Stomatal Development in Wild-Type, mpk6^−/− MPK3RNAi, and Rescued mpk3^−/− mpk6^−/− Seedlings.
(A), (C), and (E) Seedlings at 7 d after germination.
(B), (D), and (F) Confocal images of the abaxial epidermis of developing cotyledons.
Cell outlines were visualized with propidium iodide staining. The distribution of stomata on the cotyledons of wild-type seedlings follows the one-cell spacing rule. By contrast, cotyledons of mpk6^−/− MPK3RNAi and rescued mpk3^−/− mpk6^−/− seedlings are covered with excessively clustered stomata. Bars = 1 mm in (A), (C), and (E) and 10 μm in (B), (D), and (F).

Gain-of-Function MAPKK Suppresses Stomatal Development through Endogenous MPK3 and MPK6.
(A) Without Dex induction, stomatal development in Dex-inducible GVG-Nt-MEK2^DD transgenic seedlings is the same as that in the wild type (cf. Figure 2A).
(B) With Dex (0.02 μM) induction, epidermal cells of GVG-Nt-MEK2^DD transgenic seedlings rarely undergo asymmetric cell divisions, and no stomatal differentiation is observed.
(C) and (D) Loss of function of either MPK3 (C) or MPK6 (D) reverses the no-stomate phenotype of GVG-Nt-MEK2^DD transgenic seedlings.
Bars = 10 μm.

Time-Course Analysis of Epidermal Development in GVG-Nt-MEK2^DD Seedlings and the Rescued mpk3^−/− mpk6^−/− Seedlings.
(A) Without Dex induction, stomatal development in GVG-Nt-MEK2^DD seedlings is the same as in the wild type. With Dex (0.02 μM) induction, epidermal cells rarely undergo asymmetric cell divisions, and no stomatal differentiation is observed.
(B) In wild-type seedlings, stomatal development follows the one-cell spacing rule. However, in the rescued mpk3^−/− mpk6^−/− seedlings, meristematic precursor cells undergo ectopic cell divisions that produce near-isodiametric progeny, which eventually all differentiate into stomata.
The abaxial epidermis of developing cotyledons was imaged by confocal microscopy after staining with propidium iodide. dpg, days after germination. Bars = 10 μm.

Stomatal Development and Patterning Defects in MKK4 and MKK5 Loss-of-Function Seedlings.
Confocal images of the abaxial epidermis of developing cotyledons at 7 d after germination.
(A) Stomatal development follows the one-cell spacing rule in vector control transgenic plants.
(B) and (C) MKK4RNAi (B) and MKK5RNAi (C) transgenic seedlings have stomatal clusters with two to three stomata.
(D) Cotyledons of tandem MKK4-MKK5RNAi transgenic seedlings are covered by stomata; no jigsaw puzzle–like pavement cells are observed. The inset shows a representative MKK4-MKK5RNAi transgenic seedling.
Bars = 10 μm except for the inset in (D), where the bar = 1 mm.

Epistatic Interaction of YDA, Nt-MEK2^DD, MPK3, and MPK6.
(A) Loss of function of YDA results in a clustered-stomata phenotype.
(B) Gain-of-function GVG-Nt-MEK2^DD suppresses the clustered stomata phenotype in yda^−/−. The GVG-Nt-MEK2^DD yda^−/− double mutant is labeled as DDyda^−/− for simplicity.
(C) yda^−/− plants have an extremely dwarfed stature, compact rosette leaves, and an abnormal clustered inflorescence.
(D) DDyda^−/− plants are approximately the same size as the control DD (GVG-Nt-MEK2^DD) plant and have normal expanded rosette and extended inflorescence development.
(E) MPK3 and MPK6 are activated in the ΔN-YDA mutant, as shown by in-gel kinase assay of MAPK activity. Ler-0, Landsberg erecta.
Bars = 10 μm in (A) and (B) and 1 cm in (C) and (D).

Expression of Stomatal Cell Fate Marker Genes in Loss- and Gain-of-Function MAPK Cascade Mutants.
(A) In the wild type, expression of the stomatal cell fate marker gene ERL1:GUS is restricted to the progeny of cells that divide asymmetrically (meristemoids, GMCs, young guard cells, neighbor cells/stomata lineage ground cells, and mature guard cells).
(B) In the rescued mpk3^−/− mpk6^−/−, ERL1:GUS expression is detected in all of the small near-isodiametric cells.
(C) Stomatal cell fate genes are upregulated in the MKK4-MKK5RNAi loss-of-function mutant and downregulated in the GVG-Nt-MEK2^DD gain-of-function mutant (induced by 0.02 μM Dex). The levels of mRNA in pooled seedlings (>30) were determined by quantitative RT-PCR. After being normalized to EF1α, the relative levels to those in control seedlings are shown. Three biological repeats were performed, and similar results were obtained. Col-0, Columbia.
(D) A model depicts the function of the YDA-MKK4/MKK5-MPK3/MPK6 cascade in regulating asymmetric cell division and coordinating cell fate specification. This MAPK cascade functions as a rheostat-like molecular switch in coordinating stomatal cell fate specification. In the gain-of-function (GOF) MAPK cascade, high MAPK activity suppresses the asymmetric cell division of the MMC and the differentiation of stomata. In wild-type plants, the MMC undergoes normal asymmetric cell division; the coordinated cell fate specification between meristemoids and neighbor cells helps maintain the one-cell spacing rule. The loss-of-function (LOF) MAPK cascade disrupts the orientation, the frequency, and the polarity of asymmetric cell divisions, resulting in cell fate coordination defects and clustered stomata.