(A) Schematic of key stages in stomatal development mediated by bHLHs SPCH, MUTE and FAMA. Cell types are labeled as: meristemoid mother cell (MMC), meristemoid (M), stomatal lineage ground cell (SLGC), guard mother cell (GMC), guard cell (GC), pavement cell (PC). (B and C) Expression of FAMA and RBR in GMCs and GCs. Confocal images of 5-days post germination (dpg) cotyledon of FAMAp:GFP-FAMA (B, in green) and RBRp:RBR-CFP (C, in green). Inset in (B) is a fama mutant GMC at 10-dpg. Cell outlines (purple) were visualized with propidium iodide. (D and E) Reduction in RBR level leads to extra divisions in GCs. Confocal images of a co-suppressed RBRp:RBR-CFP line (D) and FAMAp:amiRBR expressing a CDKA1;1 reporter (green) (E). Yellow arrowheads in (D) indicate ectopic cell divisions. Cell outlines (white) were visualized with propidium iodide. (F) ClustalW2-based protein alignment of the LxCxE motif among FAMA relatives. (G and H) FAMA interacts with RBR in vivo and in vitro through its LxCxE motif. Representative images (G, left) and quantified data (G, right; rep: replicate) of Bimolecular Fluorescence Complementation (BIFC) analysis between FAMA and RBR. Pairs of CYCD/CYCDLGK-RBR and FAMA-bHLH93 (Ohashi-Ito and Bergmann, 2006) were used as controls. (H) Yeast two-hybrid interaction assays between FAMA and RBR. (I) Complementation of seedling lethality in fama mutants by FAMALGK (FAMAp:FAMALGK; fama). (J–L) Diversity of GC defects in adaxial cotyledon epidermis of 12-dpg FAMALGK. DIC images of a mature GC showing strong phenotype (J, false red colors indicate different GC units within another) and a broader view of GCs with different defects (K). Key: ectopic asymmetric divisions (arrowheads), new GC units (asterisks), properly spaced divisions and GC units (brackets). Inset shows a lobed GC. (L) Quantitation of different classes of GC defects (cartoons on Y-axis) in FAMALGK at 6, 9 and 12-dpg. Bars represent the percentages of each class over all GCs on adaxial cotyledons. All images are at the same magnification (including insets in B and K). Scale bar, 10 μm.