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. 2009 Nov;21(11):3506–3517. doi: 10.1105/tpc.109.070110

Novel and Expanded Roles for MAPK Signaling in Arabidopsis Stomatal Cell Fate Revealed by Cell Type–Specific Manipulations[C],[W]

Gregory R Lampard a,1, Wolfgang Lukowitz b, Brian E Ellis c, Dominique C Bergmann a,2
PMCID: PMC2798322  PMID: 19897669

Abstract

Mitogen-activated protein kinase (MAPK) signaling networks regulate numerous eukaryotic biological processes. In Arabidopsis thaliana, signaling networks that contain MAPK kinases MKK4/5 and MAPKs MPK3/6 function in abiotic and biotic stress responses and regulate embryonic and stomatal development. However, how single MAPK modules direct specific output signals without cross-activating additional downstream processes is largely unknown. Studying relationships between MAPK components and downstream signaling outcomes is difficult because broad experimental manipulation of these networks is often lethal or associated with multiple phenotypes. Stomatal development in Arabidopsis follows a series of discrete, stereotyped divisions and cell state transitions. By expressing a panel of constitutively active MAPK kinase (MAPKK) variants in discrete stomatal lineage cell types, we identified a new inhibitory function of MKK4 and MKK5 in meristemoid self-renewal divisions. Furthermore, we established roles for MKK7 and MKK9 as both negative and (unexpectedly) positive regulators during the major stages of stomatal development. This has expanded the number of known MAPKKs that regulate stomatal development and allowed us to build plausible and testable subnetworks of signals. This in vivo cell type–specific assay can be adapted to study other protein families and thus may reveal insights into other complex signal transduction pathways in plants.

INTRODUCTION

Mitogen-activated protein kinase (MAPK) signaling networks are found in all eukaryotic organisms and regulate fundamental aspects of biology, including but not limited to cell division, initiation of developmental pathways, response to abiotic and biotic stresses, and triggering programmed cell death (reviewed in Widmann et al., 1999; Chen and Thorner, 2007; Colcombet and Hirt, 2008). In plants, MAPK networks regulate a similar array of processes, but genomic sequence data have revealed that, in comparison to other eukaryotes, plant genomes encode enlarged gene families of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs (Ichimura et al., 2002; Hamel et al., 2006). Furthermore, large-scale gene expression studies indicate that many of these genes are broadly expressed throughout the plant (Schmid et al., 2005; Schmidt, 2007). These extended gene families may have evolved to allow plants, which are sessile, to sense and respond to a continuous flux of environmental conditions. In support of this, the majority of plant MAPK signaling components studied to date have been associated with responses to abiotic and biotic stresses (Colcombet and Hirt, 2008).

Constitutive and ectopic modulation of MAPK signaling pathways in plants is typically associated with pleiotropic phenotypes and/or is lethal (Jin et al., 2003; Liu et al., 2003; Popescu et al., 2009). These effects may be the result of indiscriminate activation of multifunctional kinases that have discrete functions in different cell types. Therefore, to both characterize specific functions of MAPK networks in plants and learn how signal integrity is maintained within these networks, it was necessary to devise a system that allows for cell type–specific modulation of MAPK signaling while providing an accessible means to analyze the effects of these changes.

Stomatal development is an ideal system to study discrete aspects of MAPK signaling networks. Stomata are specialized structures found in the epidermis of aerial tissues of land plants and are the primary conduit for gas and water exchange. MAPK signaling has roles in conveying intrinsic developmental cues to regulate stomatal development and relaying extrinsic environmental signals that influence stomatal physiology and development. For example, MAPK signaling networks are positive regulators of environmental stress–induced stomatal closure and negative regulators of stomatal development (Bergmann et al., 2004; Wang et al., 2007; Neill et al., 2008). Besides influencing stomatal behavior, environmental conditions are also capable of influencing stomatal development (Coupe et al., 2006; Casson and Gray, 2008; Casson et al., 2009). In Arabidopsis thaliana, increased carbon dioxide levels typically decrease the overall stomatal density (number of stomata per unit area) (Coupe et al., 2006), whereas high light intensities increase the stomatal index (number of stomata relative to the total number of cells per unit area; Casson et al., 2009). The MAPK network that contains MKK4/5 and MPK3/6 regulates both the responses to environmental conditions and overall stomatal development (Bergmann et al., 2004; Wang et al., 2007; Colcombet and Hirt, 2008; Lampard et al., 2008). Linking a stress-activated MAPK module to the negative regulation of developmental processes is not surprising; plants arrest development in response to abiotic and biotic stresses as evidenced by stress-induced downregulation of developmentally associated gene expression (Kultz, 2005; Baena-Gonzalez et al., 2007; Baena-González and Sheen, 2008). Thus, using a common MAPK module to regulate both stress responses and stomatal development could allow rapid modulation of developmental processes in response to stresses.

Arabidopsis stomatal development follows a stereotyped pathway regulated by several receptor proteins, putative ligands, a MAPK signaling module consisting (at a minimum) of a MAPKKK,YODA (YDA), two MAPKKs, MKK4/5, and two MAPKs, MPK3/6, and a series of transcription factors (Figure 1) (Bergmann et al., 2004; Ohashi-Ito and Bergmann, 2006; Bergmann and Sack, 2007; Hara et al., 2007; MacAlister et al., 2007; Pillitteri et al., 2007; Wang et al., 2007; Kanaoka et al., 2008). This particular MAPK module, which we refer to as the YDA MAPK module for simplicity, negatively regulates stomatal development, as illustrated by findings showing that artificial activation of the YDA MAPK module at both MAPKKK and MAPKK levels results in the plant creating an epidermis consisting only of pavement cells (Bergmann et al., 2004;Wang et al., 2007). Conversely, inhibition of the MAPK module using null alleles or inducible RNA interference constructs correlates with stomatal overproliferation and clustering (Bergmann et al., 2004; Wang et al., 2007). These results indicate that the YDA MAPK module functions to inhibit entry into the stomatal lineage.

Figure 1.

Figure 1.

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Diagram of Stomatal Lineage Development and Gene Expression Patterns.

Arabidopsis stomatal development follows a three-step stereotyped pathway that involves a series of asymmetric and symmetric cell divisions. Entry into the stomatal lineage is negatively regulated by a MAPK module containing YDA (MAPKKK), MKK4 and MKK5 (MAPKKs), and MPK3 and MPK6 (MAPKs). Progression through the developmental pathway is positively influenced by sequentially acting bHLH transcription factors, SPCH, MUTE, and FAMA, which regulate entry (1), progression through (2), and terminal differentiation of guard cell development (3), respectively. The expression of green fluorescent protein (GFP)-tagged transcriptional reporters of each of SPCH (A), MUTE (B), and FAMA (C) coincides with each major developmental transition. Bars = 50 μm.

