Cell fate transitions during stomatal
development
Laura Serna*
Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha. Real Fa´brica de Armas, Avda. Carlos III, Toledo,
Spain
Stomata, the most influential components in gas
exchange with the atmosphere, represent a revealing
system for studying cell fate determination. Studies in
Arabidopsis thaliana have demonstrated that many of the
components, functioning in a signaling cascade, guide
numerous cell fate transitions that occur during stomatal
development. The signaling cascade is initiated at the
cell surface through the activation of the membrane
receptors TOO MANY MOUTHS (TMM) and/or ERECTA
(ER) family members by the secretory peptide EPIDERMAL
PATTERNING FACTOR1 (EPF1) and/or a substrate
processed proteolytically by the subtilase STOMATAL
DENSITY AND DISTRIBUTION1 (SDD1) and transduced
through cytoplasmic MAP kinases (YODA (YDA), MKK4/
MKK5, and MPK3/MPK6) towards the nucleus. In the
nucleus, these MAP kinases regulate the activity of the
basic helix-loop-helix (bHLH) proteins SPEECHLESS
(SPCH), MUTE, and FAMA, which act in concert with
the bHLH-Leu zipper protein SCREAM (SCRM) (and/or
its closely related paralog, SCREAM2). This article
reviews current insights into the role of this signaling
cascade during stomatal development.
Keywords: epidermis; stomata
Introduction
Gas exchange between the plant and the atmosphere is
regulated through the ‘‘stoma’’, which is a Greek word
meaning mouth or opening. Stomata are microscopic pores
found in the epidermal tissue of most aerial organs of all
terrestrial plants. Each stomatal pore is bounded by two guard
cells. The pore opening (and gas exchange) depends on
changes in the turgor of the guard cells, which is regulated by
the flow of water and ions between the guard cells and the
adjacent epidermal cells.(1)
Gain in turgor of the guard cells
triggers stomatal opening, whereas loss of turgor induces its
closure. Gas exchange depends not only on the opening and
closing of the stomata but also on their density and spatial
distribution within the epidermal tissue.
In A. thaliana, the first sign of stomatal development is an
asymmetric cell division from a protodermal cell called the
meristemoid mother cell(2,3)
(Fig. 1). This cell division
produces a small triangular cell, the meristemoid, and a
larger cell. Meristemoids can divide asymmetrically, in an
inward spiral, up to three times and always yield a larger cell
and a smaller meristemoid that maintains its stem cell activity.
The meristemoid, after these asymmetric cell divisions, loses
its stem cell character and adopts a rounded shape giving rise
to the guard mother cell. The guard mother cell undergoes a
symmetric cell division that produces the paired guard cells.
The paired guard cells and their adjacent epidermal cells
make up the primary stomatal complex. The larger cells that
result from the asymmetric divisions and that come into
contact with the stoma (or its precursor) can either enter into
the stomatal pathway or become pavement cells. When they
enter into the stomatal pathway, they copy the developmental
program of the meristemoid mother cell, giving rise to
secondary complexes. Higher complex order can also be
formed.
Wild-type stomata almost never develop directly next to
each other, as would be expected if they were randomly
distributed, which indicates that there must be an underlying
patterning mechanism (Fig. 2). The inward-spiraling nature of
the reiterative asymmetric divisions that the meristemoids
undergo tends to result in the stomata being surrounded by a
full complement of clonally related nonstomatal cells, which
helps in preventing the development of stomatal clusters(4,5)
(Fig. 1). However, it has been demonstrated that the
orientation of the cell division plane in cells entering the
stomatal pathway that contact a stoma or its precursor
(spacing divisions) places the new meristemoid (satellite
meristemoid) away from the pre-existing stoma, ensuring a
minimal distance of one cell between stomata neighbors(6)
(Fig. 1). It seems that a combination of several strategies
ensures that guard cells never make direct contact with their
neighbors, guaranteeing their function.(2,3)
During the last few years, the understanding of the
stomatal development and patterning in a molecular context
has been advanced by the cloning of a large number of
relevant genes (Table 1). These include those encoding for
extracellular molecules,(4,7)
cell membrane components(8–10)
DOI 10.1002/bies.200800231 Review article
*Correspondence to: L. Serna, Facultad de Ciencias del Medio Ambiente,
Universidad de Castilla-La Mancha. Real Fa´brica de Armas, Avda. Carlos III,
s/n, 45071 Toledo, Spain.
