Current Biology 19, 229234, February 10, 2009 2009 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2008.12.046

Report
phytochrome B and PIF4 Regulate
Stomatal Development in Response
to Light Quantity
Stuart A. Casson,1 Keara A. Franklin,2 Julie E. Gray,3
Claire S. Grierson,1 Garry C. Whitelam,2,4
and Alistair M. Hetherington1,*
1School of Biological Sciences
University of Bristol
Woodland Road
Bristol BS8 1UG
UK
2Department of Biology
University of Leicester
University Road
Leicester LE1 7RH
UK
3Department of Molecular Biology and Biotechnology
University of Sheffield
Firth Court, Western Bank
Sheffield S10 2TN
UK

Summary
Stomata are pores on the surfaces of leaves that regulate
gas exchange between the plant interior and the atmosphere
[1]. Plants adapt to changing environmental conditions in
the short term by adjusting the aperture of the stomatal
pores, whereas longer-term changes are accomplished by
altering the proportion of stomata that develop on the leaf
surface [2, 3]. Although recent work has identified genes
involved in the control of stomatal development [4], we
know very little about how stomatal development is modulated by environmental signals, such as light. Here, we
show that mature leaves of Arabidopsis grown at higher
photon irradiances show significant increases in stomatal
index (S.I.) [5] compared to those grown at lower photon irradiances. Light quantity-mediated changes in S.I. occur in red
light, suggesting that phytochrome photoreceptors [6]
are involved. By using a genetic approach, we demonstrate
that this response is dominated by phytochrome B and also
identify a role for the transcription factor, PHYTOCHROMEINTERACTING FACTOR 4 (PIF4) [7]. In sum, we identify
a photoreceptor and downstream signaling protein involved
in light-mediated control of stomatal development, thereby
establishing a tractable system for investigating how an environmental signal modulates stomatal development.
Results and Discussion
Increased Photon Irradiance Mediates Changes
in Stomatal Development
Light is one of the most important signals controlling plant
development [8]. For example, increased photon irradiance
results in significant increases in stomatal index (S.I., the ratio
of the number of stomata in a given area divided by the total

*Correspondence: alistair.hetherington@bristol.ac.uk
4Deceased

number of stomata and other epidermal cells in that same

area) [9, 10], indicating that light influences stomatal development. To investigate the molecular basis of this response,
we first established that stomatal indices in Arabidopsis
accessions Col-0 and Ws were higher in plants grown at
175 mmol m22 s21 compared with plants grown at 50 mmol m22
s21 white light (Figure 1A). In both accessions, plants grown at
175 mmol m22 s21 had stomatal and epidermal cell densities
higher than those observed at 50 mmol m22 s21 (Figure S1A
available online). The increase in stomatal index between
plants grown at these photon irradiances must, therefore, be
due to a proportionally greater increase in stomatal numbers,
and, hence, it can be concluded that light signals positively
influence stomatal fate. It should be noted that, in these and
all subsequent experiments, stomatal spacing was not
affected, and no clustering was observed. We hypothesized
that this change in the developmental program of leaf
epidermal cells in response to light quantity could be mediated
by plant photoreceptors. These belong to three main families:
the red and far-red light-sensing phytochromes and the blue
and UV-A light-sensing cryptochromes and phototropins
[11]. To investigate whether phytochromes were involved in
the light-mediated control of stomatal development, we
measured the stomatal index of plants grown under different
photon irradiances of monochromatic red light (conditions in
which only the phytochromes and not other plant photoreceptors are active). We showed that the stomatal index of plants
grown under the higher photon irradiances of red light was
significantly higher than that grown at the lower irradiance
(Col-0 p value = 3.5 3 1025; Ws p value = 2.2 3 1023) (Figure 1B). For the Ws accession, the cell density data mimicked
the results observed in white light, whereas, in Col-0, only
stomatal density increased significantly at the higher photon
irradiance of red light, suggesting accession-specific differences in cell division activity in the different light regimes,
which may include a differential response between the two
accessions to blue light (Figure S1B). Taken together, these
results indicate that the phytochromes have a role to play in
mediating changes in stomatal development in response to
changes in light quantity.
phytochrome B Is the Dominant Photoreceptor Required
for Light Quantity-Mediated Changes in Stomatal
Development
In Arabidopsis, there are five members of the phytochrome
gene family (PHYA to PHYE) [12]. To investigate which member
or members are involved in the regulation of stomatal development, we measured stomatal indices in mutants defective in
the separate phytochrome genes. We did not investigate
stomatal development in phyE mutants. This is because the
only available alleles of this gene are in the Ler accession
[13]. Given that the ERECTA gene encodes a putative
leucine-rich receptor and erecta mutants are characterized
by, among other abnormalities, alterations to stomatal density
and leaf morphology compared with the appropriate control
[14, 15], we felt it was inappropriate to work on this material. In
addition, S.I. in the Ler accession (15.2% at 175 mmol m22 s21)
was significantly lower than Col-0 or Ws under all photon