More recent work indicates that the YDA MAPK module prevents entry into the stomatal lineage by regulating the phosphorylation state of the basic helix-loop-helix (bHLH) protein SPEECHLESS (SPCH) (Lampard et al., 2008). After entry into the stomatal lineage, stomatal development is regulated by the related and sequentially expressed bHLHs, MUTE and FAMA (Figures 1A to 1C), which like SPCH, function as positive regulators of development (Ohashi-Ito and Bergmann, 2006; MacAlister et al., 2007; Pillitteri et al., 2007; Lampard et al., 2008). While Wang et al. (2007) identified components that function downstream of YDA (MKK4/5 and MPK3/6) to regulate stomatal development, the approach of non-cell type–specific and simultaneous induction of MAPK activity did not enable them to assign discrete MAPK functions to specific stomatal lineage cell types. Here, we describe a targeted approach to address the issue of cell type specificity in MAPK signaling. We have used the promoters of the genes encoding the stomatal bHLH proteins SPCH, MUTE, and FAMA to individually express a constitutively active (CA) YDA variant (CA-YDA) and a panel of CA-MAPKKs beginning in either meristemoid mother cells (MMCs), meristemoid cells, or guard mother cells (GMCs). This strategy enabled us to activate MAPK signaling in specific stomatal lineage cell types and has resulted in our identification of functions for MAPK signaling as both a negative and a positive regulator during the major stages of stomatal development. The 26 separate MAPK pathway manipulations described here have both expanded the repertoire of known MAPKKs affecting stomatal development and have allowed us to propose plausible and testable subnetworks of signal components. Because the cell type–specific in vivo assay used in this study can readily be adapted to the study of other protein families, it has the potential to deconvolve other similarly complex signal transduction pathways in plants.

RESULTS

Macroscopic yda Seedling Phenotypes Are Separable

A major limitation of studying MAPK signaling in plants has been that the typical modes of analysis (observing phenotypes associated with either loss-of-function mutants or those arising from plants with constitutively activated MAPK signaling networks) do not allow cell-specific resolution of function. In addition, ubiquitous perturbation of broadly expressed, multifunctional proteins can induce misleading phenotypic defects that may accumulate over time. For example, yda loss-of-function plants show dramatic stomatal clustering phenotypes (Bergmann et al., 2004; Wang et al., 2007). However, these plants also display hyperactivation of MPK3/6, embryonic malformations, and severe dwarfism (G.R. Lampard, unpublished data; Bergmann et al., 2004; Lukowitz et al., 2004). Therefore, to identify specific functions of YDA (and associated downstream signaling modules) in regulating stomatal development, we needed to separate the range of phenotypes associated with altering YDA signaling. Mosaic approaches, in which genes are selectively over- or inactivated in specific tissues, have aided in the resolution of complex phenotypes (for example, in Drosophila melanogaster; Xu and Rubin, 1993; Blair, 2003), but these techniques are both technically challenging in plants and ill-suited for the specific lineage relationships among the stomatal precursors.

We hypothesized that we could analyze the effects of diminished YDA signaling specifically in cells about to enter the stomatal lineage (MMCs) using the SPCH promoter to express a dominant-negative YDA construct (SPCHpro:DN-YDA; see Methods). Phenotypic analysis of 10-d-old seedlings revealed that like yda null plants (Figure 2B), SPCHpro:DN-YDA plants also have excessive and clustered stomata (Figure 2C). However, as would be predicted from the SPCH expression pattern (Figure 1A), not all cells in the epidermis are affected, and the seedlings do not show the dwarfism associated with a systemic lack of YDA (Figure 2A). When YDA signaling was activated beginning in MMCs using a constitutively active variant of YDA (SPCHpro:CA-YDA), the resulting transgenic plants created an epidermis devoid of guard cells (Figure 2E). This result was identical to the phenotype produced by YDApro:CA-YDA (Bergmann et al., 2004). However, the additional developmental phenotypes associated with broad activation of YDA, such as partially fused cotyledons, were not observed in the SPCHpro:CA-YDA plants, indicating that induced YDA signaling was confined to the stomatal lineage (Figure 2A) (Bergmann et al., 2004; Lukowitz et al., 2004). Individuals within these transgenic populations display variability in the strength of this phenotype, and the strongest phenotypic classes (completely lacking stomata) die before producing progeny. Therefore, in this and all subsequent experiments, we characterized phenotypes in large T1 populations where every individual represented an independent transformation event. In SPCHpro:CA-YDA T1 transgenics, 16/79 plants had no stomata, while the remaining plants had reduced or normal numbers of stomata. These data suggested that manipulation of MAPK signaling by expressing dominant-negative or constitutively active kinase variants under the control of cell type–specific promoters could allow us to study discrete aspects of MAPK signaling without inducing pleiotropic phenotypes.

Figure 2.

Figure 2.

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Phenotypes Associated with Altered YDA Signaling in Seedlings 10 d Postgermination.

(A) Ubiquitous, constitutive activation of YDA (left) produces dwarfed seedlings with partial fusions of cotyledons; yda null seedlings (middle) are also dwarfed with dark-green, upturned cotyledons. By contrast, seedlings expressing a SPCHpro:DN-YDA construct (right) appear similar to the wild type (inset).

(B) to (G) Confocal images of propidium iodide–stained cotyledons 10 d postgermination (DPG).

(B) and (C) Cotyledons of yda-null seedlings show massive overproliferation and clustering of guard cells (B), whereas SPCHpro:DN-YDA plants show clustering of guard cells in a pattern consistent with signaling being diminished only in MMCs (C).

(D) Expression of FAMApro:DN-YDA in GMCs inhibits stomatal development and creates caterpillar-like structures.

(E) and (F) Activation of YDA in beginning in MMCs ([E]; SPCHpro:CA-YDA) or meristemoids ([F]; MUTEpro:CA-YDA) blocks stomatal development and results either in an epidermis consisting of only epidermal pavement cells (E) or a combination of epidermal pavement cells and meristemoid-like cells (F).

(G) FAMApro:CA-YDA promotes stomatal overproliferation and clustering, an effect opposite to FAMApro:DN-YDA.

Bars = 5 mm in (A) and 50 μm in (B) to (G).

[See online article for color version of this figure.]

Cell Type–Specific Activation of the YDA Signaling Pathway Reveals YDA Functions in Each Stage of Stomatal Development

Because the genes that have been reported to comprise the YDA signaling module (YDA, MKK4/5, and MPK3/6) are expressed throughout the stomatal lineage (Bergmann et al., 2004), we sought to determine if the YDA signaling module was capable of regulating additional aspects of stomatal development. To address this, we also used the MUTE and FAMA promoters to initiate expression of CA-YDA in meristemoids and GMCs, respectively.