E-mail: laura.serna@uclm.es
BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc. 865
Figure 1. Genetic control of the progressive steps of stomatal development. SDD1, EPF1, TMM, members of the ER family receptor-like kinases
(ER, ERL1, and ERL2), YDA and MKK4/MKK5-MPK3/MPK6 repress the entry into the stomatal pathway. In contrast, SPCH starts stomatal
development by inducing the first asymmetric division, giving rise to the first meristemoid. Two or three divisions after the formation of the first
meristemoid, MUTE drives the last asymmetric cell division producing the guard mother cell. The FLP/MYB88 and FAMA then drive the symmetric
division that gives rise to the two guard cells. Both TMM and SDD1, among other genes, also maintain the stem cell character of the meristemoids
and orient the cell division plane of meristemoid mother cells that contact stomata so that the new meristemoids form away from the pre-existing
stomata. SCRM and SCRM2, in a dosage-dependent manner, specify the actions of SPCH, MUTE, and FAMA. Adapted from.(16–18,36)
Figure 2. Stomatal phenotype of wild-type and mutant plants. Wild-type stomata are spaced by intervening cells. The gpa mutant exhibits a
reduced stomatal index. In contrast, the agb1 mutant exhibits an increased stomatal index. In sdd1, tmm, epf1, flp-1, flp-1 myb88, er erl1erl2, loss
of function of MPK3/MPK6 (or MKK4/MKK5) and yda, stomata develop adjacent to one another. The spch, mute, and fama mutants fail to develop
mature stomata. The scrm, scrm scrm2/þ, and scrm scrm2 mutants phenocopy fama, mute, and spch, respectively. AGL16 does not regulate
stomatal pattern (not shown). Adapted from.(16,36)
Review article L. Serna
866 BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc.
and cytoplasmic(11,12)
and nuclear factors.(13–18)
Here, I
review current insights into the role played by these genes and
the interplay among them.
The switch from protodermal to
meristemoid mother cell identity
Entry into the stomatal pathway is under the control of
several genes, which also control additional steps in the
stomatal pathway (see the next sections). Analysis of
stomatal development through serial imprints has shown
that mutations in the TOO MANY MOUTHS (TMM) or
STOMATAL DENSITY AND DISTRIBUTION (SDD1) genes,
encoding for a plasma membrane-anchored leucine-rich
repeat-receptor-like protein (LRR-RLP) and a subtilisinrelated
extracellular protease, respectively,(4,8)
result in
an increased number of protodermal cells entering the
stomatal pathway.(4,6,19)
This indicates that these genes
negatively regulate stomatal pathway initiation.(4,6,19)
Studies
of epidermal patterning in fixed samples have shown an
inverse correlation between the stomatal density and the level
Table 1. Genes regulating stomatal development in Arabidopsis.
