Bergmann, D. (2006). Arabidopsis FAMA controls the final Proliferation/Differentiation switch during stomatal development. Plant Cell 18(10), 2493-2505.

Pillitteri, L. J., Sloan, D. B., Bogenschutz, N. L., Torii, K. U. (2007). Termination of asymmetric cell division and differentiation of stomata. Nature 445(7127), 501-505.

Shpak, E. D., McAbee, J. M., Pillitteri, L. J., Torii, K. U. (2005). Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309(5732), 290-293.

Wang, H., Ngwenyama, N., Liu, Y., Walker, J. C., Zhang, S. (2007). Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19(1), 63-73.