Novel and Expanded Roles for MAPK Signaling in Arabidopsis...

Novel and Expanded Roles for MAPK Signalingin Arabidopsis Stomatal Cell Fate Revealed by CellType–Specific Manipulations C W

Gregory R. Lampard,a,1 Wolfgang Lukowitz,b Brian E. Ellis,c and Dominique C. Bergmanna,2

a Department of Biology, Stanford University, Stanford, California 94305b Department of Plant Biology, University of Georgia, Athens, Georgia 30602cMichael Smith Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4

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 relation-

ships between MAPK components and downstream signaling outcomes is difficult because broad experimental manip-

ulation 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 en-

larged 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 asso-

ciated with responses to abiotic and biotic stresses (Colcombet

and Hirt, 2008).

Constitutive and ectopic modulation of MAPK signaling path-

ways in plants is typically associatedwith 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 activa-

tion 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 environ-

mental signals that influence stomatal physiology and develop-

ment. 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;

1Current address: Department of Biology and Health Sciences, PaceUniversity, 861 Bedford Road, Pleasantville, NY 10570.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantphysiol.org) is: Dominique C.Bergmann ([email protected]).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.070110

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

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The Plant Cell Preview, www.aspb.org ã 2009 American Society of Plant Biologists 1 of 12

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 downregu-

lation of developmentally associated gene expression (Kultz,

2005; Baena-Gonzalez et al., 2007; Baena-Gonzalez 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), twoMAPKKs, MKK4/5, and twoMAPKs,

MPK3/6, anda 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 develop-

ment, 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.

More recent work indicates that the YDA MAPK module

prevents entry into the stomatal lineage by regulating the phos-

phorylation 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).

Figure 1. 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 mm.

2 of 12 The Plant Cell

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 simul-

taneous induction ofMAPK 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), meriste-

moid 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 subnet-

works 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 asso-

ciated with either loss-of-function mutants or those arising from

plants with constitutively activatedMAPK signaling networks) do

not allow cell-specific resolution of function. In addition, ubiqui-

tous perturbation of broadly expressed, multifunctional proteins

can induce misleading phenotypic defects that may accumulate

over time. For example, yda loss-of-function plants show dra-

matic stomatal clustering phenotypes (Bergmann et al., 2004;

Wang et al., 2007). However, these plants also display hyper-

activation 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 mod-

ules) 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 dimin-

ished 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 as-

sociated 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 popula-

tions where every individual represented an independent trans-

formation 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 pheno-

types.

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 ad-

dress 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 expres-

sion, and complete inhibition of stomatal development occurred

in 12/68 T1 transgenics. We attempted to study the phenotype

associatedwith diminished YDA activity in these cells by express-

ing 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 fromYDA activation inmeristemoids

(MUTEpro:CA-YDA) were consistent with previously reported

functions of YDA as a negative regulator of stomatal

MAPK Regulation of Stomatal Cell Fate 3 of 12

development. The expanded capability of YDA to regulate addi-

tional 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 begin-

ning in GMCs via the expression of a FAMApro:CA-YDA trans-

gene 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

Figure 2. 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 mm in (B) to (G).


was activated in GMCs, diminished YDA signaling arrested

stomatal development prior to guard cell formation; the epider-

mis of T3 progeny contained caterpillar-like structures strongly

reminiscent of those observed in fama null or flp myb88mutants

(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 down-

stream MAPKKs regulate specific stages (entry, progression,

and terminal differentiation of stomata). First, we sought to

determine the extent towhichMKK4 andMKK5activity 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 pro-

moters. As with YDA, activation of either MKK4 (SPCHpro:CA-

MKK4) or MKK5 (SPCHpro:CA-MKK5) beginning in MMCs pre-

vents 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).

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 cluster-

ing. However, expression of neither transgene affected guard

cell development (Figures 3C and3F). For each construct, >75 T1

lines were screened and despite verification of transgene ex-

pression (as detected by yellow fluorescent protein [YFP] fluo-

rescence in GMCs and young guard cells; see Supplemental

Figure 1 online), only wild-type stomatal patterns were detected.

