Molecular Profiling of Stomatal Meristemoids Reveals New
Component of Asymmetric Cell Division and Commonalities
among Stem Cell Populations in Arabidopsis
C W OA
Lynn Jo Pillitteri,a,b,1Kylee M. Peterson,a,1Robin J. Horst,a,1and Keiko U. Toriia,c,d,2
aDepartment of Biology, University of Washington, Seattle, Washington 98195
bBiology Department, Western Washington University, Bellingham, Washington 98225
cHoward Hughes Medical Institute, University of Washington, Seattle, Washington 98195
dPrecursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Tokyo 102-0075 Japan
The balance between maintenance and differentiation of stem cells is a central question in developmental biology.
Development of stomata in Arabidopsis thaliana begins with de novo asymmetric divisions producing meristemoids,
proliferating precursor cells with stem cell–like properties. The transient and asynchronous nature of the meristemoid has
made it difficult to study its molecular characteristics. Synthetic combination of stomatal differentiation mutants due to
loss- or gain-of-function mutations in SPEECHLESS, MUTE, and SCREAM create seedlings with an epidermis overwhelm-
ingly composed of pavement cells, meristemoids, or stomata, respectively. Through transcriptome analysis, we define and
characterize the molecular signatures of meristemoids. The reporter localization studies of meristemoid-enriched proteins
reveals pathways not previously associated with stomatal development. We identified a novel protein, POLAR, and
demonstrate through time-lapse live imaging that it exhibits transient polar localization and segregates unevenly during
meristemoid asymmetric divisions. The polar localization of POLAR requires BREAKING OF ASYMMETRY IN THE
STOMATAL LINEAGE. Comparative bioinformatic analysis of the transcriptional profiles of a meristemoid with shoot and
root apical meristems highlighted cytokinin signaling and the ERECTA family receptor-like kinases in the broad regulation of
stem cell populations. Our work reveals molecular constituents of stomatal stem cells and illuminates a common theme
among stem cell populations in plants.
Plant stem cells continuously produce new organs to sustain
indeterminate growth. To this end, these cell populations must
ensure stem cell survival through self-renewal and produce
daughter cells destined for specialization. The two major stem
of plant stem cells (Clark et al., 1993; Di Laurenzio et al., 1996;
outside the meristems, such as procambium cells and stomatal
meristemoids, share distinct properties with meristematic stem
cells and are likely to provide additional insight into cell self-
renewal in plants (Fisher and Turner, 2007; Pillitteri et al., 2007a).
Meristemoids represent a transitional state in the cell lineage
producing stomata, valves on the plant epidermis (Figure 1A)
(Sachs, 1991). Specifically, meristemoids have transient self-
renewing capacity, and their differentiation can be blocked in the
absence of a key regulator (Bergmann and Sack, 2007; Pillitteri
and Torii, 2007).
A central mechanism for stem cell maintenance and the gen-
eration of cellular diversity in both plants and animals is through
asymmetric cell division, which ensures that the two daughter
cells maintain separate identities (Abrash and Bergmann, 2009;
division during development can occur through signals from
surrounding neighbors (extrinsic control) or, alternatively, intrinsic
polarity within the cell can trigger partitioning of cell fate determi-
nants (intrinsic control) (Abrash and Bergmann, 2009). Due to the
tractability and accessibility of the epidermis, stomatal develop-
ment has emerged as a model to study asymmetric division and
cellular self-renewal. In Arabidopsis thaliana, stomatal develop-
ment initiates from a subset of protodermal cells, termed meris-
temoid mother cells (MMCs) (Figure 1A). An MMC undergoes an
meristemoid. The meristemoid reiterates several rounds of asym-
metric cell divisions, producing neighboring nonstomatal cells
(stomatal lineage ground cells [SLGCs]) prior to differentiating into
a guard mother cell (GMC). The GMC undergoes a single sym-
metric division and terminally differentiates into a set of paired
guard cells (GCs) that constitute a stoma (Bergmann and Sack,
2007; Peterson et al., 2010) (Figure 1A).
1These authors contributed equally to this work.
2Address correspondence to email@example.com.
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.plantcell.org) is: Keiko U. Torii
CSome figures in this article are displayed in color online but in black
and white in the print edition.
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
The Plant Cell, Vol. 23: 3260–3275, September 2011, www.plantcell.org ã 2011 American Society of Plant Biologists. All rights reserved.
combinatorial activities and heterodimerization of the basic helix-
loop-helix (bHLH) transcription factors SPEECHLESS (SPCH),
MUTE, FAMA, SCREAM (SCRM), and SCRM2 (Ohashi-Ito and
Bergmann, 2006; MacAlister et al., 2007; Pillitteri et al., 2007a;
Kanaoka et al., 2008). SPCH, MUTE, and FAMA act as molecular
division of MMCs and entry into the stomatal lineage, whereas
MUTE triggers the differentiation of meristemoids into GMCs
(MacAlister et al., 2007; Pillitteri et al., 2007a, 2008). FAMA is
required for the proper differentiation of GCs (Ohashi-Ito and
Bergmann, 2006). Knockout mutations in SPCH, MUTE, or FAMA
result in a complete block of stomatal lineage progression at their
respective points of action (Figure 1A). SCRM and SCRM2, two
paralogous and partially redundant bHLH proteins, integrate the
three cell state transitional events for stomatal differentiation by
forming heterodimers with SPCH, MUTE, and FAMA (Kanaoka
et al., 2008) (Figure 1A).
divisions makes this cell type an attractive system to understand
the molecular and cellular basis of self-maintenance and differ-
entiation in plants. To date, the molecular character of meriste-
moids remains unclear due to the transient nature and low
density of meristemoids on the epidermal surface. Here, we
report genome-wide transcriptional profiling of meristemoids
through targeted enrichment of stomatal cell states enabled
through combinatorial, synthetic mutants of stomatal differenti-
ation. Pairwise comparisons of transcriptomes among mutant
seedlings that develop an epidermis composed solely of pave-
ment cells, meristemoids, and SLGCs or GCs identified new
genes that are highly and specifically expressed in the stomatal
lineage (Figure 1B). Meristemoid-enriched gene products in-
clude those involved in cell–cell signaling, hormone metabolism
and signaling, and the cell cycle, as well as cell polarity compo-
nents associated with the meristemoid state. A survey of com-
monly upregulated genes in meristemoids and other stem cell
cell proliferation and differentiation within the SAM and stomatal
cell lineages. Through this transcriptome analysis, we identify a
protein, POLAR, that is polarly localized and unequally segre-
gated during asymmetric cell divisions of the stomatal lineage.
Combined, our study enables the isolation of a unique gene set
actively expressed during the transient meristemoid cell state
and provides high-quality data for building a gene regulatory
network. Furthermore, our study reveals the molecular character
of the meristemoid and illuminates common themes in gene
expression among plant stem cells.