Gene name Mutant phenotype Proposed wild-type function Protein encoded
STOMATAL DENSITY AND
DISTRIBUTON 1 (SDD1)
Increased stomatal index, some
stomatal clusters
Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms,
orients spacing divisions, reduces the
number of higher order complexes
Subtilisin-related
protease
EPIDERMAL PATTERNING
FACTOR1 (EPF1)
Stomatal clusters Orients spacing divisions, represses
stomatal initiation
Secretory peptide
ERECTA (ER) family Almost all epidermal cells
are GCs
Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms
Leucine-rich repeatreceptor-like
kinases
TOO MANY MOUTHS
(TMM)
Stomatal clusters Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms,
orients spacing divisions, reduces the
number of higher order complexes
Leucine-rich repeatreceptor-like
protein
YODA (YDA) Almost all epidermal
cells are GCs
Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms,
orients spacing divisions
Mitogen-activated
protein kinase kinase
kinase
MITOGEN-ACTIVATED
PROTEIN KINASE
KINASE (MKK4/MKK5)
Almost all epidermal
cells are GCs
Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms,
orients spacing divisions
Mitogen-activated
protein kinase kinase
MITOGEN-ACTIVATED
PROTEIN KINASE
(MPK3/MPK6)
Almost all epidermal
cells are GCs
Represses stomatal initiation, promotes
stem cell fate maintenance of the Ms,
orients spacing divisions
Mitogen-activated
protein kinase
SPEECHLESS (SPCH) Only jigsaw-puzzle-shaped
pavement cells
Drives the first asymmetric cell division bHLH protein
MUTE Ms aborted after excessive
asymmetric cell divisions
Represses stem cell fate of Ms and
promotes GMC formation
bHLH protein
FAMA Clusters of GMCs or immature
GCs
Drives the transition from the GMC to the
GCs
bHLH protein
SCREAM (SCRM) Clusters of GMCs or immature
GCs
Specifies the actions of SPCH, MUTE
and FAMA
bHLH-leucine
zipper protein
SCREAM2 (SCRM2) None; scrm scrm2 phenocopies
spch; scrm scrm2/þ
phenocopies mute
Specifies the actions of SPCH, MUTE,
and FAMA
bHLH-leucine
zipper protein
FOUR LIPS (FLP) Stomatal clusters with arrested
GCs
Drives the transition from the GMC to the
GCs
R2R3 MYB protein
MYB88 None; enhances flp phenotype Drives the transition from the GMC to the
GCs
R2R3 MYB protein
AGAMOUS-LIKE16
(AGL16)
Reduced higher-order stomatal
complexes
Promotes the formation of higher order
complexes
MADS box protein
G PROTEIN a-SUBUNIT
(GPA1)
Reduced stomatal index Promotes the formation of higher order
complexes and reduces the formation
of primary complexes
Heterotrimeric G protein
G PROTEIN b-SUBUNIT
(AGB1)
Increased stomatal index Reduces the formation of higher order
complexes and promotes the
formation of primary complexes
Heterotrimeric G protein
M, meristemoid; GMC, guard mother cell; GC, guard cells. Phenotypes of leaves and/or cotyledons.
L. Serna Review article
BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc. 867
of expression of EPIDERMAL PATTERNING FACTOR1
(EPF1), with plants that exhibit higher expression levels
producing no stomata and being infertile as a consequence of
the absence of stomata.(7)
This indicates that EPF1, which
encodes a secretory peptide, also represses stomatal
initiation.(7)
This finding is supported by the epf1 mutant,
which exhibits an increased stomatal density with some
stomatal clusters.(7)
Analysis of epidermal patterning in fixed
plants also revealed that members of the ERECTA family
[ERECTA (ER), ERECTA LIKE1 (ERL1), and ERL2], which
control several aspects of plant growth and also its responses
to biotic and abiotic signals,(20,21)
also repress the entry into
the stomatal pathway, although in a redundant manner.(9,22)
The three members of the ER family encode for members of
the membrane leucine-rich repeat-receptor-like kinases
(LRR-RLK).(21,23)
Cells in plants with a permanently active
version of the mitogen-activated protein kinase kinase kinase
(MAPKKK) YODA (YDA) do not initiate stomatal development,
which indicates that stomatal initiation requires a reduction in
or suppression of YDA activity.(11)
In agreement with this
finding, the number of meristemoid mother cells is increased
in the yda mutant.(11)
YDA also controls cell fate determination
during plant embryonic development with most yda
mutants dying at the embryo stage and those that survive
remaining very small and fuzzy.(24)
Similar to yda, loss of