Current Biology Vol 19 No 3

230

Figure 1. Stomatal Index Changes in Response to Light Quantity

(A) The S.I. of mature leaves of Arabidopsis accessions Col-0 and Ws grown
in white light at either 50 mmol m22 s21 or 175 mmol m22 s21. Mean values are
shown with error bars indicating SEM. Asterisks indicate significant difference in S.I. between plants grown at the different light quantities. Col-0
p value = 2.7 3 1027; Ws p value = 4.9 3 1027.
(B) The S.I. of mature leaves of Arabidopsis accessions Col-0 and Ws grown
in monochromatic red light at either 65 mmol m22 s21 or 130 mmol m22 s21.
Mean values are shown with error bars indicating SEM. Asterisks indicate
significant difference in S.I. between plants grown at the different light
quantities. Col-0 p value = 3.5 3 1025; Ws p value = 2.2 3 1023.

irradiances. Furthermore, it did not show a robust response

between the different photon irradiances, suggesting that the
er mutation (or other accession-specific factors) may be dominant to the light quantity signal.
When grown in white light at 175 mmol m22 s21 or 50 mmol
22 21
m s , neither single mutants in phyA and phyC nor a phyAC
double mutant showed any significant difference in stomatal
index compared with the Col-0 wild-type (also see below).
By way of contrast, at the higher photon irradiance, plants
defective in phyB, either as the single mutant or the phyBC
and phyAB double mutants (phyAB p value = 0.001; data not
shown), showed a significant reduction in stomatal index
compared with the Col-0 wild-type. Consistent with the
suggestion that phyB, but not phyA or phyC, contributes
significantly to the control of stomatal development, at the
photon irradiances used in these experiments, we found that
stomatal indices in the phyBC double mutants (and phyAB;
data not shown) were not significantly different than those in
the phyB single mutant (Figure 2A).
To investigate whether phyD has a role to play in this
response, we examined the stomatal index of the Arabidopsis
Ws accession, which is naturally defective in phyD [16]. An
introgressed PHYD line ([16]; Figure 2B, Ws+D) did not show
a significant difference to the Ws control, indicating that, as
with phyA and phyC mutants in Col-0, phyD does not appear
to contribute significantly to this response under these conditions. Furthermore, no significant change in stomatal index

Figure 2. phytochrome B Is Required to Mediate Changes in S.I. in

Response to Light Quantity
(A) The S.I. of mature leaves of phytochrome mutants, accession Col-0,
grown in white light at 50 mmol m22 s21 or 175 mmol m22 s21. Mean values
are shown with error bars indicating SEM. Asterisks indicate significant
difference in S.I. compared to the control (Col-0) under the same light conditions. phyB p value = 0.014; phyBC p value = 0.049.
(B) The S.I. of mature leaves of phytochrome mutants, accession Ws, grown
in white light at 50 mmol m22 s21 or 175 mmol m22 s21. Mean values are
shown with error bars indicating SEM. Asterisks indicate significant difference in S.I. compared to the control (Ws) under the same light conditions.
phyBD p value = 7.0 3 1024 at 175 mmol m22 s21 and 4.8 3 1024 at
50 mmol m22 s21. phyBCD p value = 8.9 3 1024 at 175 mmol m22 s21 and
5.1 3 1024 at 50 mmol m22 s21.