Transcriptional Profiling of Stomatal Lineage Cells Enabled
by Combinatorial Mutations in Stomatal
in the stomatal lineage, we took advantage of mutant resources
identified in our previous studies, which enable targeted enrich-
ment of pavement cells, meristemoids, and GCs. To assess the
transcriptional profile of meristemoids, we used a synthetic
mutant carrying both scrm-D and mute. Meristemoids of mute
mutants never differentiate into GMCs or transdifferentiate into
pavement cells (Pillitteri et al., 2007a). By contrast, the gain-of-
function scrm-D mutation triggers constitutive initiation and
differentiation of the stomatal lineage, resulting in the production
of stomata over the entire epidermis (Kanaoka et al., 2008)
(Figure 1B). Therefore, the combined scrm-D mute mutations
Figure 1. Stomatal Cell Lineage Transitions and Cell Enrichment
(A) Diagram of the sequential cell state transitions during stomatal
development specified by the combinatorial and sequential actions of
the stomatal bHLH genes. A protodermal cell (P) can differentiate into a
pavement cell (PC) or undergo a transition to become an MMC (purple).
An MMC enters the stomatal lineage through an asymmetric division to
create a meristemoid (M; blue). Meristemoids have transient stem cell–
like properties that can undergo several rounds of amplifying divisions
before differentiating into a GMC (light green). GMCs divide symmetri-
cally to produce two GCs (dark green), which form a mature stoma. The
point of action is indicated for each bHLH gene (colored arrows).
(B) Strategy for comparing molecular signatures associated with each
epidermal cell state/type. Images are of abaxial cotyledon epidermis of
spch, scrm-D mute, and scrm-D mutant seedlings at 5 d after germina-
tion. Colors coincide with those described in (A). Bars = 10 mm.
Molecular Signature of Meristemoids3261
produce a synthetic phenotype of an epidermis composed
almost entirely of meristemoids (Figure 1B). The scrm-D mutant
spch mutant, characterized by an epidermis composed solely of
pavement cells, was used as a genotype devoid of any stomatal
cell lineage–derived transcripts (MacAlister et al., 2007; Pillitteri
et al., 2007a) (Figure 1B). Pairwise comparisons of transcrip-
tomes among three biological replicates of spch, scrm-D mute
double mutants, scrm-D, and the wild type using Affymetrix
ATH1 expression arrays were performed in an effort to maximize
the signal-to-noise ratio between molecular signatures associ-
constitutive differentiation of stomata, very young seedlings 5 d
after germination were used for profiling (see Supplemental
Figure 1 online). To minimize diurnal effects on gene expression
(Harmer et al., 2000), all seedlings were harvested in the middle
of the light period.
Gene Expression Clustering Reveals Dramatic Changes in
Transcript Accumulation during Cell State Transition
To assess genotype- or cell type–specific trends in gene ex-
pression, we performed a K-means cluster analysis using nor-
malized expression values from each individual replicate of four
different genotypes (Figure 2). Genes were prefiltered using the
variance filter implemented in the MeV software to remove those
that showed no differential expression across the genotypes
(Saeed et al., 2003). This yielded 2000 genes with the highest
were built from a maximum of 200 iterations using a Pearson
in one or two genotypes was identified (see Supplemental Data
Sets 1 to 3 online).
Gene cluster I showed robust change associated with the
significant upregulationinthescrm-Dmutebackground, butless
in scrm-D (Figure 2; see Supplemental Data Set 1 online). Within
this group are all genes known to be highly expressed in
meristemoids, including TOO MANY MOUTHS (TMM), ERECTA-
LIKE1 (ERL1), ERL2, STOMATAL DENSITY AND DISTRIBU-
TION1 (SDD1), SPCH, MUTE, SCRM, SCRM2, BREAKING OF
ASYMMETRY IN THE STOMATAL LINEAGE (BASL), and EPI-
DERMAL PATTERNING FACTOR2 (EPF2).
Gene cluster V includes genes highly upregulated in GCs
(scrm-D) and to a lesser extent in scrm-D mute: 407 genes are
represented in this cluster (Figure 2; see Supplemental Data Set
2 online). These likely represent genes implicated in guard cell
development or function, as well as GMC differentiation. For
instance, KAT1 and KAT2, two genes encoding GC-specific
et al., 1995), are represented in this cluster (Figure 2). FAMA and
EPF1, a signaling peptide expressed in late meristemoids and
GMCs to enforce stomatal spacing (Hara et al., 2007), are also in
this cluster (Figure 2). No FAMA transcripts were detected in the
scrm-D mute population, supporting the previous finding that in
the absence of MUTE, stomatal precursor cells will not progress
to GMC and express FAMA (Pillitteri et al., 2007a). By contrast,
the presence of EPF1 transcripts in scrm-D mute implies that
EPF1 is not directly controlled by MUTE. Genes in clusters I and
V show lower expression in the wild type and fall under the
detection limit in spch (Figure 2), consistent with the absence of
stomatal lineage cells in spch. Combined, the results emphasize
that our strategy enabled us to document robust changes in
states, which are normally asynchronously transient and cannot
be effectively captured in the wild-type background.
Gene Categories Overrepresented in Each Epidermal
To identify genes with statistically significant enrichment in each
mutant background, we used a significance of microarrays
analysis using a Q-value cutoff of <0.005 and a two- or three-
Using these stringent criteria, we identified gene signatures for
each background compared with the wild type (see Supple-
mental Data Sets 4 to 6 online). We first performed overrepre-
sentation analysis (Usadel et al., 2006) to determine the gene
categories enriched in each background (see Supplemental
Figure 2 online). No overrepresentation of genes involved in
photosynthesis, carbohydrate, or central tricarboxylic acid
metabolism was observed. A moderate overrepresentation of
stress-related genes was found in spch and scrm-D, but not in
scrm-D mute. This indicates that the alteration of epidermal
phenotype did not significantly affect overall metabolism or
stress level of the seedlings. Whereas cell wall biosynthesis
genes were mainly upregulated in scrm-D, they were down-
deposition at the stomatal pore in scrm-D and the low degree of
cell expansion in scrm-D mute.
We also found that signaling components, such as receptor
kinases, were greatly enriched in the scrm-D mute background
but not in spch or scrm-D. This is in accordance with the well-
secreted ligands, membrane receptors, and mitogen-activated
kinase cascades are critical for proper stomatal patterning and
spacing (Geisler et al., 2000; Bergmann et al., 2004; Shpak et al.,
2005; Hara et al., 2007, 2009; Hunt and Gray, 2009).
Molecular Profiling Reveals Genes Highly Enriched
The significance of microarray analysis identified 527 genes
significantly deregulated (277 up/250 downregulated) in the
scrm-D mute background. The 2-kb upstream promoter regions
of this group of genes were enriched in the E-box (CANNTG) cis-
regulatory sequence known to be bound by bHLH DNA binding
domains (P = 10210; Athena data mining tool; O’Connor et al.,
2005). This is in contrast with the 74 genes deregulated (39
upregulated/35 downregulated) in the spch background, where
no significant enrichment of the E-box element was detected.