Plants expressing the MUTEpro:CA-YDA construct also fail to produce mature stomata (Figure 2F). Whereas activation of YDA signaling via SPCHpro:CA-YDA blocked entry into the stomatal lineage, activation of YDA beginning in meristemoids (MUTEpro:CA-YDA) arrests stomatal development at a later stage. Here, while being devoid of guard cells, the epidermis is comprised of both epidermal pavement cells and smaller, meristemoid-like cells (Figure 2F). As with SPCHpro:CA-YDA expression, a range of phenotypes was associated with MUTEpro:CA-YDA expression, and complete inhibition of stomatal development occurred in 12/68 T1 transgenics. We attempted to study the phenotype associated with diminished YDA activity in these cells by expressing a DN-YDA variant beginning in meristemoids (MUTEpro:DN-YDA). However, we were unable to recover transformants among >100,000 seeds from three independent transformations.

The phenotypes resulting from YDA activation in meristemoids (MUTEpro:CA-YDA) were consistent with previously reported functions of YDA as a negative regulator of stomatal development. The expanded capability of YDA to regulate additional stages of stomatal development led us to hypothesize that YDA activity in GMCs would also inhibit stomatal development. However, contrary to our predictions, activation of YDA beginning in GMCs via the expression of a FAMApro:CA-YDA transgene promoted excess guard cell formation. Small clusters of guard cells were observed throughout the epidermis, which appeared otherwise normal (Figure 2G). These surprising results were confirmed by reducing YDA signaling with FAMApro:DN-YDA. Consistent with the results obtained when YDA signaling was activated in GMCs, diminished YDA signaling arrested stomatal development prior to guard cell formation; the epidermis of T3 progeny contained caterpillar-like structures strongly reminiscent of those observed in fama null or flp myb88 mutants (Figure 2D) (Lai et al., 2005; Ohashi-Ito and Bergmann, 2006).

Activation of MKK4 and MKK5 Inhibits Stomatal Development at Multiple Stages

Given these additional roles of YDA in regulating stomatal development identified here, we next questioned which downstream MAPKKs regulate specific stages (entry, progression, and terminal differentiation of stomata). First, we sought to determine the extent to which MKK4 and MKK5 activity regulates each developmental stage. Constitutively active versions of MKK4 and MKK5 (CA-MKK4 and CA-MKK5, respectively) were created by substituting the phosphorylatable S/T residues with phosphomimic E/D residues (see Methods; Popescu et al., 2009). Each of CA-MKK4 and CA-MKK5 was expressed in wild-type Columbia-0 (Col-0) plants during discrete stages of stomatal development using the SPCH, MUTE, or FAMA promoters. As with YDA, activation of either MKK4 (SPCHpro:CA-MKK4) or MKK5 (SPCHpro:CA-MKK5) beginning in MMCs prevents entry into the stomatal lineage (Figures 3A and 3D), and expression of either construct beginning in meristemoids (MUTEpro:CA-MKK4 or MUTEpro:CA-MKK5) results in a buildup of arrested meristemoid-like cells (Figures 3B and 3E). These results are consistent with previously described functions of a Nicotiana tabacum MEK2 (the putative tobacco ortholog of MKK4 and MKK5) transgene and mutations in each of MKK4 and MKK5 in regulating stomatal development downstream of YDA (Wang et al., 2007).

Figure 3.

Figure 3.

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MKK4 and MKK5 Activity Influences Multiple Stages of Stomatal Development.

Activation of MKK4 and MKK5 signaling at specific stages of stomatal development using the SPCH, MUTE, and FAMA promoters revealed additional inhibitory functions of MKK4 and MKK5 on stomatal development. Gray-shaded cells in the top panel show the expression pattern of each of the SPCH, MUTE, and FAMA promoters, which are active in MMCs, meristemoids, and GMCs, respectively.

(A) to (F) Confocal images of propidium iodide–stained 10-DPG cotyledons. SPCHpro:CA-MKK4 (A) and SPCHpro:CA-MKK5 (D) blocked entry into the stomatal lineage, and MUTEpro:CA-MKK4 (B) and MUTEpro:CA-MKK5 (E) result in cells arresting with meristemoid morphology. Expression of neither FAMApro:CA-MKK4 (C) nor FAMApro:CA-MKK5 (F) affected stomatal development. Bars = 50 μm.

[See online article for color version of this figure.]

We next expressed CA-MKK4 and CA-MKK5 beginning in GMCs (FAMApro:CA-MKK4 and FAMApro:CA-MKK5); here, we expected that activation of MKK4 or MKK5 in GMCs would, like YDA activation, induce guard cell overproliferation and clustering. However, expression of neither transgene affected guard cell development (Figures 3C and 3F). For each construct, >75 T1 lines were screened and despite verification of transgene expression (as detected by yellow fluorescent protein [YFP] fluorescence in GMCs and young guard cells; see Supplemental Figure 1 online), only wild-type stomatal patterns were detected.

Design and Construction of a CA-MAPKK Panel

Although we have shown that YDA can both inhibit and promote specific transitions during stomatal development, MKK4 and MKK5 appear to be downstream kinases only during the first two (inhibitory) stages. These results raise two important questions. First, do additional MAPKKs function downstream of YDA to regulate early stages of stomatal development? Second, if other MAPKKs are involved in stomatal development, which ones function downstream of YDA in GMCs?

To answer these questions, we expanded our MAPKK test panel to include MAPKKs whose broad expression patterns included developing leaf tissues, that had previously been demonstrated to mediate abiotic stress responses, and for whom cognate downstream MAPKs had been described. Database queries of publicly available microarray and MPSS data sets indicated that each of MKK1, MKK2, MKK4, MKK5, MKK7, and MKK9 are expressed in leaf tissue (see Supplemental Table 1 online) and have been implicated in stress responses. Interestingly, these MAPKKs are all capable of phosphorylating MPK3 and/or MPK6 (Colcombet and Hirt, 2008; Popescu et al., 2009). As an additional test of the specificity of this system, we also included MKK6, which has not been reported to be capable of phosphorylating MPK3 or MPK6 in vivo. MKK6 allowed us to determine whether expressing any CA-MAPKK would create abnormal stomatal phenotypes. As with the other kinases in this panel, MKK6 is broadly expressed in leaf tissue (see Supplemental Table 1 online).

Additional MAPKKs Can Inhibit Stomatal Development at Multiple Stages

To examine the extent of MAPKK regulation over stomatal development, each member of our CA-MAPKK panel was expressed beginning in MMCs and meristemoids using the SPCH and MUTE promoters, respectively. Initiating MKK7 and MKK9 overactivity in MMCs prevents guard cell formation as reflected by an epidermis consisting only of epidermal pavement cells (similar to MKK4 and MKK5; Figures 4J and 4M). Expression of CA-MKK6 in MMCs had no effect on guard cell development (Figure 4G). Thus, the relationship between MAPK signaling and guard cell development does not appear to be due to nonspecific MAPK signaling defects within the stomatal lineage but, interestingly, correlates with the ability to phosphorylate MPK3 and MPK6.

Figure 4.

Figure 4.

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Expression of CA-MKKs in Multiple Stages of the Stomatal Lineage Identifies New Regulators of Development.

