Stomata regulate gas exchange and are distributed across the leaf epidermis with characteristic spacing. Arabidopsis stomata are produced by asymmetric cell divisions. Mutations in the gene TOO MANY MOUTHS (TMM) disrupt patterning by randomizing the plane of formative asymmetric divisions and by permitting ectopic divisions. TMM encodes a leucine-rich repeat-containing receptor-like protein expressed in proliferative postprotodermal cells. TMM appears to function in a position-dependent signaling pathway that controls the plane of patterning divisions as well as the balance between stem cell renewal and differentiation in stomatal and epidermal development.

Wild-type stomata are spaced by intervening cells, a pattern disrupted in the Arabidopsis mutant too many mouths ( tmm ). To determine the mechanism of wild-type spacing and how tmm results in pattern violations, we analyzed the behavior of cells through time by using sequential dental resin impressions. Meristemoids are stomatal precursors produced by asymmetric division. We show that wild-type patterning largely results when divisions next to a preexisting stoma or precursor are oriented so that the new meristemoid is placed away. Because this placement is independent of cell lineage, these divisions may be oriented by cell-cell signaling. tmm randomizes this orientation and releases a prohibition on asymmetric division in cells at specific locations, resulting in stomatal clusters. TMM is thus necessary for two position-dependent events in leaves: the orientation of asymmetric divisions that pattern stomata, and the control of which cells will enter the stomatal pathway. In addition, our findings argue against most previous hypotheses of wild-type stomatal patterning.

The two guard cells of a stoma are produced by a single symmetric division just before terminal differentiation. Recessive mutations in the FOUR LIPS (FLP) gene abnormally induce at least four guard cells in contact with one another. These pattern defects result from a persistence of precursor cell identity that leads to extra symmetric divisions at the end of the cell lineage. FLP is likely to be required for the correct timing of the transition from cell cycling to terminal differentiation. FLP encodes a two-repeat (R2R3) MYB protein whose expression accumulates just before the symmetric division. A paralogous gene, MYB88, overlaps with FLP function in generating normal stomatal patterning. Plants homozygous for mutations in both genes exhibit more severe defects than flp alone, and transformation of flp plants with a genomic MYB88 construct restores a wild-type phenotype. Both genes compose a distinct and relatively basal clade of atypical R2R3 MYB proteins that possess an unusual pattern of amino acid substitutions in their putative DNA binding domains. Our results suggest that two related transcription factors jointly restrict divisions late in the Arabidopsis thaliana stomatal cell lineage.

Homeobox genes are master regulatory genes that specify the body plan and control development of many eukaryotic organisms, including plants. We isolated and characterized a cDNA designated ATML1 (for Arabidopsis thaliana meristem L1 layer) that encodes a novel homeodomain protein. The ATML1 protein shares high sequence homology inside and outside of the homeodomain with both the Phalaenopsis O39 and the Arabidopsis GLABRA2 (GL2) homeodomain proteins, which together define a new class of plant homeodomain-containing proteins, designated HD-GL2. The ATML1 gene was first expressed in the apical cell after the first asymmetric division of the zygote and continued to be expressed in all proembryo cells until the eight-cell stage. In the 16-cell proembryo, the ATML1 gene showed a distinct pattern of expression, with its mRNA becoming restricted to the protoderm. In the torpedo stage of embryo development, ATML1 mRNA disappeared altogether but reappeared later only in the L1 layer of the shoot apical meristem in the mature embryo. After germination, this L1 layer-specific pattern of expression was maintained in the vegetative shoot apical meristem, inflorescence, and floral meristems, as well as in the young floral organ primordia. Finally, ATML1 mRNA accumulated in the protoderm of the ovule primordia and integuments and gradually became restricted in its expression to the endothelium surrounding the embryo sac. We propose that ATML1 may be involved in setting up morphogenetic boundaries of positional information necessary for controlling cell specification and pattern formation. In addition, ATML1 provides an early molecular marker for the establishment of both apical-basal and radial patterns during plant embryogenesis.

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