The finding is consistent with the notion that combinatorial
actions of master regulatory bHLH proteins specify meristemoid
3262The Plant Cell
It is important to address whether the arrested meristemoid-like
cells constituting the epidermis of scrm-D mute share molecular
(Figure 2). We first introduced representative markers of early and
late meristemoids, EPF2pro:erGFP (for green fluorescent protein)
and MUTEpro:GFP, respectively, to scrm-D mute (Figure 3). Indeed,
both reporter constructs showed high and nearly constitutive ex-
pression in the epidermis of scrm-D mute (Figures 3B and 3D).
Consistent with the reporter activity, among the top signatures
(based on fold increase) in scrm-D mute are genes known to be
highly expressed in meristemoids and regulate meristemoid de-
velopment: MUTE (79-fold increase), EPF2 (39-fold increase),
TMM (22-fold increase), ERL1 (22-fold increase), SPCH (20-fold
increase), BASL (17-fold increase), and ERL2 (7.5-fold increase)
(Figure 3E). A quantitative RT-PCR (qRT-PCR) analysis of these
genes further verified the microarray results (Figure 3F). It is worth
Figure 2. K-Means Clustering of Genes Showing Differential Expression in Stomatal Differentiation Mutants.
The log2expression levels are plotted against the three replicates of each genotype (the wild type, spch, scrm-D mute, and scrm-D). Genes were
prefiltered using the variance filter implemented in the MeV software to yield the 2000 genes showing highest variance across the 12 arrays. Cluster I:
genes upregulated in scrm-D mute and to a lesser extent in scrm-D (meristemoid and GMC/GC enriched, respectively). Black, TMM; green, EPF2; red,
ARR16; blue, CLE9; yellow, POLAR (At4g31805). WT, wild type. Cluster II: genes upregulated in spch (pavement cell–only epidermis). Highlighted in
black is a representative gene (Orp4C, At5g57240) of this cluster. Cluster III: genes with reduced expression in scrm-D mute and scrm-D. Highlighted in
black is IAA7 (At3g23050), which shows the representative expression pattern in this cluster. Cluster IV: genes expressed most strongly in the wild type
and scrm-D. Highlighted in black is a representative gene (integral membrane family protein, At5g44550) of this cluster. Cluster V: genes upregulated in
scrm-D (GMC/GC-enriched). Black, CHX20, a cation/H+exchanger of GCs; blue, FAMA; green, EPF1.
Molecular Signature of Meristemoids3263
noting thatmute displaysthe samephenotype asa transcriptional
null allele, mute-2 (Pillitteri et al., 2008), but accumulates mis-
spliced transcripts with premature termination codons (see Sup-
mute epidermal cell population has the molecular characteristics
of meristemoids, which validates our approach and provides the
basis for further characterization of genes enriched in this cell
Reporter Expression and Cellular Localization of
To gain insight into the roles of meristemoid-enriched genes, we
chose 14 genes previously uncharacterized in relation to stoma-
designed constructs containing up to 2.5 kb of the 59 upstream
regulatory sequence for each gene driving the expression of
the full-length coding region fused to GFP. Alternatively, for
those genes whose translational fusions did not result in detect-
able GFP signals, transcriptional fusion constructs were pro-
duced. They include the following: At1g26600 (CLE9; 30-fold),
At4g31805 (misannotated as WRKY family transcription factor
[see below]; 21-fold), At5g13220 (JAZ10; 15-fold), At2g40670
(ARR16; 14-fold), At3g17640 (leucine-rich repeat [LRR] protein;
9-fold), and At5g62210 (EMBRYO-SPECIFIC PROTEIN3 [ESP3];
>20-fold in scrm-D mute compared with spch) (see Supplemen-
tal Table 1 online).
Of the 14 reporter constructs, 10 showed expression patterns
in the stomatal lineage (Figures 4A to 4K).Some arecomponents
of signaling pathways that have not been assigned to stomatal
development. JAZ10-GFP was found in the nucleus through all
transitional states of the stomatal lineage but not in fully differ-
of jasmonate signaling (Chung and Howe, 2009). ARR16-GFP
showed strong and specific expression in meristemoids (Figure
4B). ARR16 belongs to the A-type Arabidopsis response regu-
lator (ARR) family acting downstream in cytokinin signaling (Kiba
et al., 2002). Interestingly, a gene coding for the cytokinin
catabolic enzyme At4g29740 (CKX4) (Werner et al., 2006) is also
enriched in scrm-D mute and its transcriptional GFP fusion
exhibited expression in meristemoids (Figure 4C). GFP transla-
tional fusions of At1g33930, a protein with a GTP binding motif,
and At3g17640 are also strongly detected in the cytoplasm of
meristemoids (Figures 4D and 4E). CLE9, one of the CLE peptide
genes, and At5g07280 (EMS1), an LRR–receptor-like kinase
(RLK) regulating microsporogenesis (Zhao et al., 2002; Jun et al.,
2010), showed strong expression in meristemoids and GMCs as
GFP transcriptional fusions (Figures 4F and 4G).
Figure 3. Arrested Meristemoids Constituting the Epidermis of scrm-D
mute Express Meristemoid Markers.
EPF2 and MUTE promoter activity (green) is detected only in early
stomatal lineage cells and not in mature stomata. Bars = 10 mm.
(C) and (D) MUTEpro:GFP in the wild type (C) and scrm-D mute (D). Cell
peripheries were highlighted with either propidium iodide or FM4-64
(E) Known regulators of meristemoid development are highly expressed
in scrm-D mute. Shown is a heat map showing the absolute expression
levels in the wild type and scrm-D mute (meristemoid enriched). All three
replicates are shown to demonstrate consistency.
(F) qRT-PCR verification of a subset of stomatal regulators known to be
expressed in meristemoids. Error bars represent the SE (n = 3).
3264 The Plant Cell
Three proteins displayed unique expression patterns. ESP3,
which is predicted to be membrane anchored via glycosylphos-
phatidylinositol (Borner et al., 2002), displayed patchy plasma
membrane localization across the epidermis, including pavement
cells, but showed strong stomatal lineage–specific expression on
internal membranes (Figure 4H). At1g52910 (unknown protein)
exhibited a characteristic, vesicular localization and strong accu-
mulation at the cell plate of dividing meristemoids and GMCs
(Figure 4I, arrow), suggesting that this protein may be involved in
the cytokinesis of stomatal lineage cells. Lastly, At4g31805 ex-
hibited an intriguing localization, with strong GFP signal detected
in the cytoplasm of meristemoids and cell periphery in SLGCs
(Figure 4J). Strikingly, At4g31805-GFP was localized polarly in
SLGCs, distalto thenewly divided meristemoid and paralleltothe
dynamic behavior of this protein during asymmetric divisions
POLAR (for POLAR LOCALIZATION DURING ASYMMETRIC DI-
VISION AND REDISTRIBUTION).