function of MITOGEN-ACTIVATED PROTEIN KINASE4
(MKK4)/MKK5 or of their downstream MPK3/MPK6 increases
the number of cells entering the stomatal pathway, and
activation of MKK4/MKK5-MPK3/MPK6 switches off stomatal
development initiation.(12)
MKK4 and MKK5 are also
upstream kinases for MPK3 and MPK6 in responses to
multiple stress stimuli.(25–27)
Mutations in these loci promote the entry into the stomatal
pathway (Fig. 1), but they produce different stomatal
phenotypes in mature organs (Fig. 2). Mutations in TMM or
EPF1 lead to many single stomata being replaced by stomatal
clusters.(7,28)
Mutations in SDD1 mainly increase the stomatal
index ([stomatal density/(stomatal density þepidermal cell
density)] Â 100, where epidermal cells include guard cells as
well as others that constitute the epidermis) but also produce
some stomatal clusters.(4)
In both yda and er erl1 erl2 almost
all epidermal cells are guard cells.(11,21)
Similarly, silencing of
MPK3 and MPK6 or MKK4 and MKK5 genes results in the
entire epidermal tissue being composed of stomata.(12)
In addition to these negative regulators of stomatal pathway
initiation, positive ones have also been identified. Mutations in
the SPEECHLESS (SPCH) gene, which encodes a member of
the basic helix-loop-helix (bHLH) family of transcriptional
factors, produce an epidermal tissue consisting of jigsawpuzzle-shaped
pavement cells(16)
(Fig. 2). This suggests that
SPCH drives the first asymmetric cell division that initiates
stomatal development(16)
(Fig. 1). Supporting such as role, the
overexpression of SPCH increases the number of cells
entering the stomatal pathway.(16,17)
The INDUCER OF
CBF EXPRESSION1 (ICE also named SCREAM) and
SCREAM2 (SCRM2) genes also redundantly initiate stomatal
lineage with scrm scrm2 double loss-of-function mutants
phenocopying the spch epidermal phenotype(18)
(Figs. 1
and 2). In agreement with this positive role in stomatal
development, gain-of-function mutations in either SCRM or
SCRM2 increase the number of stomata, producing stomata in
direct contact.(18)
SCRM and SCRM2 also encode nuclear,
closely related bHLH-leucine zipper proteins.(18,29)
G protein signaling can be transmitted through the
activated Ga, the Gbg dimer, and/or the heterotrimeric
complex. G protein a-subunit (GPA1) and G protein b-subunit
(AGB1) encode the a-subunit and b-subunit, respectively, of
the G protein.(30,31)
GPA1 positively regulates stomatal
formation, with gpa1 loss-of-function mutant exhibiting a
reduced stomatal index (Fig. 2), whereas plants overexpressing
the constitutively active form of GPA1 show an increased
stomatal index compared with that of wild-type plants.(10)
In
agreement with the positive role of GPA1 in stomatal
formation, plants with an elevated steady-state pool of
activated GPA1 (rgs1) exhibit an increased stomatal index
compared with that of both wild-type and gpa1 plants.(10)
In
contrast with the gpa1 mutant, plants with an inactive form of
the G protein b-subunit (agb1) exhibit an increased stomatal
index (Fig. 2), which suggests that Gbg dimer represses
stomatal formation.(10)
In accord with this view, overexpression
of the AGB1 subunit reduces the stomatal index.(10)
These
results can be also attributable to the lack of GPA1. However,
because the stomatal index in agb1 is higher than that in rgs1
or in plants overexpressing GPA1, the increased stomatal
index in agb1 most probably has not resulted from the positive
role of the active form of GPA1.(10)
Thus, the antagonistic
action between GPA1 and AGB1 in stomatal initiation indicates
that both Ga and Gbg can transduce signals to downstream
effectors. This is supported by the stomatal density in the
double gpa1 agb1 mutant, which exhibits the mean value of
the single gpa1 and agb1 mutants.(10)
Driving the number of asymmetric
cell divisions
Many of the genes that control the switch from protodermal to
meristemoid mother cell identity (SDD1, TMM, members of
the ER-family, YDA, MKK4/MKK5, MPK3/MPK6, SPCH,
SCRM, and SCRM2) also regulate the number of asymmetric
cell divisions that occur before stoma formation. Analysis of
serial imprints and of fixed specimens has shown that guard
mother cell identity is prematurely assumed, after the first or
second asymmetric cell division, in both tmm and sdd1
mutants.(4,6,32)
In the yda mutant, almost all epidermal cells
are guard cells.(11)