was observed in phyAD/CD/ACD mutants (accession Ws),

indicating that these family members are not acting redundantly in this response. This is significant given that, in Col-0,
the stomatal index of phyAC mutant is reduced, but not significantly, compared with the control. Therefore, by examining
both accessions, we conclude that phyA and phyC are unlikely
to be acting in concert to redundantly regulate this response.
However, as was observed in Col-0, mutants defective in
phyB (phyBD and phyBCD, p values = 7.0 3 1024 and 8.9 3
1024, respectively) did show a significant reduction in stomatal
index when grown in white light at 175 mmol m22 s21, verifying
the requirement for phyB in mediating this response. Interestingly, unlike Col-0, phyB mutants in the Ws accession also
showed significantly reduced stomatal indices at the lower
photon irradiance, suggesting that other accession-specific
factors may control the sensitivity of this response to light
quantity. In the Ws accession, it is apparent that the reduction
in stomatal indices observed in genotypes defective in phyB is
due to increases in epidermal cell number because stomatal
density is relatively stable (Figures S2C and S2D). This is not
so evident in the Col-0 accession in which the general trend
was for reduced stomatal and epidermal cell densities in all

Control of Stomatal Development by phytochrome B

231

Figure 3. PIF4 Functions in a phytochromeDependent Manner to Mediate Changes in

Stomatal Index
(A) The S.I. of mature leaves of pif mutants,
accession Col-0, grown in white light at 50 mmol
m22 s21 or 175 mmol m22 s21. Mean values are
shown with error bars indicating SEM. Asterisks
indicate significant difference in S.I. compared
to the control (Col-0) under the same light conditions. pif4 p value = 3.5 3 1023; pif4 pif5 p value =
0.041.
(B) The S.I. of mature leaves of plants grown in
monochromatic red light at 130 mmol m22 s21.
Mean values are shown with error bars indicating
SEM. Asterisks indicate significant difference in
S.I. compared to the control. pif4 (Col-0) p value =
5.6 3 1023; phyB (Col-0) p value = 1.1 3 1025;
phyBD (Ws) p value = 6.9 3 1026.
(C) S.I. of mature leaves of plants grown in
white light at 175 mmol m22 s21. Mean values
are shown with error bars indicating SEM.
Asterisks indicate significant difference in S.I.
compared to the control. pif4 p value = 0.01; phyB p value = 3.1 3 1026; phyB pif4 p value = 2.2 3 1028. s indicates that the pif4 epidermal phenotype
is not the same as phyB, whereas = indicates that phyB and phyB pif4 mutants are phenotypically indistinguishable.

phytochrome mutants grown at 175 mmol m22 s21 compared

with the wild-type control (Figures S2A and S2B). The reduced
stomatal indices of the phyB mutant genotypes at 175 mmol
m22 s21 compared with those of the Col-0 wild-type is, therefore, likely to be due to either a proportionally smaller lightinduced increase in stomatal number or a proportionally
greater light-induced increase in epidermal cell number in
phyB mutant genotypesor due to both of these possibilities
operating in parallel. What is evident is that, whereas the
phytochrome mutants in the two accessions show different
cell density responses, the common output is a reduction in
stomatal index in phyB mutant genotypes.
From these results and those from the plants grown in
monochromatic red light, it is possible to conclude that light
quantity-induced changes in stomatal index and, therefore,
stomatal development are mediated, at least in part, by the
phytochrome family of photoreceptors, with phyB assuming
the dominant role. Whereas mutations in PHYA, PHYC, and
PHYD do have effects on cell density, significant changes in
stomatal index are not observed, and, therefore, they do not
appear to contribute significantly under these conditions,
although it is possible that they work redundantly in combination with phyE, which was not tested in this study.
PIF4 Acts in a phytochrome-Dependent Manner to Mediate
Changes in Stomatal Index
Following photoactivation, phyB has been shown to translocate from the cytoplasm to the nucleus [17, 18], a step that is
crucial with respect to phyB function [19]. Within the nucleus,
active phytochromes have been shown to interact with the
phytochrome-interacting factors (PIFs), which consist of
several closely related bHLH transcription factors [20]. Several
studies have now confirmed mechanisms and regulatory roles
for PIFs in regulating phytochrome signaling [7, 2127]. Given
their role in phytochrome signaling, we used mutants defective
in PIF3, PIF4, PIF5, and PIF6 to assess whether these PIFs
mediate changes in stomatal index in response to light. The
stomatal indices of pif3, pif5, and pif6 mutants were not significantly different from those of the wild-type (Col-0) at the
higher photon irradiance. By contrast, when grown at the
higher irradiance and in comparison to Col-0, pif4 (p value =
3.5 3 1023) and a pif4 pif5 double mutant (p value = 0.041)

