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. 2007 Jan;19(1):63-73.
doi: 10.1105/tpc.106.048298. Epub 2007 Jan 26.

Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis

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Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis

Huachun Wang et al. Plant Cell. 2007 Jan.

Abstract

Stomata are specialized epidermal structures that regulate gas (CO(2) and O(2)) and water vapor exchange between plants and their environment. In Arabidopsis thaliana, stomatal development is preceded by asymmetric cell divisions, and stomatal distribution follows the one-cell spacing rule, reflecting the coordination of cell fate specification. Stomatal development and patterning are regulated by both genetic and environmental signals. Here, we report that Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MPK3) and MPK6, two environmentally responsive mitogen-activated protein kinases (MAPKs), and their upstream MAPK kinases, MKK4 and MKK5, are key regulators of stomatal development and patterning. Loss of function of MKK4/MKK5 or MPK3/MPK6 disrupts the coordinated cell fate specification of stomata versus pavement cells, resulting in the formation of clustered stomata. Conversely, activation of MKK4/MKK5-MPK3/MPK6 causes the suppression of asymmetric cell divisions and stomatal cell fate specification, resulting in a lack of stomatal differentiation. We further establish that the MKK4/MKK5-MPK3/MPK6 module is downstream of YODA, a MAPKKK. The establishment of a complete MAPK signaling cascade as a key regulator of stomatal development and patterning advances our understanding of the regulatory mechanisms of intercellular signaling events that coordinate cell fate specification during stomatal development.

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Figures

Figure 1.
Figure 1.
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).
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
Gain-of-Function MAPKK Suppresses Stomatal Development through Endogenous MPK3 and MPK6. (A) Without Dex induction, stomatal development in Dex-inducible GVG-Nt-MEK2DD 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-MEK2DD 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-MEK2DD transgenic seedlings. Bars = 10 μm.
Figure 4.
Figure 4.
Epistatic Interaction of YDA, Nt-MEK2DD, MPK3, and MPK6. (A) Loss of function of YDA results in a clustered-stomata phenotype. (B) Gain-of-function GVG-Nt-MEK2DD suppresses the clustered stomata phenotype in yda−/−. The GVG-Nt-MEK2DD 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-MEK2DD) 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).
Figure 5.
Figure 5.
Time-Course Analysis of Epidermal Development in GVG-Nt-MEK2DD Seedlings and the Rescued mpk3−/− mpk6−/− Seedlings. (A) Without Dex induction, stomatal development in GVG-Nt-MEK2DD 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.
Figure 6.
Figure 6.
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-MEK2DD 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.

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