Because asymmetric divisions contribute
Review article L. Serna
868 BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc.
to the formation of pavement cells, we infer that in the yda
mutant, guard mother cell identity is also prematurely
assumed. The same argument can be applied to the
er erl1 erl2 triple mutant or to plants silencing MPK3 and
MPK6 or MKK4 and MKK5, whose epidermis consists
basically of paired guard cells.(9)
In addition, erl1, erl2 and
erl1erl2 mutants seem to exhibit a reduction in the number of
larger cells that surround the stoma (or its precursor), which
arise from the asymmetric divisions.(9)
This suggests that both
ERL1 and ERL2 maintain the stem cell character of the
meristemoids, preventing guard mother cell formation.(9,22)
Either direct or indirect observations indicate that the affected
genes in these mutants maintain the stem cell character of the
meristemoids in wild-type plants (Fig. 1).
SPCH may also play roles other than that related with the
entry into the stomatal pathway. However, the fact that cells in
the spch mutant do not enter into the stomatal pathway limits
further characterization of hypothetical SPCH functions.
Interestingly, a missense mutation affecting the carboxy
terminus of SPCH reduces but does not repress stomata
formation.(16)
The analysis of static pictures from the pedicel
epidermis showed that SPCH mutation reduces the number
of sister cells of the meristemoids that surround every stoma.
This led to the proposal that SPCH, in addition to promoting
the first asymmetric cell division during stomatal development,
also maintains the stem cell activity of the meriste-
moids.(16)
Whether SPCH also plays this role in the leaf and/or
cotyledon remains to be established.
Normal stomatal development requires a delicate balance
between the maintenance of meristemoid cell activity and its
differentiation into guard mother cell. The genes described
here maintain the stem cell character of the meristemoids.
However, how do meristemoids lose their stem cell properties
to differentiate into guard mother cells? The loss-of-function
mute mutant does not develop paired guard cells but forms
meristemoids that abort after excessive asymmetric cell
divisions (Fig. 2), indicating that MUTE negatively regulates
stem cell activity and promotes guard mother cell forma-
tion(16,17)
(Fig. 1). MUTE encodes a bHLH protein,(17)
and it is
also required for the production of the hydathode pore.(33)
Both
SCRM and SCRM2 are also required to drive the transition
from meristemoid to guard mother cell (Fig. 1), with scrm
scrm2/þ mutant exhibiting an identical phenotype to mute(18)
(Fig. 2). The reduced stomatal index of the gpa1 loss-offunction
mutant, together with the increased index of plants
overexpressing the constitutively active form of GPA1,
suggests that GPA1 represses the stem cell fate of the
meristemoids.(10)
AGB1 modulates this process in the
opposite direction to that of GPA1.(10)
As previously discussed,
both Ga and Gbg probably transduce signals to downstream
effectors regulating the stem cell fate of the meristemoids.
Stopping cell divisions: From the guard
mother cell to the guard cells
A group of four genes controls the last and symmetric
cell division in the stomatal pathway that gives rise to the
two guard cells from the guard mother cell (Fig. 1). Two of
them, FOUR LIPS (FLP) and MYB88, encode R2R3 MYB
regulators with very similar sequences and structures.(13)
Mutations in FLP gene (severe flp alleles) induce stomatal
clusters with arrested guard cells(13,28)
(Fig. 2) and, although
mutations in MYB88 induce no apparent stomatal pheno-
type,(13)
plants homozygous for mutations in both genes
show more and larger clusters than flp alone, with some
developmentally arrested guard cells.(13)
This suggests
that they play redundant functions during stomatal
development. The complementation of the flp phenotype by
expression of a genomic MYB88 construct supports such a
conclusion.(13)
A combination of techniques, including monitoring the
cell division pattern through time and the determination of
cell identity by cytological (wall thickenings) and molecular
(KAT gene expression) markers, has led to the proposal that
in the flp mutant the daughter cells of the guard mother cells
divide symmetrically one or more times before assuming
guard cell identity.(13)
This indicates that the FLP gene drives
the transition from the guard mother cell to the guard cells.