Cellular Dynamics of POLAR Protein That Is Polarly
Localized and Unequally Segregated during Asymmetric
Cell Division of the Stomatal Lineage
The polar localization of POLAR in SLGCs (Figures 4J and 4K)
resembles that of BASL (Dong et al., 2009). Unlike GFP-BASL,
which remains in the nucleusin meristemoids (Dong et al., 2009),
however, POLAR-GFP displays no nuclear localization in any
stage of stomatal development. Based on publicly available data
sets, POLAR and BASL display little or no detectable expression
in other asymmetrically dividing tissues, such as roots (see
Supplemental Figure 4 online). To resolve the cellular dynamics
of POLAR during asymmetric divisions of the stomatal lineage,
we developed a time-lapse imaging technique (Figure 5; see
Supplemental Movie 1 online). Mature embryos were dissected
at germination, and cotyledons were subjected to time-lapse
imaging at 30-min intervals over 72 h to capture the real-time
movement of this protein in relation to cell division during
stomatal development (see Methods). During the process of
imaging, cotyledons developed normally and eventually formed
The time-lapse imaging revealed that during germination,
POLAR-GFP initially appears in a subset of protodermal cells,
which are likely MMCs, both in the cytosol and at the cell
division occurs, POLAR-GFP becomes dynamically localized at
the cell cortex distal to the division plane (Figures 5B, 5E, and
5H). Clear polar localization of POLAR distal to the division plane
was observed in all asymmetrically dividing cells of the stomatal
inonly oneofthe daughtercells,which ispredictiveofthe cellthat
will continue to divide asymmetrically. When the larger daughter
Figure 4. Localization in Stomatal Cell Lineages and Expression Levels.
Genes represent those upregulated in the scrm-D mute background. All
lines carry a C-terminal GFP protein fusion (green) of the indicated gene
driven by its native promoter, except for CLE9 and EMS1, which carry the
native promoter driving GFP.
(A) to (K) Confocal images of wild-type seedling leaf epidermis. Images
are of the abaxial surface, except (E) (adaxial surface). Cell peripheries
were highlighted with either propidium iodide or FM4-64 (magenta).
Arrow in (I) indicates cell plate. Asterisks in (K) indicate asymmetrical
localization of At4g31805-GFP; arrowheads indicate location of division.
Bars = 10 mm in (A) to (I) and 25 mm in (J).
(L) Heat map representing the degree of upregulation of each indicated
gene in scrm-D mute compared with the wild type. Three replicates are
shown to demonstrate consistency. Scale represents absolute expres-
sion of each gene.
(M) qRT-PCR verification of selected meristemoid-enriched genes iden-
tified in this study. Values are relative to expression of corresponding
genes in the wild type. Error bars represent the SE (n = 3).
Molecular Signature of Meristemoids3265
cell (SLGC) retains POLAR expression, localization is directed to
the periphery of the cell adjoining the adjacent meristemoid or
GMC, correlated with a new satellite meristemoid being placed
away from the existing one (see Supplemental Movie 1 online).
Expression of POLAR appears to mark stomatal lineage cells with
undergo asymmetric entry division but underwent pavement cell
differentiation or, alternatively, differentiated into GMCs without
asymmetric divisions immediately lost POLAR-GFP expression
(see Supplemental Figure 5 online). Likewise, once a meristemoid
terminated asymmetric division and differentiated into a GMC,
POLAR-GFP expression rapidly diminished (Figures 5I to 5K).
Asymmetric Localization of POLAR Is Disrupted by Loss of
Function in BASL
To understand whether a known regulator of asymmetric cell
divisions in the stomatal cell lineage influences the polar local-
ization of POLAR, we next investigated cellular dynamics of
POLAR-GFP in basl using time-lapse imaging (Figure 6; see
Supplemental Movie 2 online). The basl mutant is characterized
by a loss of physical asymmetry during division and concomitant
loss of cell fate asymmetries, resulting in clustered stomata
(Dong et al., 2009). Asymmetric localization of POLAR-GFP was
nonasymmetric division of the GFP-expressing cell (Figures 6D
and 6H). Duringthe division of stomatal lineage cells in basl, both
divide (Figures 6E to 6H). This is in sharp contrast with the wild
type, where only one cell from a division retains expression of
POLAR-GFP and continues asymmetric division (Figures 5E to
after division are absent in the basl background (Dong et al.,
2009). The observed perturbation of POLAR-GFP localization in
the basl background suggests that a functional BASL protein is
required for POLAR-GFP asymmetry. Some cells in the basl
background may also express POLAR-GFP without dividing for
over 24 h, a much longer duration than we observed in the wild
type, which may indicate that the regulation of this protein is
perturbed by the loss of BASL. The asymmetric division defects
in basl, POLAR-GFP exhibits normal expression dynamics and
polar localization (see Supplemental Movie 2 online).
Protein Structure and Loss-of-Function Allele of POLAR
Although itis annotated as WRKY transcription factor (The Arab-
idopsis Information Resource: http://www.arabidopsis.org/),
sequence analysis does not reveal any similarity to the WRKY
domain or any other recognizable structural motifs or domains
aside from itsmoderatesimilaritytoanArabidopsis myosin-heavy
Figure 5. Polar Localization of POLAR Precedes the Asymmetric Division of Stomatal Cell Lineages.
Real-time imaging was performed using wild-type cotyledons expressing a C-terminal GFP protein fusion of POLAR driven by the native promoter
(POLARpro:POLAR-GFP). Individual fields from imaging are shown over time as indicated ([A] to [K]). Cell membranes (magenta) are visualized with the
mCherry plasma membrane marker pm-RB. POLAR-GFP displays localized, transient accumulation at the cell periphery distal to the plane of division
prior to asymmetric divisions of stomatal cell lineage (MMC and meristemoids), then is redistributed following asymmetric division. Asterisk indicates
polar localization. Arrowhead indicates the plane of division. SLGCs resulting from the asymmetric division are numbered (K). hpg, hours post
germination. Bar = 20 mm. See also Supplemental Movie 1 online.
3266The Plant Cell
chain-like protein (At5g10890: 29% identity/49% similarity). Sec-
ondary structure analysis predicts a predominantly alpha-helical
structure with a coiled-coil region at the C terminus (Lupas et al.,
1991). We did not identify homologs of POLAR outside of plants.
To unravel the function of POLAR, we analyzed two T-DNA
insertion alleles (SALK_112914 and SALK 142820). The RT-PCR
analysis detected no POLAR mRNA accumulation (see Supple-
mental Figure 6 online), indicating that the insertions lead to the
loss/reduction of POLAR transcripts. These insertion lines did not
reveal discernable growth or developmental phenotypes, sug-
gesting that POLAR may be functionally redundant.
Meristemoid-Enriched Transcriptome Includes Specific
Members of Core Cell Cycle Regulators
Meristemoids exhibit a rare capacity for reiterating asymmetric
cell divisions, which is lost with the cell state transition to GMCs:
GMCs instead undergo a symmetric division and differentiation.