Gray-shaded cells in the top panel show the expression pattern of each of the SPCH, MUTE, and FAMA promoters, which initiate expression in MMCs, meristemoids, and GMCs, respectively.

(A) to (O) Confocal images of propidium iodide–stained 10-DPG cotyledons, including CA-MKK1, CA-MKK2, CA-MKK6, CA-MKK7, and CA-MKK9. Expression beginning in MMCs of SPCHpro:CA-MKK1 (A), SPCHpro:CA-MKK2 (D), or SPCHpro:CA-MKK6 (G) had no effect on stomatal development, while SPCHpro:CA-MKK7 (J) and SPCHpro:CA-MKK9 (M) blocked entry into the stomatal lineage. Similarly, MUTEpro:CA-MKK1 (B), MUTEpro:CA-MKK2 (E), and MUTEpro:CA-MKK6 (H) had no effect on stomatal development, whereas MUTEpro:CA-MKK7 (K) and MUTEpro:CA-MKK9 (N) arrested cells before they became GMCs. FAMApro:CA-MKK1 (C), FAMApro:CA-MKK2 (F), and FAMApro:CA-MKK6 (I) again showed no effect on stomatal development. However, FAMApro:CA-MKK7 (L) and FAMApro:CA-MKK9 (O) resulted in guard cell overproliferation and severe clustering. Bars = 50 μm.

[See online article for color version of this figure.]

Due to their relationships with stress signaling involving MPK3 and MPK6, we speculated that MKK1 or MKK2 activity in MMCs would also prevent stomatal development. However, we found that plants expressing either MKK1 or MKK2 with the SPCH promoter retained normal guard cell patterning in each of >50 T1 lines scored for each construct (Figures 4A and 4D). Transgene expression was verified by the appearance of YFP fluorescence in MMCs (see Supplemental Figure 1 online).

When expressed beginning in meristemoids, CA-MKK7 and CA-MKK9, like CA-MKK4 and CA-MKK5, cause the plant to create an epidermis consisting of epidermal pavement cells and clusters of small cells that appear morphologically similar to meristemoids (Figures 4K and 4N). However, expression of MUTEpro:CA-MKK1 or MUTEpro:CA-MKK2, as confirmed by the presence of YFP fluorescence (see Supplemental Figure 1 online), had no effect on guard cell development (Figures 4B and 4E). Similarly, MUTEpro:MKK6 expression did not impair stomatal patterning (Figure 4H). Thus, we identified two additional MAPKKs (MKK7 and MKK9) that are capable of inhibiting the first two stages of stomatal development.

MKK7 and MKK9 Positively Influence the GMC to Guard Cell Transition

We then turned to the outstanding question of which MAPKK(s) could act downstream of YDA in promoting the differentiation of guard cells. As seen in earlier stages, MKK1, MKK2, or MKK6 activity again appears to have no function in guard cell development. The stomatal pattern in transgenic plants expressing FAMApro:CA-MKK1, FAMApro:CA-MKK2, or FAMApro:CA-MKK6 appeared wild-type (Figures 4C, 4F, and 4I). In all cases, expression was confirmed by observation of YFP fluorescence in GMCs and young guard cells (see Supplemental Figure 1 online).

By contrast, expression of FAMApro:CA-MKK7 or FAMApro:CA-MKK9 resulted in a phenotype resembling the most severe of the FAMApro:CA-YDA plants: gross overproduction of stomata and the formation of guard cell clusters that protrude from the epidermis of the leaves (Figures 4L and 4O). Thus, it appears that like YDA, MKK7 and MKK9 can influence development at the GMC to guard cell stages and, moreover, their activity promotes rather than inhibits guard cell proliferation at the terminal stages of stomatal development. Transgene expression was verified by fluorescence from the CA-MKK7-YFP construct within GMCs and young guard cells (see Supplemental Figure 1 online).

Guard Cell Tumor Formation Caused by Activation of MKK9 Results in SPCH Transcription

The guard cell overproliferation phenotype observed upon activation of YDA, MKK7, or MKK9 in GMCs is consistent with a positive role of the YDA MAPK module in regulating terminal guard cell development. Because misexpression of MUTE can result in stomatal overproliferation independently of SPCH activity (essentially bypassing this first step in the pathway; MacAlister et al., 2007; Pillitteri et al., 2007), we tested whether the guard cells that comprise the MKK9-induced stomatal clusters develop by following the normal stomatal development pathway. We assayed this by examining if the developing guard cells displayed SPCH expression using a SPCH transcriptional reporter (SPCHpro:nGFP; MacAlister et al., 2007). Because of the similarity in the phenotypes produced by YDA, MKK7, and MKK9, we followed only the effects of CA-MKK9. FAMApro:CA-MKK9 plants displayed SPCHpro:nGFP reporter activity in the clusters of cells that would become guard cells (Figure 5A). This suggests that the guard cell overproliferation phenotype involves resetting of the program back to the beginning of the normal stomatal development pathway.

Figure 5.

Figure 5.

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Analysis of Stomatal Development Regulated by MKK9 Activity in GMCs.

(A) Confocal images of propidium iodide–stained 5-DPG cotyledons. Immature cells that will eventually become mature guard cell clusters express SPCHpro:nGFP.

(B) to (D) Confocal images of propidium iodide–stained 10-DPG cotyledons.

(B) flp myb88 double mutants display caterpillar-like structures that tend to contain parallel division planes and a single stomate at one end.

(C) and (D) flp myb88 FAMApro:CA-MKK9 plants produce caterpillar-like structures that both divide in multiple orientations (C) and are found in clusters (D).

Bars = 50 μm.

[See online article for color version of this figure.]

MAPKs Acting in the Pathway Downstream of MAPKKs

MPK3 and MPK6 function downstream of MKK4 and MKK5 in broadly regulating stomatal development (Wang et al., 2007). As with MKK4 and MKK5, MKK7 and MKK9 each phosphorylate MPK3 and MPK6 and are thus capable of signaling through these kinases to regulate entry and progression through the stomatal lineage. However, no positive roles for MPK3 or MPK6 in regulating stomatal development have been reported. To determine whether MPK3 and MPK6 function downstream of the YDA module in promoting terminal guard cell development, we expressed CA-MKK9 under the control of the FAMA promoter in previously established mpk3 (SALK_100651) and mpk6 (SALK_074003) T-DNA insertion lines. Since MKK7 and MKK9 both phosphorylate MPK3 and MPK6 and are very similar in amino acid sequence (79.5% identity; 88.8% similarity), we chose to further examine the effects of only MKK9 activity in GMCs. There were no differences in the ability of the CA-MKK9 transgene to promote guard cell overproliferation in mpk3 (38/40) or mpk6 (38/38) null lines relative to wild-type Col (32/32). This is consistent with MPK3 and MPK6 functioning redundantly in the regulation of guard cell development but also with MKK9 signaling through different or additional MAPKs in GMCs.