The presence of arrested guard cells in both flp myb88 and
severe flp alleles suggests that FLP may also drive guard
cell differentiation. Alternatively, the role for these genes
in guard cell differentiation might be only indirect due to
cell packing constraints or the dilution of intrinsic positive
regulators of guard cell differentiation.(13)
If this is true, flp
and flp myb88 should show full guard cell differentiation
in single stomata. The presence of fully differentiated
guard cells placed in the border line of the clusters would
support the hypothesis that packing limits guard cell
differentiation and lead to discarding the hypothesis that
dilution of the intrinsic regulators prevents guard cell
differentiation.
Plants lacking detectable FAMA expression form clusters
of guard mother cells or immature guard cells instead of
single and mature stomata(14)
(Fig. 2). We therefore
conclude that the FAMA gene, which encodes a protein
belonging to the bHLH family of transcriptional factors, also
controls the symmetrical cell division of the guard mother cell
and guard cell differentiation.(14)
The loss-of-function
mutation in SCRM also induces the formation of groups
of guard mother cells or immature guard cells instead of
single and fully differentiated stomata.(18)
This indicates that
SCRM plays a role similar to that of FAMA and FLP/
MYB88(18)
(Fig. 1).
L. Serna Review article
BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc. 869
Higher order complexes and spacing
divisions
Primary stomatal complexes can give rise to higher order
complexes derived from satellite meristemoids.(2)
The MADS
box gene AGAMOUS-LIKE16 (AGL16) promotes the formation
of higher order stomatal complexes and is subject to
miRNA regulation.(15)
The number of higher order stomatal
complexes increases in plants expressing miRNA824-resistant
AGL16 mRNA and decreases in agl16-1 mutant or
miR824-overexpressing plants.(15)
This result and the fact
that both AGL16 and miR824 locate to stomatal complexes,
but have no overlapping pattern, suggest that in wild-type
stomatal development the down-regulation of AGL16 by
miR824 reduces the number of higher order stomatal
complexes.(15)
The reduced number of higher order complexes
in gpa1 indicates that GPA1 promotes the formation of
higher order complexes. GPA1 also reduces the formation of
primary complexes.(10)
Like that for the stomatal index, AGB1
counteracts the action of GPA1.(10)
In contrast to AGL16 and
GPA1 function, both TMM and SDD1 reduce the number of
higher order complexes, with tmm and sdd1 mutants
developing an increased number of higher order stomatal
complexes.(4,6)
When cells in contact with a stoma (or its precursor cell)
enter into the stomatal pathway, their division planes are
orientated, so that the new meristemoids are placed away
from the pre-existing stomata (or stomatal precursors)(3)
(Fig. 1). Analysis of serial imprints has shown that, in both
tmm and sdd1 mutants, the orientation of the cell division
plane of these meristemoid mother cells is random, which
produces meristemoids adjacent to the pre-existing sto-
mata.(4,6)
Studies of epidermal patterning in fixed plants have
also shown that mutations in YDA, EPF1, MKK4/MKK5 or
MPK3/MPK6 also trigger the formation of ectopic meriste-
moids.(7,11,12)
Therefore, TMM, SDD1, YDA, EPF1, and
MKK4/MKK5- MPK3/MPK6 orient the cell division plane of
meristemoid mother cells, preventing the development of the
meristemoids in contact with the stomata or their precursors.
Because this is the main mechanism that prevents stomatal
cluster formation,(6)
these genes play a relevant role in
stomatal pattern formation. Although no additional genes
have been shown to be involved in this process, it does not
exclude the possibility that genes such as the ER family, for
example, can play a role in such a process.
A model for stomatal development
Membrane receptors transmit information from the cell
surface to the interior cells. Interestingly, TMM lacks a
cytoplasmic domain.(8)
Based on well-characterized systems
as prediction models, it has been proposed that TMM, which
locates to the plasma membrane, physically interacts with a
partner that would provide the cytoplasmic domain required to
transmit the signal across the membrane.(8,19)
The molecular
nature of the members of the ER-family and their domains of
promoter induction, which overlap with that of TMM in
stomatal lineage cells, suggest that they may be the proposed
partner(s)(8,21,23,34)
(Fig. 3). The activated receptor complexes
signal to the MKK4/MKK5-MPK3/MPK6 module via
YDA.(11,12)
The activation of this signaling cascade negatively
regulates SPCH activity, repressing stomatal initiation.