Figure 3. 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 andMKK5 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 mm.


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 dem-

onstrated 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. Inter-

estingly, 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 Supple-

mental 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 ex-

pressed 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 toMKK4andMKK5; Figures 4Jand4M).ExpressionofCA-

MKK6 in MMCs had no effect on guard cell development (Figure

4G). Thus, the relationship betweenMAPK 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.

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 addi-

tional 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 develop-

ment. 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, ex-

pression 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 themost 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 acti-

vation 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 ac-

tivity (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 clus-

ters 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.

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


Figure 4. 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 mm.


with MKK4 and MKK5, MKK7 and MKK9 each phosphorylate

MPK3 andMPK6 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 regu-

lating 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 inmpk3 (38/40)

ormpk6 (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 signal-

ing 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 trans-

genic lines, which may be due in part to functional redundancy

among downstreamMAPKs. Because these results do not allow

us to distinguish between MPK3/MPK6 functioning redundantly

downstream of MKK9 in GMCs and/or additional MAPKs func-

tioning 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.

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 MUTEwas 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, func-

tion at approximately the same stage as FAMA, and are both in

vitro substrates of MPK3 andMPK6 (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

Figure 5. Analysis of Stomatal Development Regulated by MKK9 Ac-

tivity 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 coty-

ledons.

(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 mm.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



MPK3 SALK_100651 AT3G45640

MPK6 SALK_074003 AT2G43790

MPK7 SALK_038863 AT2G18170


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

The FAMApro:CA-MKK9 transgene was introduced into plants homozy-

gous 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.


excess cell division in the epidermis of these plants. In flp myb88

doublemutants, 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

numerousdirections (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 intercon-

nected. 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 asso-

ciated 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), andMKK4, MKK5, MKK7, andMKK9 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 afore-

mentioned 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 acti-

vating 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. taba-

cum 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 regu-

lating stomatal development.

Here, we have shown that MAPK networks can be dissected

by modulating the expression of individual components in spe-

cific stomatal lineage cell types at both theMAPKKK andMAPKK

levels. For example, expression of a DN-YDA construct using the

SPCH promoter results in stomatal clustering and overprolifera-

tion 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 asso-

ciated 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 overprolif-

eration associated with YDA overactivity at the same stage. This

suggests that while each of the aforementioned MAPKKs can

function downstreamof YDA, signal integrity in specific cell types

may bemaintained 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 perturbingMAPK 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 YDAmodule (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 devel-

opment 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-

Gonzalez and Sheen, 2008), suggesting that plants divert

resources from these processes to better manage stress re-

sponses. 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 re-

versible) 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. Con-

sistent 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 ofMKK7 orMKK9

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, thesemodifications 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 eliminateMKK7 and/orMKK9 activity in stomatal lineage cells.

The specificity within the MAPK module whereby MKK7 and

MKK9 but not MKK4 or MKK5 activity promotes terminal differ-

entiation 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 pro-

teins. In trying to determine if additional MAPKs function down-

stream ofMKK7 and/orMKK9 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 redun-

dantly 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 tar-

gets 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 transi-

tioned through the early stages of stomatal development are now

differently competent to respond to MAPK signaling. This last

Figure 6. 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 develop-

ment at the meristemoid-to-GMC transition. Activation of YDA, MKK7, or

MKK9 beginning in GMCs results in stomatal clustering and overprolif-

eration, 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 down-

stream 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.


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) de-

velopment. This system is readily adaptable to study additional

signaling networks in Arabidopsis and complements the pro-

teome scale in vitrowork 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 encodingMAPKs 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 Supplemen-

tal 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, andCA-MKK6were containedwithin 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-TOPOplasmid 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) usingGateway 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 DNB89 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 YODAandMPKs andMKKs 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.

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.

Received July 14, 2009; revised September 9, 2009; accepted October

15, 2009; published November 6, 2009.

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