To establish whether additional MAPKs were involved in stomatal development downstream of MKK9 in GMCs, we assayed the stomatal clustering phenotype induced by MKK9 activity in GMCs in plant lines carrying T-DNA insertions within coding regions of 14/20 of the Arabidopsis MAPK genes (Table 1). Guard cell clustering was not blocked in any of these transgenic lines, which may be due in part to functional redundancy among downstream MAPKs. Because these results do not allow us to distinguish between MPK3/MPK6 functioning redundantly downstream of MKK9 in GMCs and/or additional MAPKs functioning downstream of MKK9, it remains unclear which MAPKs function downstream of MKK9 during the guard mother cell to guard cell transition and subsequent guard cell differentiation.

Table 1.

Single T-DNA Insertion Lines Tested for Their Ability to Block the Phenotypic Consequences of FAMApro:CAMKK9 Expression

Gene T-DNA Insertion Line Accession Number
MPK1 SALK_63847C AT1G10210
MPK1 SALK_122198C AT1G10210
MPK2 SALK_047422C AT1G59580
MPK3 SALK_100651 AT3G45640
MPK6 SALK_074003 AT2G43790
MPK7 SALK_038863 AT2G18170
MPK8 SALK_219553C AT1G18150
MPK10 SALK_026099C AT3G59790
MPK11 SALK_049352C AT1G01560
MPK14 SALK_018940C AT4G36450
MPK15 SALK_046143C AT1G73670
MPK16 SALK_059737C AT5G19010
MPK18 SALK_069399C AT1G53510
MPK19 SALK_075213C AT3G14720
MPK20
 SALK_090004
 AT2G42880
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The FAMApro:CA-MKK9 transgene was introduced into plants homozygous for each T-DNA insertion listed to determine which, if any, potential mpk-mutant lines blocked the ability of MKK9 activity to promote the formation of guard cell clustering and overproliferation. Neither guard cell overproliferation nor patterning defects was blocked in any line.

Targets of MAPKs

SPCH regulates the first cell state transition in the stomatal lineage and was shown to be a target of MPK3 and MPK6 (Lampard et al., 2008). In these assays, however, neither FAMA nor MUTE was found to be an in vitro substrate of either MPK3 or MPK6 (Lampard et al., 2008). Given the novel role of MAPK signaling in regulating the GMC to guard cell transition, we directed our experiments toward identifying targets of the MAPK module during this last stage of stomatal development.

FLP and MYB88 are related MYB transcription factors, function at approximately the same stage as FAMA, and are both in vitro substrates of MPK3 and MPK6 (Feilner et al., 2005; data not shown). flp myb88 double mutants display defective terminal guard cell differentiation, and they form caterpillar-like stomatal lineage structures, often with a single stoma at one end of the chain of cells (Lai et al., 2005). The FAMApro:CA-MKK9 construct was introduced into flp myb88 double mutant plants (Figures 5C and 5D). While guard cell formation was blocked in these plants, consistent with the pattern observed in flp myb88 plants, there were differences in the caterpillar structures generated by excess cell division in the epidermis of these plants. In flp myb88 double mutants, these structures generally show parallel division planes (Figure 5B), whereas in flp myb88 FAMApro:CA-MKK9 plants, the division planes within the structures were oriented in numerous directions (Figure 5C). Furthermore, multiple caterpillar-like structures were clustered next to each other (Figures 5C and 5D), consistent with an additive effect of the FAMApro:CA-MKK9 transgene on the flp myb88 double mutant phenotype.

DISCUSSION

Using Cell Type–Specific Promoters to Activate MAPK Signaling Networks

Large-scale genomic and proteomic studies have illustrated that eukaryotic signaling pathways are complex and vastly interconnected. Plant MAPK signaling networks are no exception. Each of the MAPK components used in this study is multifunctional and broadly expressed: YDA has functions in stomatal and embryonic patterning (Bergmann et al., 2004; Lukowitz et al., 2004; Wang et al., 2007), MKK1 and MKK2 are generally associated with cold stress and pathogen responses (Teige et al., 2004; Meszaros et al., 2006; Brader et al., 2007; Gao et al., 2008; Qiu et al., 2008), and MKK4, MKK5, MKK7, and MKK9 have each been reported to function in hormone, stress, and pathogen signaling networks (Dai et al., 2006; Zhang et al., 2007; Colcombet and Hirt, 2008; Xu et al., 2008; Yoo et al., 2008). In addition, MPK3 and MPK6 are downstream targets of each of the aforementioned MAPKKs and can be activated in response to a wide range of stimuli (reviewed in Colcombet and Hirt, 2008). Because of the lethal effects of broadly activating MAPKKs and/or the multiple phenotypes associated with altered MAPK signaling in multiple or ectopic cell types, it has been particularly difficult to identify how these signaling components function in specific physiological and/or developmental contexts.

Chemically inducible gene expression techniques that allow quantitative induction of transgene expression have allowed some circumnavigation around the lethal effects of widely activating MAPK networks (Wang et al., 2007). In fact, they have been instrumental in providing details regarding MAPK signaling and its role in stomatal development: A dexamethasone-inducible MEK2 (the putative tobacco ortholog of MKK4 and MKK5) construct was used to activate MPK3 and MPK6 signaling in Arabidopsis, revealing inhibitory functions of MKK4/MKK5 and MPK3/MPK6 in regulating stomatal development (Wang et al., 2007). However, dexamethasone-induced activation of N. tabacum MEK2 arrested stomatal development by inhibiting entry into the stomatal lineage, which prevented the identification of the additional regulatory functions of MKK4 and MKK5 in regulating stomatal development.

Here, we have shown that MAPK networks can be dissected by modulating the expression of individual components in specific stomatal lineage cell types at both the MAPKKK and MAPKK levels. For example, expression of a DN-YDA construct using the SPCH promoter results in stomatal clustering and overproliferation but does not show the severe growth defects characteristic of yda-null plants (Figure 2). Activation of MKK4 or MKK5 in MMCs inhibited entry into the stomatal lineage (likely due to SPCH phosphorylation by MPK3 and MPK6) without inducing the rapid hypersensitive response–like cell death typically associated with general overexpression of MKK4/5 activity (Jin et al., 2003; Popescu et al., 2009). The revelation that discrete effects of MAPKK activation can be effectively studied by inducing activity in specific cell types provided the impetus to analyze the roles of MKK4, MKK5, as well as other MKKs in regulating stomatal development.

We have also used this system to investigate both functional specificity among MAPK networks and the extent to which MAPK signaling impacts stomatal development. Besides entry into the stomatal lineage, MKK4, MKK5, MKK7, and MKK9 can each negatively regulate the meristemoid-to-GMC transition. However, only MKK7 and MKK9 activity beginning in GMCs is capable of phenocopying the stomatal clustering and overproliferation associated with YDA overactivity at the same stage. This suggests that while each of the aforementioned MAPKKs can function downstream of YDA, signal integrity in specific cell types may be maintained via additional mechanisms, such as tethering via scaffold proteins. Since activity of MKK1 or MKK2, which each phosphorylate MPK3 and/or MPK6, does not influence stomatal development, it is reasonable to conclude that in addition to the specificity occurring at the MAPK substrate level (Lampard et al., 2008), MAPK signaling specificity occurs at the MAPKKK-MAPKK level. Therefore, this report also validates the usefulness of perturbing MAPK signaling in specific stomatal cell types to identify novel functions of MAPK signaling in vivo.