Because primary complexes can give rise to higher order
complexes when their cells resulting from the asymmetric
divisions copy the meristemoid mother cell program,(2,3)
the
induction of SPCH activity should start in the precursor cell of
the primary complex and should progress through the
precursor cells of secondary (and higher order) complexes.
This signaling cascade also maintains the stem cell character
of the meristemoids by repressing MUTE activity, and guides
the cell division orientation of cells entering the stomatal
pathway in contact with stomata (or their precursors). Both
mechanisms contribute to the stomatal pattern, with the
second one being the main mechanism preventing stomatal
cluster formation.(2,3)
This cascade, therefore, controls not
only stomatal initiation but also its distribution in the epidermal
layer. However, how are these receptors activated? A
possibility is that either an SDD1-processed molecule or
the EPF1 peptide, which are expressed in stomatal lineage
cells,(7,35)
diffuse from the stomatal lineage cell and are
recognized by the extracellular domain of the hypothetical
heterodimer(s).(4,7)
Both extracellular molecules seem to be
involved in at least the entry into the stomatal pathway and
placing meristemoids away from existing stomata.(4,7)
However,
the differences in the stomatal phenotypes of plants with
mutations in the different genes that have a role in this
signaling cascade indicate that the reality is probably more
complex than this simple model.
Genetic analyses support the relationship between these
factors. For example, the finding that constitutive SDD1
expression reduces the number of stomata in the epidermis of
wild-type plants (and of sdd1 and flp1 mutants), but is unable
to suppress the tmm-1 stomatal phenotype, suggests that
TMM acts downstream of SDD1.(35)
The stomatal pattern of
the epf1 tmm double mutant, which is similar to that of tmm
single mutant, supports that TMM also functions downstream
of EPF1.(7)
This is in agreement with the fact that the stomatal
pattern of 35S::EPF1 plants is dependent on the TMM gene
(and ER-family genes).(7)
Interestingly, the effect of epf1 sdd1
on the stomatal pattern is additive, indicating that EPF1 and
SDD1 function independently, and arguing against the theory
that SDD1 cleaves and activates the EPF1 peptide.(7)
This is
supported by the fact that the 35S::EPF1 stomatal pattern
does not depend on SDD1: the number of stomata in
35S::EPF1 wild-type plants is similar to those in 35S::EPF1
Review article L. Serna
870 BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc.
sdd1.(7)
The epidermal patterning phenotype of tmm, yda, and
er erl1 erl2 is epistatic to 35S::EPF1, which suggests that
EPF1 acts upstream of TMM, YDA, and ER family genes.(7)
The fact that the constitutive activation of YDA can dominantly
suppress the defects of tmm suggests that YDA acts
downstream of TMM.(11)
The gain-of-function Nt-MEK2, the
tobacco homolog of MKK4 and MKK5, rescues the yda
stomata clustered phenotype, suggesting that YDA plays a
role upstream of MKK4/MKK5-MPK3/MPK6.(12)
In agreement
with this finding, MPK3 and MPK6 are activated in
constitutively active YDA plants.(12)
The genetic interactions
among TMM and members of the ER family seem to be more
complex. The stomatal phenotypes arising from combinations
of mutants in the TMM gene with single and multiple mutants
in the ER family support the hypothesis that the affected
proteins are part of one physical complex, but in several
organs they can also be explained by TMM acting independently
of the ER family, e.g., in stems.(8,9)
Genetic analysis also supports the fact that these signaling
components act upstream of SPCH, MUTE, and FAMA.(36)
Moreover, an elegant set of experiments has shown that
MPK3 and MPK6 phosphorylate to SPCH in vitro and
modulate its activity in vivo.(37)
In addition, all the phosphorylation
sites are contained within the 93-amino acid MAPK
target domain of SPCH.(37)
SPCH, MUTE, and FAMA exhibit
a sequential expression pattern, with SPCH being expressed
in all protodermal cells,(16,17)
MUTE mainly in meriste-
moids(16,17)
and FAMA in guard mother cells and developing
guard cells.(14)
The proteins encoded by these genes exert
their actions through dimer formation with both SCRM and