We therefore investigated whether cell cycle regulatory genes
were enriched in meristemoids. Our analysis revealed that 17
core cell cycle genes were represented in gene cluster I (Figures
2 and 7). Among them, we observed the reporter b-glucuroni-
dase (GUS) expression of CYCLIN DEPENDENT KINASE B2;1
(CDKB2;1), CYCB1;2, and CYCA2;3 (Figures 7A to 7C), all of
which showed high promoter activity in stomatal lineage cells
and have higher levels of transcript accumulation in the scrm-D
mute background compared with spch or scrm-D (Figures 7E
and 7F). It has been reported that ERECTA family receptor
kinases, which regulate organ growth and stomatal patterning,
affect expression of cyclin A2 genes (Pillitteri et al., 2007b). We
further examined the reporter activity of the remaining cyclin A2
genes and found that CYCA2;2 is upregulated in scrm-D mute
(Figure 7F) and CYCA2;2pro:GUS is expressed in a subset of
meristemoids (Figure 7D), potentially those making a transition
Molecular Signatures of Meristemoids and Meristems:
Commonalities and Distinctions
Comparison of transcriptome profiles among different microarray
experiments can identify unexpected molecular commonalities
among specific cell types. Our meristemoid-enriched transcrip-
tome provides the opportunity to compare the meristemoid state
with the true stem cell population in the SAM and RAM. For this
purpose, we obtained publicly available transcriptome data de-
rived from isolated cells expressing the SAM-specific markers
CLAVATA3 (CLV3; stem cell domain), WUSCHEL (WUS; rib mer-
istem), and FILAMENTOUS FLOWER (FIL; peripheral zone of the
floral meristem) (Yadav et al., 2009) as well as data from two
et al., 2008) and root tips(Sena etal., 2009). These data sets were
compared with our meristemoid-enriched transcript (scrm-D
mute) profile (Figure 8). Overrepresentation analysis showed that
genes classified as transcription factors (RNA regulation of tran-
scription) and receptor kinases are equally overrepresented in all
stem cell populations included in this meta-analysis (see Supple-
mental Figure 7 online), highlighting the importance of transcrip-
tional regulation and cell–cell signaling in maintenance and
function of meristematic cells. Alternatively, these trends may
reflect general characteristics of actively dividing cells.
We identified relatively little overlap in upregulated genes
between scrm-D mute and individual SAM populations: 11, 3,
and 6 genes for the FILpro, CLV3pro, and WUSproarrays, respec-
tively. This is far less than the overlap between the CLV3proarray
andthe WUSpro(149genes)andFILproarrays(119genes) (Figure
8A). However, we identified 19 genes that are commonly
upregulated in the all three SAM populations and scrm-D mute
Figure 6. Localization of POLAR in the basl Mutant Defective in Asymmetric Division.
Real-time imaging was performed using basl cotyledons expressing POLARpro:POLAR-GFP. Individual fields from imaging are shown over time as
indicated ([A] to [H]). Loss-of-function basl mutants have diffuse peripheral POLAR-GFP expression that is not strongly polarized. Arrowheads indicate
symmetrical division plane ([D] and [H]) relative to the parent cell. Asterisks indicate asymmetric division. hpg, hours postgermination. Bar = 20 mm. See
also Supplemental Movie 2 online.
[See online article for color version of this figure.]
Molecular Signature of Meristemoids3267
(see Supplemental Table 2 online). As expected, several cell
division–related proteins were commonly upregulated: At3g19590
(BUDDING UNINHIBITED BY BENZYMIDAZOL [BUB3.1]),
At2g06510 (REPLICATION PROTEIN A [RPA1A]), and At1g20930
(CDKB2;2). The most notable among the commonly upregulated
genesare two members ofthe ERECTAfamily ofgenes,ERL1and
Among all arrays, ERL1 (At5g62230) shows eightfold to 22-fold
upregulation and ERL2 (At5g07180) a sevenfold to 35-fold in-
crease. The finding underscores the known roles of the ERECTA
family in promoting cell proliferation during shoot and flower de-
velopment as well as restricting stomataldifferentiation (Torii et al.,
1996; Shpak et al., 2004, 2005; Pillitteri et al., 2007b; Hord et al.,
2008). The LRR-RLK gene EMS1 was also transcriptionally en-
riched in scrm-D mute, WUSpro, and FILpro(19-, 14-, and 24-fold,
respectively). Enrichment in CLV3prowas statistically insignificant
(Q = 0.017) but likely a false negative due to our stringent cutoffs.
EMS1 regulates anther cell differentiation (Zhao et al., 2002) by
physical interaction with the small peptide TAPETUM DETERMI-
NANT1 (TPD1) (Jia et al., 2008). A role for the TPD1/EMS1 ligand
receptor system in the SAM has not been documented.
Comparison of scrm-D mute to transcriptional profiles from root
tips revealed 12 genes commonly enriched in both root tip exper-
iments and scrm-D mute (Figure 8B). Among them is SCHIZORIZA
(SCZ), a heat shock family transcription factor, which regulates
asymmetric cell divisions in the root stem cell niche (ten Hove et al.,
2010). SCZ is also transcriptionally enriched in scrm-D mute and
specifically expressed in meristemoids during stomatal develop-
ment (Figures 8C to 8E), suggesting an exciting potential role for
SCZ in asymmetric cell division in the stomatal lineage.
Here, we present the transcriptome profile of the meristemoid, a
stem cell–like properties. Producing plants highly enriched in
specific epidermal cell types (pavement cells, meristemoids, or
GCs forming stomata) is possible using loss- and gain-of-function
state transitional events within the stomatal cell lineages (Mac-
Alisteretal.,2007;Pillitterietal., 2007a;Kanaokaetal., 2008).Cell
type–specific profiling has been widely performed using physical
isolation of cells via laser capture microdissection (Kerk et al.,
2003; Nakazono et al., 2003; Day et al., 2007) or protoplasting fol-
lowed by fluorescence-activated cell sorting (FACS) (Iyer-Pascuzzi
and Benfey, 2010). Protoplasting and FACS were used for the cell
type–specific expression profiling of the SAM used in our meta-
analysis (Yadav et al., 2009). These approaches, while accurately
isolating specific cell types of interest from wild-type plants,
Figure 7. Analysis of Cell Cycle Regulatory Gene Expression in the
(A) to (D) All lines carry the GUS coding sequence driven by the native
promoter of each indicated gene. Shown are differential interference
contrast microscope images of abaxial leaf epidermis from wild-type
seedlings expressing the following reporter lines: CDKB2;1, At1g76540
(A); CYCB1;2, At5g06150 (B); CYCA2;3, At1g15570 (C); and CYCA2;2,
At5g11300 (D). Arrowheads indicate meristemoids; asterisks indicate
GMCs; (+) indicates stomata. Bar = 10 mm.
(E) Heat map representing the degree of upregulation of each indicated
gene in the scrm-D mute background compared with the wild type. All
three replicates of both genotypes are shown to demonstrate consis-
tency. Scale represents absolute expression of each gene.
(F) qRT-PCR of selected core cell cycle regulators in spch, scrm-D mute,
and scrm-D. Gene names are indicated. Values are relative to the Col
wild-type background. Error bars represent the SE (n = 3).