Negative Regulation of Stomatal Development by MAPK Signaling

The effector(s) downstream of YDA in meristemoids remains unknown. MUTE is not an in vitro substrate of MPK3 or MPK6, suggesting it is not the direct target of the YDA module (Lampard et al., 2008). Recently it was reported that MUTE is an in vitro target of another stress and pathogenesis-associated MAPK, MPK4, and that MPK4 is phosphorylated by MKK1 and MKK2 (Popescu et al., 2009). We demonstrated that expression of CA-MKK1 or CA-MKK2 had no effect on the transition regulated by MUTE. Therefore, either MPK4 is not a target of MKK1 or MKK2 in stomatal lineage cells or MUTE phosphorylation status does not correlate with the arrested stomatal development phenotype observed upon activation of YDA, MKK4, MKK5, MKK7, or MKK9.

By revealing that additional stress-associated MAPKKs (MKK7 and MKK9) can regulate stomatal development, we have highlighted an important question: why is stomatal development inhibited by stress-associated MAPKs? Several reports indicate that the expression of genes linked to metabolism and growth is downregulated upon exposure of plants to stress and pathogens (reviewed in Baena-Gonzalez et al., 2007; Baena-González and Sheen, 2008), suggesting that plants divert resources from these processes to better manage stress responses. It is plausible that activation of the MPK3/6 module by environmental or pathogenic stimuli would be coupled with arrested stomatal development. The rapid (and potentially reversible) block to development made possible by employing broadly expressed, stress-responsive kinases may, in fact, be especially well suited to fine-tune the stomatal lineage. The stomata on a mature leaf develop from many independent precursor cells; it is estimated that two-thirds of the cells in the Arabidopsis leaf epidermis are capable of producing stomata (Geisler et al., 2000). Each of these cells has the potential to transit through the precursor stages with independent timing. Transient stresses can block development of subsets of the lineage without compromising the ability of the leaf to ultimately make stomata and be photosynthetically active, while chronic stresses will eventually be able to affect the entire lineage.

A Novel, Positive Role for MAPK Signaling in Regulating Stomatal Development

We unexpectedly observed that activation of YDA in GMCs correlated with the appearance of clustered guard cells. Consistent with this, we also observed that inhibition of YDA signaling in GMCs corresponded with the appearance of cell patterns similar to those seen in fama null plants and a general lack of mature guard cells in the epidermis. Activation of MKK7 or MKK9 but not MKK4 or MKK5 resulted in the formation of large clusters of mature guard cells. Therefore, it appears that there is a branch point in the YDA MAPK signaling module with MKK7 and MKK9 specifically functioning downstream of YDA in GMCs (Figure 6). We attempted to assay loss of MKK7/9 function by engineering changes in the MKK9 kinase domain equivalent to those shown in the DN version of tobacco NQK1 (Soyano et al., 2003). However, these modifications did not result in phenotypic effects nor did expression of a synthetic microRNA construct dually targeting MKK7 and MKK9 in GMCs. It remains to be seen whether these results point to additional genetic redundancy in the signaling module or reflect technical limitations in our ability to eliminate MKK7 and/or MKK9 activity in stomatal lineage cells.

Figure 6.

Figure 6.

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Model of MAPK Regulation of Stomatal Development.

The activation of four MKKs, MKK7 MKK9, MKK4, and MKK5, can prevent entry into the stomatal lineage, likely by signaling through MPK3 and MPK6 to influence the phosphorylation status of SPCH. Each of the aforementioned MAPKKs is also capable of blocking stomatal development at the meristemoid-to-GMC transition. Activation of YDA, MKK7, or MKK9 beginning in GMCs results in stomatal clustering and overproliferation, highlighting a novel, positive function of MAPK signaling in regulating terminal guard cell differentiation. Because each of these MAPKKs mediates abiotic and/or biotic stress responses, there is a potential for crosstalk between environmental and developmental cues. The integration point could lie at the level of upstream activators, downstream targets, or interaction partners. Alternatively, the MAPKKs functioning at each step of stomatal development may be regulated solely by developmental cues. While MPK3 and MPK6 are likely downstream MAPKs, this has only been experimentally verified for the entry stage, and it is probable that additional MAPKK participate, especially at the guard cell differentiation stage. The substrates of the MAPK modules in meristemoids and GMCs remain to be identified.

The specificity within the MAPK module whereby MKK7 and MKK9 but not MKK4 or MKK5 activity promotes terminal differentiation of guard cells may lie in the substrate specificity for each of the MAPKKs, or it could be due to the presence of additional specificity-determining factors, such as scaffold proteins. In trying to determine if additional MAPKs function downstream of MKK7 and/or MKK9 in GMCs, we assayed the ability of MKK9 activity to promote guard cell clustering in single putative loss of function mutants for 14 of the 20 MAPKs (Table 1). Guard cell clustering was not blocked in any of these lines, suggesting that either multiple MAPKs can function at least partially redundantly in promoting guard cell development, or this process is regulated by a yet to be identified MAPK. We note that this developmental question may only be addressed by cell type–specific manipulations because reduction of MPK3 and MPK6 expression throughout the plant (and therefore early in the stomatal lineage where the MAPK signals are inhibitory) will induce stomatal clusters (Wang et al., 2007).

An intriguing and unexpected result revealed by our studies is the flip in behavior of the MAPK signaling system between the negative regulation of the first stages and the positive regulation of the final stage of stomatal development. This change can be considered from several perspectives. At a physiological level, the inverted behavior could be tied to a threshold effect. Upon encountering a biotic or abiotic challenge, it may be favorable to complete guard cell development once GMCs have formed instead of just arresting their development. At the level of signaling cascades, the ultimate development-promoting targets of the MAPKs in the early stages (e.g., SPCH; Lampard et al., 2008) may be repressed by phosphorylation, whereas the targets at later stages may be activated. Finally, in terms of cell fate and commitment, it is possible that cells that have transitioned through the early stages of stomatal development are now differently competent to respond to MAPK signaling. This last hypothesis fits with the observation that the GMCs induced to restart the pathway express SPCH but do not then immediately arrest and will instead continue developing into guard cells. In addition, a diminished or differential ability to respond to normal developmental cues could explain why these reset cells form clusters and do not adhere to the one-cell-spacing rule of normal guard cell development.