SCRM2, which are broadly expressed in the epidermis.(18)
Although SPCH and SCRM/SCRM2 are broadly expressed in
the epidermal tissue, their expression patterns are not
completely overlapping. SPCH is expressed in the entire
protoderm,(16,17)
and was the first of the regulatory genes to
be transcribed. Kanaoka et al.(36)
have proposed that SPCH
just confers the competency to enter into the stomatal
pathway and that SCRM and SCRM2 are required to initiate
stomatal development. In addition, these bHLH factors
autoregulate their own expression and activate that of one
Figure 3. Model for stomatal development. At the plasma membrane, the leucine-rich repeat receptor-like protein (LRR-RLP) TMM, which
lacks a cytoplasmic domain, physically interacts with members of the ER family of LRR-receptor-like kinases (LRKs). Either the EPF1 peptide or a
molecule processed by the SDD1 subtilase is recognized by the extracellular domain of the hypothetical heterodimer. The activated TMM-ER
complex signals to the MKK4/MKK5-MPK3/MPK6 module via YDA. Such activation negatively regulates the activities of the bHLH heterodimers
(SPCH-SCRM/SCRM2 and/or MUTE-SCRM/SCRM2) triggering a normal stomatal development. In addition, SPCH positively autoregulates its
own transcription, and it is also required to maintain the wild-type expression levels of FAMA and MUTE. SCRM/SCRM2 is also required to
maintain its/their expression and also to activate both MUTE and FAMA expression. SPCH and SCRM/SCRM2 regulate each other’s
expression?, unknown interactions or components.
L. Serna Review article
BioEssays 31:865–873, ß 2009 Wiley Periodicals, Inc. 871
another (Fig. 3). Such interactions might serve to reinforce the
decision to differentiate and confer stability to the phenotype.
Strikingly, the mechanism regulating stomatal development
seems to be similar to that in animal systems:(18,38–42)
skeletal
muscle differentiation depends on the interaction of several
broadly expressed bHLH proteins with bHLH proteins
exhibiting more restricted expression patterns.
It is largely known that some MYB proteins regulate their
activity by physically interacting with proteins belonging to the
bHLH family.(43)
However, FLP/MYB88 and FAMA, in spite of
having overlapping expression patterns and playing similar
roles,(13,14)
do not contain the amino acid signatures required
for the interaction between members of these two families.(44)
Consistently, FLP and MYB88 fail to physically interact with
FAMA.(14)
It also seems that neither protein is required for the
transcriptional activation of the other.(14)
It is therefore likely
that FAMA and FLP/MYB88 control the transition from the
guard mother cell to the guard cell independently.
Some link between G proteins and the signaling cascade
has been found. Certainly, GPA1 positively regulates SPCH
and MUTE expression. AGB1 plays a reverse role.(10)
The
transcriptional regulation of ER, YDA, TMM, and FAMA is not
regulated by these genes.(10)
Conclusion
Evidence is accumulating that helps to resolve the central
question on how stomata are specified and patterned.
Perhaps the main conclusion emerging from the study of
stomatal development is that many genes regulate several
developmental stages. How the discrimination between the
genes that must be activated in each developmental state
functions is at present unknown. The study of stomatal
development has unraveled not only stomatal lineage-specific
processes but also processes that are common to many other
cells in the regulation of proliferation versus differentiation or
the orientation of the cell division plane. Comparison of the
signaling network reviewed here with other networks
regulating these common processes in other cell types will
help to deepen our knowledge of these processes. Most of the
results to date concern the signaling cascade from SDD1/
EPF1 to the bHLH proteins involved in stomatal development,
and further work will be necessary to extend it. Future
challenges include, for example, unraveling the targets of the
bHLH heterodimers.
Acknowledgments: This work was supported by grants from
both the Communities Council of Castilla-La Mancha (grant,
PCI08-0041-1136) and the Ministry of Science and Innovation
of Spain (grant, BIO2008-02149).
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