3268 The Plant Cell
sectioning and protoplast isolation, that could affect gene expres-
sion. In addition, the FACS process relies on the availability of GFP
markers that specifically and exclusively mark the cell types of
Using spch, scrm-D mute, and scrm-D mutants, we were able
to isolate RNA directly without further manipulations to perform
pairwise transcriptome comparisons. Using mutant back-
grounds invites two major questions: whether the identity of
the cells enriched in these mutants is truly normal and whether
physiological consequences due to the mutations deregulate
transcripts unrelated to the enriched cell types. We did not find
significant alterations in stress- or photosynthesis-related tran-
scripts in the scrm-D mute population. Top signatures from this
population include genes known to be highly and/or specifically
expressed in meristemoids and regulate stomatal development,
and SCRM2 (Figure 3E). Furthermore, a new gene associated
powerful alternative to unravel molecular signatures associated
with this transient cell state.
Coexpression programs (ATTED-II: http://atted.jp/) (Obayashi
et al., 2011) cluster only two of our 10 more closely analyzed
genes, POLAR and CLE9, with known stomatal regulatory net-
works; the other eight genes identified in this analysis with
stomatal-specific expression (Figure 4) were not predicted to be
coexpressed with stomatal regulators. This inconsistency dem-
onstrates the importance of continued transcriptome profiling in
controlling developmental processes. Such discoveries provide
a framework of how the molecular mechanisms driving cell
division maintenance may be partially conserved among stem
Cytokinin Signaling May Play a Universal Role in Stem
We found that the cytokinin response genes ARR16, CKX4, and
CLE9 are strongly upregulated in scrm-D mute, and their ex-
pression in stomatal lineage cells was confirmed by reporter
analysis (Figure 4). CKX4 encodes a cytokinin oxidase, which
degrades and thus inactivates cytokinin (Schmu ¨lling et al., 2003),
and ARR16 is a member of the A-type ARR family, which function
Figure 8. Integrative Analysis of Transient and Permanent Stem Cell Populations.
(A) and (B) Venn diagrams showing the distribution of unique and shared genes upregulated among scrm-D mute, CLV3, FIL, or WUS expressing cells
(A) as well as among scrm-D mute and root tips zone1 and root tips (B).
(C) qRT-PCR analysis showing that SCZ, a known regulator of asymmetric cell division within a root meristem, is highly upreguated in scrm-D mute and
downregulated in spch. Error bars represent the SE (n = 3).
(D) and (E) Expression of SCZ promoter driving cyan fluorescent protein (CFP). SCZ promoter is active in stelar tissue within the root meristem (D) as
well as in meristemoids (E). Bars = 10 mm in (D) and 20 mm in (E).
Molecular Signature of Meristemoids3269
a two-component signaling pathway, where cytokinin receptors,
ARABIDOPSIS HISTIDINE KINASE (AHK2, 3, and 4) (Inoue et al.,
2001; Yamada et al., 2001), relay a phosphate group to ARR
proteins. AHK3 has been shown to display stomatal lineage–
specificepidermal expression(Stolzetal., 2011). AlthoughT-DNA
insertion lines of ARR16 and CKX4 did not confer discernible
phenotypes, our finding implies that downregulation of cytokinin
signaling and/or metabolism may play a role in the transition from
proliferation to differentiation states of stomatal cell lineages.
Recently, a molecular link between cytokinin signaling and
CLE peptide-mediated stem cell maintenance has been reported.
represses the transcription of Type-A ARRs (ARR5, ARR6,
ARR7, and ARR15), which are required for proper meristem
function (Leibfried et al., 2005). Similarly, during root vascular
development, one of the CLE peptides, CLE10, inhibits proto-
xylem differentiation by repressing Type-A ARRs (ARR5 and
ARR6) (Kondo et al., 2011). Based on the known roles of the CLE
peptides in stem cell maintenance and high expression of CLE9
in meristemoids (Figure 4F) (Jun et al., 2010), it is attractive to
hypothesize that CLE9 may act as a signaling molecule for
meristemoid maintenance. Our results, in combination with the
expanding evidence of crosstalk between cytokinin and CLE
signaling in other stem cell populations, suggest that integration
broad hormone signals into local changes of gene expression
and, ultimately, cell behavior.
Asymmetric Division and Segregation of Cell Fate in the
Stomatal development serves as a model to study de novo
asymmetric cell divisions in plants. BASL was the only known
proteintobedistributed unevenlyduringtheasymmetric division
of stomatal lineages (Dong et al., 2009). Our result adds a
potential component to the BASL-mediated pathway. POLAR
exhibits a striking, transient polar distribution during asymmetric
cell division, and this polar localization requires functional BASL.
in protein–protein interactions (Burkhard et al., 2001) and shows
sequence similarity to myosin heavy chain–like protein (see
Supplemental Figure 6 online). Based on the protein structure
and localization patterns, it is tempting to hypothesize that
POLAR associates with BASL, components of a complex that
includes BASL, or cytoskeletal components to mark the site for
rapid cell expansion, which facilitates the cell fate separation of
SLGC from meristemoid. POLAR is evenly distributed at the cell
periphery in meristemoids where BASL is known to be exclu-
sively localized in the nucleus (Dong et al., 2009). This supports
the idea that sequestering BASL in the nucleus is a mechanism
preventing polar cell expansion, a process involving the dynamic
relocation of POLAR to the cell periphery. The null allele of
POLAR does not exhibit stomatal patterning defects like basl,
implying the presence of functional redundancy. Indeed, the
closest homolog of POLAR, At5g10890, which is not on the
ATH1 expression array, is also highly expressed in scrm-D mute
(88-fold upregulation), while being downregulated in spch (14-
fold downregulation) (Figure 4M). The extent of up- and down-
regulation of At5g10890 in the meristemoid-enriched popula-
tions is similar to that of POLAR (149-fold upregulation in scrm-D
mute; 17-fold downregulation in spch) (Figure 4M).
It is known that the bHLH transcription factor SPCH initiates
the meristemoid state together with SCRM/SCRM2 (MacAlister
et al., 2007; Pillitteri et al., 2007a; Kanaoka et al., 2008). Thus,
genes driving asymmetric division of meristemoids may be
downstream targets of these bHLH proteins. A coexpression
gene network places both BASL and POLAR directly connected
to SPCH and closely connected to SCRM (see Supplemental
Figure 8 online). Further experiments should address the possi-
bility of direct induction.
Cell Cycle Regulators and Stomatal Cell Divisions
Stomatal cell lineages undergo stereotypical cell divisions, from
entry and amplifying divisions of meristemoids to the eventual
symmetric divisions of GMCs. Our analysis revealed that pro-
moter activity of CDKB2;1, CYCB1;2, CYCA2;2, and CYCA2;3
was highly specific to stomatal lineage cells (Figure 7). Consis-
tent with a possible direct role in stomatal development, all three
cyclin genes examined have been shown to interact directly with
CDKB1;2 and CDKB2;1 in yeast and in planta (Van Leene et al.,
2011). A direct link between CYCA2;3 and epidermal patterning
was recently reported using ectopic overexpression analysis
where the ectopic overexpression of both CYCA2;3 and
CYCB1;1 resulted in increased epidermal cell division similar to
a SPCH overexpression phenotype (Boudolf et al., 2009). Single
overexpression of either regulator failed to confer a clear phe-
notype, suggesting that they function together as a complex
(Boudolf et al., 2009). However, CYCD4, which is known to
regulate stomatal cell lineage divisions specifically in the hypo-
transcriptome. Plants encode significantly more cell cycle reg-
ulatory proteins than other eukaryotic organisms, presumably
due to a sessile existence and subsequent need for plasticity
during development. Our profiling and expression analysis add
to the growing evidence supporting the idea that overlapping
expression of plant core cell cycle regulators is a key component
driving cell division and differentiation (Engler et al., 2009).