We have described a novel in vivo assay to address specificity issues inherent in many signaling networks. This has resulted in the identification of new roles for specific MAPK members at multiple time points throughout plant epidermal (stomatal) development. This system is readily adaptable to study additional signaling networks in Arabidopsis and complements the proteome scale in vitro work on kinases and substrates (for example, Popescu et al., 2009). Ultimately, we will benefit from developing other tools to activate and inactivate genes during stomatal development, possibly by incorporating chemically inducible gene expression systems and by a thorough characterization of how stomatal development is affected following acute and chronic stresses.

METHODS

Plant Materials

Col-0 was used as the wild type, and all transgenic plants were created using this background unless otherwise noted. Plant lines containing T-DNA inserts in coding regions of genes encoding MAPKs are described in Table 1. Additional lines and alleles used in this work are as follows: yda-Y295 (Bergmann et al., 2004) and flp myb88 (Lai et al., 2005).

Construction of CA-MKKs and Promoter-Specific Expression Constructs

cDNA sequences corresponding to MKK2, MKK5, MKK7, and MKK9 in pCR8 (Lee et al., 2008) were used as starting templates. Stop codons for each gene were removed by amplifying the coding sequences using Accuprime Pfx (Invitrogen) and the oligonucleotides listed in Supplemental Table 2 online. Following amplification, each amplicon was cloned into pENTR-D-TOPO (Invitrogen). Constitutively active variants of each MAPKK were constructed by conversion of the S/TXXXXXS/T MAPKK activation motif to E/DXXXXXE/D using the QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocols using oligonucleotides listed in Supplemental Table 2 online and as described (Popescu et al., 2009). cDNA sequences encoding each of CA-MKK1, CA-MKK4, and CA-MKK6 were contained within pCR8, and the stop codons from each sequence were removed as described.

For expression with the SPCH, MUTE, and FAMA promoters, we ligated the previously described 2.5-, 1.5-, and 2.5-kb sequences into the NotI site within the pENTR-D-TOPO plasmid immediately upstream of the cDNA sequences (Ohashi-Ito and Bergmann, 2006; MacAlister et al., 2007). Each promoter:CA-MKK construct was then recombined into pHGY (Kubo et al., 2005) using Gateway LR recombinase II (Invitrogen) for transformation and subsequent expression in transgenic Arabidopsis thaliana plants. The same procedure was performed to create promoter-specific expression constructs of CA-YDA and DN-YDA, each of which initially was contained within pENTR-D-TOPO. The CA-YDA sequence contains a deletion of amino acids 185 to 322 and is a cDNA version of the previously described ΔNB89 CA-YDA construct (Lukowitz et al., 2004), and the DN-YDA variant contains a K429R substitution to eliminate the catalytic site of the kinase.

Microscopy

Confocal images were collected using a Leica SP5 confocal microscope with excitation/emission spectra of 514/520 to 540 for YFP and 565/580 to 610 for propidium iodide counterstaining. Images were processed in ImageJ (NIH).

Accession Numbers

Accession numbers for YODA and MPKs and MKKs used in this study can be found in Table 1 and Supplemental Table 1 online.

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Figure 1. Verification of Expression of CA-MKKs in Multiple Stages of the Stomatal Lineage.

  • Supplemental Table 1. Gene Expression Data for MAPKKs Used in This Study.

  • Supplemental Table 2. Oligonucleotides Used in This Study.

Supplementary Material

[Supplemental Data]
tpc.109.070110_index.html (776B, html)
[Author Profile]
tpc.109.070110v2_index.html (2.5KB, html)

Acknowledgments

We thank David Ehrhardt (Carnegie Department of Plant Biology) for the use of the confocal microscopes, Kyoko Ohashi-Ito (University of Tokyo, Japan) for building the CA- and DN-YDA constructs, and Marcus Samuel (University of Calgary, Canada) for building the CA-MKK1, CA-MKK4, and CA-MKK6 constructs. We also thank the current and past members of the Bergmann lab for their comments on and insights into this study. This work was supported by DOE-FG02-06ER15810.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Dominique C. Bergmann (dbergmann@stanford.edu).

[C]

Some figures in this article are displayed in color online but in black and white in the print edition.

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Online version contains Web-only data.

