2265 RESEARCH ARTICLE INTRODUCTION Plants conquered land approximately 400 million years ago (Edwards et al., 1998). Correlated with this expansion in habitat was the development of an epidermis that, although made highly impermeable by a lipid-rich cuticle, still permitted the exchange of external CO 2 for internal O 2 and water vapor. Microscopic epidermal valves called stomata were the structural innovations that allow this regulated exchange (Edwards et al., 1998). Stomata are present on the aerial surfaces of all large land plants. At a minimum, stomata consist of two guard cells, a pore and an underlying airspace. In many species, however, the stomatal complex includes subsidiary cells adjacent to the guard cells. Both guard and subsidiary cells are morphologically distinct from other epidermal cell types. Since their appearance in the fossil record (contemporaneous with the appearance of land plants), stomatal densities and distributions have changed significantly, but guard cell morphology has remained quite constant (Edwards et al., 1998). In general, there are only two broad classes of stomatal guard cells: the kidney-shaped cells found most plant species and the dumbbell- shaped guard cells found in grasses (Evert, 2006) (see Fig. 1A). Grass stomata, as described as early as 1881 (Campbell, 1881), have both a pair of dumbbell-shaped guard cells and associated subsidiary cells. Grass stomata are usually arranged in linear files and this final arrangement reflects the developmental process that created them (Fig. 1B). Grass stomata may be on either the top (adaxial) or bottom (abaxial) side of the expanded leaf, but in many species they are preferentially or exclusively abaxial. Grass guard cell morphology is thought to be derived from the kidney-shaped guard cells found in mosses, ferns, gymnosperms, dicots, and some monocots. For convenience, we will refer to this form as ‘dicot’. Dicot stomata often lack subsidiary cells, but they may also have two or more such cells. In contrast to the linear arrangement typical of grass stomata, dicot stomata are scattered on leaf surfaces, a pattern that reflects their ‘dispersed’ mode of development (Fig. 1B). This distribution pattern is not random, however, and stomatal architecture and patterns are valuable taxonomic characters for both living and fossilized plants (Garland, 1984; Stebbins, 1960). Developmental pathways for stomata in Arabidopsis In Arabidopsis, stomatal development requires a series of asymmetric and symmetric cell divisions in a specialized epidermal cell lineage. Stomatal development is initiated by an asymmetric division in a protodermal cell to produce a small meristemoid and a larger sister cell. The meristemoid is a self-renewing cell and can continue asymmetric divisions. However, it possesses only transient ‘stem cell-like’ properties and after one to three divisions differentiates into a guard mother cell (GMC). The GMC undergoes a single symmetric division to produce a pair of guard cells (Fig. 1B) (reviewed by Bergmann and Sack, 2007). Stomatal development proceeds roughly in an apical-basal gradient with the more mature stages near the tip, but this is not absolute because sister cells of the stomatal precursors might divide later, intercalating new stomata into areas where stomata previously formed (Fig. 1B). Stomata are formed via similar developmental mechanisms on both the abaxial and adaxial leaf surfaces. Three of the positive regulators that direct this three-step sequential stomatal development in Arabidopsis are the closely related basic helix-loop-helix (bHLH) domain transcription factors FAMA, MUTE and SPEECHLESS (SPCH) (MacAlister et al., 2007; Ohashi-Ito and Bergmann, 2006; Pillitteri et al., 2007). SPCH is expressed in many young epidermal cells and controls the first asymmetric division of protodermal cells to initiate the stomatal lineage (MacAlister et al., 2007; Pillitteri et al., 2007). MUTE is highly expressed in meristemoids and is required for termination of meristemoid stem cell identity and the transition to GMC fate (Pillitteri et al., 2007). Finally, FAMA is expressed in GMCs and regulates the last stage of stomatal development by promoting the symmetric differentiation of a GMC into a guard cell pair (Ohashi- Ito and Bergmann, 2006). FAMA, MUTE and SPCH therefore act as molecular switches controlling major cell fate transitions during stomatal development. Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses Tie Liu 1 , Kyoko Ohashi-Ito 1,2 and Dominique C. Bergmann 1, * Stomata are adjustable pores in the plant epidermis that regulate gas exchange between the plant and atmosphere; they are present on the aerial portions of most higher plants. Genetic pathways controlling stomatal development and distribution have been described in some detail for one dicot species, Arabidopsis, in which three paralogous bHLH transcription factors, FAMA, MUTE and SPCH, control discrete sequential stages in stomatal development. Orthologs of FAMA, MUTE and SPCH are present in other flowering plants. This observation is of particular interest when considering the grasses, because both the morphology of guard cells and their tissue distributions differ substantially between Arabidopsis and this group. By examining gene expression patterns, insertional mutants and cross-species complementation studies, we find evidence that FAMA function is conserved between monocots and dicots, despite their different stomatal morphologies, whereas the roles of MUTE and two SPCH paralogs are somewhat divergent. KEY WORDS: Stomata, Monocotyledon, Rice, Arabidopsis, Maize, bHLH Development 136, 2265-2276 (2009) doi:10.1242/dev.032938 1 Biology Department, 371 Serra Mall, Stanford University, Stanford, CA 94305- 5020, USA. 2 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan. *Author for correspondence (e-mail: dbergmann@stanford.edu) Accepted 24 April 2009 DEVELOPMENT