Molecular Commonalities among Stem Cell Populations
A notable feature of plant development is indeterminate growth,
where organogenesis persists throughout the life cycle via
continual activity of stem cells in the SAM and RAM. Our
meristemoid-enriched transcriptomeprovides theopportunityto
investigate the similarities and differences of molecular constit-
uents between transient and permanent stem cell populations.
The comparison of meristemoid versus SAM signatures reem-
phasizes the diverse roles of ERECTA family RLKs as regulators
coordinating cell proliferation and differentiation during shoot
and flower development as well as stomatal patterning (Torii
et al., 1996; Shpak et al., 2003, 2004, 2005; Pillitteri et al., 2007b;
3270The Plant Cell
cell divisions, while ERL1 and ERL2 inhibit the differentiation of
meristemoids to GMC and enforce stomatal spacing. Genetic
to perceive secreted signaling ligands EPF1 and EPF2 (Shpak
et al., 2005; Hara et al., 2007, 2009; Hunt and Gray, 2009).
Because TMM, EPF1, and EPF2 are not expressed in the SAM,
an exciting possibility is that the ERECTA family perceives other
EPF-LIKE (EPFL) peptides via dimerization with other receptor
partner(s). There are 11 EPFL genes in Arabidopsis (Hara et al.,
2009; Rowe and Bergmann, 2010; Rychel et al., 2010), for seven
of which the expression patterns and functions are yet unknown.
The highlighted roles of LRR-RLKs, ERECTA family, and EMS1
as commonly upregulated SAM meristemoid signatures under-
score the importance of cell–cell signaling in orchestrating stem
cell proliferation and differentiation.
In contrast with the SAM, where stem cell divisions are less
organized, Arabidopsis RAM cells undergo stereotypical asymmet-
ric divisions. Based on their positions relative to the quiescent
center, each stem cell in the RAM gives rise to a defined cell file
(Scheres, 2002). Our RAM versus meristemoid comparison re-
vealed SCZ as a commonly upregulated gene. SCZ executes
proper separation of cell fate in stem cells generating differentiated
root tissues, including epidermis, cortex, endodermis, and root cap
(ten Hove et al., 2010). The molecular mechanism by which SCZ
mediates cell fate separation is unknown, but it appears to involve
both cell-autonomous and non-cell-autonomous effects (ten Hove
et al., 2010). Our study highlights specific components involved in
asymmetric division within the stomatal lineage, BASL and POLAR,
as well as a potential common component, SCZ. Future mecha-
nistic studies of these proteins may molecularly define the com-
monalities and uniqueness of asymmetric divisions in plants.
Plant Material and Growth Conditions
Arabidopsis thaliana Columbia (Col) accession was used as the wild type.
Mutants used in the study were in the Col background. Identification
numbers for T-DNA insertional mutants of genes identified in this study
are given in Supplemental Table 3 online. All insertion lines and the
plasma membrane mCherry reporter, pm-RB (CD3-1008) (Nelson et al.,
2007), were obtained from the ABRC. The following mutants and reporter
lines were described previously: spch and mute (Pillitteri et al., 2007a),
scrm-D and scrm-D mute (Kanaoka et al., 2008), basl-2 (Dong et al.,
2009), MUTEpro:GFP (Pillitteri et al., 2008), EPF2pro:erGFP (Hara et al.,
2009), CDKB2;1pro:GUS, CycB1;2pro:GUS, CycA2;2pro:GUS, and
CycA2;3pro:GUS (gifts from Lieven de Veylder and Steffen Vanneste;
differentiation mutants. For microarray preparation, seeds of the wild
type, scrm-D, scrm-D mute, and spch were sterilized using 33% bleach
solution (bleach and 0.05% Tween 20) for 12 min and washed five times
with sterile water. Seeds were plated on 13 Murashige and Skoog media
and placed at 48C for5d. Plates were thenplaced understandardgrowth
5 d after germination.
Microarray Material Preparation
Five-day-old seedlings of the wild type, scrm-D, scrm-D mute, and spch
were collected and immediately placed in liquid nitrogen. To avoid any
complications due to circadian gene regulation, all genotypes were
harvested at the same time (1 PM) within 1 h of each other for each
replicate. spch plants were from a segregating population and were
identified by the smooth appearance of the cotyledons compared with
wild-type plants. Total RNA was extracted using the Qiagen RNeasy kit
(Qiagen) following manufacturer’s instructions. RNA purity and yieldwere
confirmed using the Agilent 2100 Bioanalyzer (Brolingen). Probe prepa-
ration and hybridization to the ATH1 GeneChip (Affymetrix) were per-
formed according to the manufacturer’s instructions by the University of
Washington Center for Array Technologies. Signal intensities from each
array were converted to raw expression .CEL data using GeneChip
operating software (Affymetrix).
Statistical Analysis of Microarrays
Our own and publicly available ATH1 microarray data were normalized
using Robin software (Lohse et al., 2010). Quality control was performed
with standard settings (Limma package using RMA normalized data with
Benjamini Hochberg Pvalue correction) (Benjamini and Hochberg, 1995).
Further statistical analysis was performed using the MultiExperiment
Viewer (MeV) software (Saeed et al., 2003). Genes with statistically
the significance of microarrays module with a Q-value cutoff of <0.005
and a twofold- or threefold-change cutoff. K-means clustering was
performed on our own microarrays using log2-transformed normalized
expression values as input. Genes were prefiltered using the variance
filter implementedin theMeV software to remove genesthat showedlittle
highest variance among the different arrays. Five clusters were built from
maximal 200 iterations using a Pearson correlation distance matrix.
Annotation of genes and classification into functional pathways (BINs)
was performed withtheMapmansoftware (Thimmet al.,2004). Pageman
software (Usadel et al., 2006) was used for overrepresentation analysis.
Verification of Microarray Results by RT-PCR and qRT-PCR
The RNA isolated from Col-0 wild-type, spch, scrm-D mute, and scrm-D
seedlings used for the microarray experiment was treated with DNase I
(Invitrogen). RNA was converted to cDNA using iScript reverse transcrip-
time PCR was performed using a CFX96 real-time PCR detection system
(Bio-Rad) using iTaq SYBR Green Supermix with ROX (Bio-Rad) and
Amplification of the ACT2 gene was used to verify equal loading of cDNA
(RT-PCR) or as an internal control in reactions with relative expression
calculated using the cycle threshold (Ct) 22DDCtmethod (qRT-PCR) (Livak
and Schmittgen, 2001). For RT-PCR, all genes were amplified in 31 cycles,
except ACT2 (30 cycles) and POLAR (35 cycles) using standard PCR
can be found in Supplemental Table 4 online.