References

  1. Baena-Gonzalez, E., Rolland, F., Thevelein, J.M., and Sheen, J. (2007). A central integrator of transcription networks in plant stress and energy signalling. Nature 448 938–942. [DOI] [PubMed] [Google Scholar]
  2. Baena-González, E., and Sheen, J. (2008). Convergent energy and stress signaling. Trends Plant Sci. 13 474–482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bergmann, D.C., Lukowitz, W., and Somerville, C.R. (2004). Stomatal development and pattern controlled by a MAPKK kinase. Science 304 1494–1497. [DOI] [PubMed] [Google Scholar]
  4. Bergmann, D.C., and Sack, F.D. (2007). Stomatal development. Annu. Rev. Plant Biol. 58 163–181. [DOI] [PubMed] [Google Scholar]
  5. Blair, S.S. (2003). Genetic mosaic techniques for studying Drosophila development. Development 130 5065–5072. [DOI] [PubMed] [Google Scholar]
  6. Brader, G., Djamei, A., Teige, M., Palva, E.T., and Hirt, H. (2007). The MAP kinase kinase MKK2 affects disease resistance in Arabidopsis. Mol. Plant Microbe Interact. 20 589–596. [DOI] [PubMed] [Google Scholar]
  7. Casson, S., and Gray, J. (2008). Influence of environmental factors on stomatal development. New Phytol. 178 9–23. [DOI] [PubMed] [Google Scholar]
  8. Casson, S.A., Franklin, K.A., Gray, J.E., Grierson, C.S., Whitelam, G.C., and Hetherington, A.M. (2009). Phytochrome B and PIF4 regulate stomatal development in response to light quantity. Curr. Biol. 19 229–234. [DOI] [PubMed] [Google Scholar]
  9. Chen, R.E., and Thorner, J. (2007). Function and regulation in MAPK signaling pathways: Lessons learned from the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1773 1311–1340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Colcombet, J., and Hirt, H. (2008). Arabidopsis MAPKs: A complex signalling network involved in multiple biological processes. Biochem. J. 413 217–226. [DOI] [PubMed] [Google Scholar]
  11. Coupe, S.A., Palmer, B.G., Lake, J.A., Overy, S.A., Oxborough, K., Woodward, F.I., Gray, J.E., and Quick, W.P. (2006). Systemic signalling of environmental cues in Arabidopsis leaves. J. Exp. Bot. 57 329–341. [DOI] [PubMed] [Google Scholar]
  12. Dai, Y., Wang, H., Li, B., Huang, J., Liu, X., Zhou, Y., Mou, Z., and Li, J. (2006). Increased expression of MAP KINASE KINASE7 causes deficiency in polar auxin transport and leads to plant architectural abnormality in Arabidopsis. Plant Cell 18 308–320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feilner, T., et al. (2005). High throughput identification of potential Arabidopsis mitogen-activated protein kinases substrates. Mol. Cell. Proteomics 4 1558–1568. [DOI] [PubMed] [Google Scholar]
  14. Gao, M., Liu, J., Bi, D., Zhang, Z., Cheng, F., Chen, S., and Zhang, Y. (2008). MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants. Cell Res. 18 1190–1198. [DOI] [PubMed] [Google Scholar]
  15. Geisler, M., Nadeau, J., and Sack, F.D. (2000). Oriented asymmetric divisions that generate the stomatal spacing pattern in arabidopsis are disrupted by the too many mouths mutation. Plant Cell 12 2075–2086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamel, L.P., et al. (2006). Ancient signals: Comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 11 192–198. [DOI] [PubMed] [Google Scholar]
  17. Hara, K., Kajita, R., Torii, K.U., Bergmann, D.C., and Kakimoto, T. (2007). The secretory peptide gene EPF1 enforces the stomatal one-cell-spacing rule. Genes Dev. 21 1720–1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ichimura, K., Shinozaki, K., Tena, G., Sheen, J., Henry, Y., Champion, A., Kreis, M., Zhang, S., and Hirt, H. (2002). Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci. 7 301–308. [DOI] [PubMed] [Google Scholar]
  19. Jin, H., Liu, Y., Yang, K.Y., Kim, C.Y., Baker, B., and Zhang, S. (2003). Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco. Plant J. 33 719–731. [DOI] [PubMed] [Google Scholar]
  20. Kanaoka, M.M., Pillitteri, L.J., Fujii, H., Yoshida, Y., Bogenschutz, N.L., Takabayashi, J., Zhu, J.K., and Torii, K.U. (2008). SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20 1775–1785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kubo, M., Udagawa, M., Nishikubo, N., Horiguchi, G., Yamaguchi, M., Ito, J., Mimura, T., Fukuda, H., and Demura, T. (2005). Transcription switches for protoxylem and metaxylem vessel formation. Genes Dev. 19 1855–1860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kultz, D. (2005). Molecular and evolutionary basis of the cellular stress response. Annu. Rev. Physiol. 67 225–257. [DOI] [PubMed] [Google Scholar]
  23. Lai, L.B., Nadeau, J.A., Lucas, J., Lee, E.K., Nakagawa, T., Zhao, L., Geisler, M., and Sack, F.D. (2005). The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage. Plant Cell 17 2754–2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lampard, G.R., MacAlister, C.A., and Bergmann, D.C. (2008). Arabidopsis stomatal initiation Is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 322 1113–1116. [DOI] [PubMed] [Google Scholar]
  25. Lee, J.S., Huh, K.W., Bhargava, A., and Ellis, B.E. (2008). Comprehensive analysis of protein-protein interactions between Arabidopsis MAPKs and MAPK kinases helps define potential MAPK signaling modules. Plant Signal. Behav. 3 1037–1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liu, Y., Jin, H., Yang, K.Y., Kim, C.Y., Baker, B., and Zhang, S. (2003). Interaction between two mitogen-activated protein kinases during tobacco defense signaling. Plant J. 34 149–160. [DOI] [PubMed] [Google Scholar]
  27. Lukowitz, W., Roeder, A., Parmenter, D., and Somerville, C. (2004). A MAPKK kinase gene regulates extra-embryonic cell fate in Arabidopsis. Cell 116 109–119. [DOI] [PubMed] [Google Scholar]
  28. MacAlister, C.A., Ohashi-Ito, K., and Bergmann, D.C. (2007). Transcription factor control of asymmetric cell divisions that establish the stomatal lineage. Nature 445 537–540. [DOI] [PubMed] [Google Scholar]
  29. Meszaros, T., Helfer, A., Hatzimasoura, E., Magyar, Z., Serazetdinova, L., Rios, G., Bardoczy, V., Teige, M., Koncz, C., Peck, S., and Bogre, L. (2006). The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin. Plant J. 48 485–498. [DOI] [PubMed] [Google Scholar]
  30. Neill, S., Barros, R., Bright, J., Desikan, R., Hancock, J., Harrison, J., Morris, P., Ribeiro, D., and Wilson, I. (2008). Nitric oxide, stomatal closure, and abiotic stress. J. Exp. Bot. 59 165–176. [DOI] [PubMed] [Google Scholar]
  31. Ohashi-Ito, K., and Bergmann, D.C. (2006). Arabidopsis FAMA controls the final proliferation/differentiation switch during stomatal development. Plant Cell 18 2493–2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Pillitteri, L.J., Sloan, D.B., Bogenschutz, N.L., and Torii, K.U. (2007). Termination of asymmetric cell division and differentiation of stomata. Nature 445 501–505. [DOI] [PubMed] [Google Scholar]
  33. Popescu, S.C., Popescu, G.V., Bachan, S., Zhang, Z., Gerstein, M., Snyder, M., and Dinesh-Kumar, S.P. (2009). MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev. 23 80–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Qiu, J.L., Zhou, L., Yun, B.W., Nielsen, H.B., Fiil, B.K., Petersen, K., Mackinlay, J., Loake, G.J., Mundy, J., and Morris, P.C. (2008). Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 148 212–222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005). A gene expression map of Arabidopsis thaliana development. Nat. Genet. 37 501–506. [DOI] [PubMed] [Google Scholar]
  36. Schmidt, E.E. (2007). The origins of polypeptide domains. Bioessays 29 262–270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Soyano, T., Nishihama, R., Morikiyo, K., Ishikawa, M., and Machida, Y. (2003). NQK1/NtMEK1 is a MAPKK that acts in the NPK1 MAPKKK-mediated MAPK cascade and is required for plant cytokinesis. Genes Dev. 17 1055–1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Teige, M., Scheikl, E., Eulgem, T., Doczi, R., Ichimura, K., Shinozaki, K., Dangl, J.L., and Hirt, H. (2004). The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol. Cell 15 141–152. [DOI] [PubMed] [Google Scholar]
  39. Wang, H., Ngwenyama, N., Liu, Y., Walker, J.C., and Zhang, S. (2007). Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 19 63–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Widmann, C., Gibson, S., Jarpe, M.B., and Johnson, G.L. (1999). Mitogen-activated protein kinase: Conservation of a three-kinase module from yeast to human. Physiol. Rev. 79 143–180. [DOI] [PubMed] [Google Scholar]
  41. Xu, J., Li, Y., Wang, Y., Liu, H., Lei, L., Yang, H., Liu, G., and Ren, D. (2008). Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis. J. Biol. Chem. 283 26996–27006. [DOI] [PubMed] [Google Scholar]
  42. Xu, T., and Rubin, G.M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117 1223–1237. [DOI] [PubMed] [Google Scholar]
  43. Yoo, S.D., Cho, Y.H., Tena, G., Xiong, Y., and Sheen, J. (2008). Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling. Nature 451 789–795. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zhang, X., Dai, Y., Xiong, Y., DeFraia, C., Li, J., Dong, X., and Mou, Z. (2007). Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance. Plant J. 52 1066–1079. [DOI] [PubMed] [Google Scholar]

Associated Data

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