Molecular Cloning and Generation of Transgenic Plants
See Supplemental Table 5 online for a list of plasmid constructs gener-
ated in this study and Supplemental Table 6 online for a list of primerDNA
sequences used for molecular cloning. Briefly, the promoter region (up to
;2.5 kb) upstream of the translation start site was cloned into the pENTR
59 TOPO (Invitrogen) vector according to the manufacturer’s instructions.
The full-length genomic coding region was amplified and cloned into the
pENTR D-TOPO vector (Invitrogen). These vectors were combined in a
three-way Gateway cloning reaction using LR Clonase II Plus (Invitrogen)
to produce a C-terminal GFP fusion driven by the endogenous promoter.
Each genomic plasmid was used in a two-way Gateway reaction to
Molecular Signature of Meristemoids3271
produce an overexpression construct driven by the cauliflower mosaic
virus 35S promoter. The Gateway-based destination vectors were pro-
vided by Tsuyoshi Nakagawa (Shimane University) (Nakagawa et al.,
2007, 2008). Generation and selection of transgenic plants and their
phenotypic analyses were performed as described by Pillitteri et al.
Confocal microscopy images were taken on either the Zeiss LSM700 or
images were false colored, and their brightness/contrast settings were
adjusted using Photoshop CS4 (Adobe). Histochemical staining for GUS
activity was performed as previously described (Sessions et al., 1999).
Time-Lapse Cotyledon Imaging
as described above and held at 48C for 2 d. All seeds were imbibed and
placed under germinating conditions within 1 h of the same time (10 AM).
Seeds were moved into room temperature and immediately dissected:
the seed coat was removed and the hypocotyl severed. Removing the
hypocotyl shows no noticeable effect on cotyledon development for the
first 4 d under our conditions. A solution of 0.5% Bacto Agar was
prepoured into chamber slides (Lab Tek II chambered 1.5 German cover
glass system; Fisher Scientific) at room temperature, and dissected
cotyledons wereplaced beneathitfor immobilizationand protection from
desiccation. The Zeiss LSM700 (inverted, 320 objective, 31 zoom) was
used to image the GFP reporter with excitation at 488 nm and a band-
pass emission filter of 470 to 500 nm at intervals of 30 min. Z-stacks of 50
to 70 slices at 0.96 mm were captured for each time point and projected
for maximum intensity. For POLAR-GFP, stable lines were crossed with
at 555 nm and collection with a short-pass 555-nm filter.
Sequence data from this article can be found in the GenBank/EMBL data
the following: POLAR (At4g31805), CDKB2;2 (At1g20930), RPA1A
(AT1G76540), CYCB1;2 (AT5G06150), CYCA2;3 (AT1G15570), CYCA2;2
(AT5G11300), IAA7 (AT3G23050), Integral membrane family protein
(AT3G24140), ICE1/SCRM (AT3G26744), SCRM2 (AT1G12860), SDD1
(AT1G69320), and SCZ (AT1G46264). The GenBank accession number
for POLAR cDNA is JN663804. The complete expression data set is
available in the National Center for Biotechnology Information Gene
Expression Omnibus (Edgar et al., 2002) under accession number
The following materials are available in the online version of this article.
Supplemental Figure 1. Images of Representative 5–Days After
Germination Seedlings Used in This Study.
Supplemental Figure 2. Summary of Overrepresented Functional
Gene Categories Associated with Cell-Enriched Genotypes.
Supplemental Figure 3. RT-PCR Data for Selected Genes.
Supplemental Figure 4. Heat Map of Absolute Expression of
Selected Stomatal Regulatory Genes in Rosettes and Roots.
Supplemental Figure 5. Expression of POLAR in a GMC Differenti-
ating without Asymmetric Divisions.
Supplemental Figure 6. T-DNA Insertion Lines and Sequence Com-
parison Coiled-Coil Domain of POLAR.
Supplemental Figure 7. Overrepresentation Analysis of Transient
and Permanent Stem Cell Populations.
Supplemental Figure 8. POLAR Coexpression Network.
Supplemental Table 1. Genes Used for Production of GFP Reporter
Supplemental Table 2. Genes Common to Transient and Permanent
Stem Cell Populations.
Supplemental Table 3. T-DNA Insertion Lines Examined in This
Supplemental Table 4. Primers Used for Transcript Amplification in
RT-PCR and qRT-PCR.
Supplemental Table 5. Reporter and Overexpression Constructs
Produced for This Study.
Supplemental Table 6. Primers Used for Cloning in This Study.
Supplemental Data Set 1. Genes Showing High Expression in scrm-D
Supplemental Data Set 2. Genes Showing High Expression in
Supplemental Data Set 3. Genes Showing High Expression in spch.
Supplemental Data Set 4. Genes Significantly Deregulated in the
Meristemoid-Enriched Background, scrm-D mute.
Supplemental Data Set 5. Genes Significantly Deregulated in the
Stomata-Enriched Background, scrm-D.
Supplemental Data Set 6. Genes Significantly Deregulated in the
Pavement Cell-Enriched Background, spch.
Supplemental Movie 1. Time-Lapse Imaging of POLAR Localization.
Supplemental Movie 2. Time-Lapse Imaging of POLAR Localization
We thank the ABRC and SIGnAL for providing clones and insertion lines
used in this study; S. Vanneste, P. Doerner, J. Murray, D. Inze ´, and L. de
Vylder for kindly providing transgenic plants expressing various cell
cycle GUS reporter lines; R. Heidstra for the SCZpro:CFP line; J.
Nemhauser for advice on designing the microarray experiments; D.
Bergmann for discussion about stomatal cell lineage profiling; and T.
Kuroha and A. Rychel for assisting construction of reporter plasmids.
The work was supported by the University of Washington Royalty
Research Fund (RRF-4098), the National Science Foundation (MCB-
0855659), and the Precursory Research for Embryonic Science and
Technology award from the Japan Science and Technology Agency to
K.U.T. L.J.P.’s research was in part supported by the startup funds from
3272The Plant Cell
Western Washington University. K.M.P. is supported by the National
Science Foundation Graduate Research Fellowship (DGE-0718124),
and R.J.H. is supported by the Deutsche Forschungssgemeinschaft
research fellowship. K.U.T. is a Howard Hughes Medical Institute–
Gordon and Betty Moore Foundation investigator.
K.U.T. conceived and directed the project. L.J.P. conducted microarray
experiments and analyzed the results with R.J.H. R.J.H. performed
bioinformatic analysis. L.J.P. and K.M.P. generated expression con-
structs and Arabidopsis reporter lines and performed analysis with
R.J.H. K.M.P. developed and performed time-lapse imaging. Thus,
L.J.P., K.M.P., and R.J.H. made uniquely equal contributions. All authors
contributed to writing the article.
Received June 26, 2011; revised September 10, 2011; accepted Sep-
tember 18, 2011; published September 30, 2011.
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