showed significantly reduced stomatal indices (Figure 3A), as

well as reduced stomatal densities (Figures S3A and S3B),
consistent with a role for PIF4 in the control of this response.
In addition, because there was no additive effect evident in
pif4 pif5 plants with regards to their stomatal index, we
conclude that PIF5 does not contribute to the control of
stomatal development under the growth conditions that we
have used (Figure 3A). As with the phytochrome mutants in
the Col-0 accession, no significant difference in stomatal index
was found in plants grown at the lower photon irradiance,
except for pif6 leaves, which showed an increase in stomatal
index (Figure 3A). Although it was not investigated further,
this latter result might indicate that, in the control of stomatal
development by light, individual PIFs may have photon irradiance-specific effects.
To confirm the role of PIF4 in regulating stomatal development in response to changes in light quantity and to determine
whether this response was dependent on phytochrome
signaling, we grew pif4 plants under red light at 130 mmol m22
s21 (leaf development was too retarded in these mutants at
lower photon irradiances to enable analysis). Under these
conditions, phyB mutants in the Arabidopsis accessions
Col-0 and Ws both showed significantly reduced stomatal
indices compared with those of their respective controls (Col0 p value = 1.1 3 1025; Ws p value = 6.9 3 1026), confirming
the dominant role of this photoreceptor in this response
(Figure 3B). pif4 mutants also showed an attenuated response
in red light (p value 5.6 3 1023), whereas pif5 mutants were
indistinguishable from the Col-0 control (Figure 3B). These
data confirm that PIF4 is required for light quantity-mediated
changes in stomatal development. We then asked whether
this response is dependent on phyB activity or whether PIF4
mediates this response in conjunction with other members of
the phytochrome gene family. The response of a phyB pif4
double mutant was examined and compared with both phyB
and pif4 mutants (see Figure S5 for plant phenotypes and
impressions). As was observed previously in both white and
red light, both of the single mutants had significantly reduced
stomatal indices at the higher photon irradiance (175 mmol
m22 s21; phyB p value = 3.1 3 1026; pif4 p value = 0.010). The
phyB pif4 mutant was found to be virtually indistinguishable
from the phyB single mutant (Figure 3C; data not shown)

Current Biology Vol 19 No 3

232

(p value 2.2 3 1028). The response of the phyB mutant is significantly different from that of the pif4 mutant, which suggests
that phyB may also influence this response via a PIF4-independent pathway. Consistent with our previous findings, at the
lower photon irradiance (50 mmol m22 s21), no significant differences were observed between the mutant genotypes and the
Col-0 wild-type (data not shown). Therefore, both phyB and
pif4 mutants show attenuated responses to the higher photon
irradiances used in this study (Figures 2, 3A3C, and S4). It
should be noted that plants deficient in phyB and PIF4 do not
show a complete inhibition of the response to changes in light
quantity. Other factors may, therefore, act either in concert or in
parallel to mediate this response, with phyB and PIF4 assuming
dominant roles. In conclusion, these data are, therefore,
consistent with PIF4 acting in a phyB-dependent manner to
modulate stomatal development in response to light signals.
The abaxial epidermal surface of Arabidopsis rosette leaves
consists mostly of either stomata or epidermal cells, predominantly tessellated pavement cells in the fully differentiated
epidermis. Plants grown at the higher photon irradiances of
both white and red light used in this study show significant
increases in stomatal index, indicating that epidermal cellfate decisions are influenced by light quantity signals. At
increased photon irradiances, light is likely to regulate
stomatal index through positive influences on stomatal fate,
negative effects on pavement cell fate, or by controlling these
two processes in parallel; whatever the mechanism, the net
result is the samean increase in stomatal index. The cell
density data for the Arabidopsis accessions used in this study,
Col-0 and Ws, indicate that both accessions respond similarly
to white light but differently to red light (Figures S1A and S1B),
whereas the stomatal index response is consistent between
the accessions and the light regimes. We have only analyzed
the mature leaf phenotypes for these two accessions. From
these data, it is not possible to determine that the developmental pathways by which these accessions respond to light
are the same. However, from analysis of the phytochrome
and pif mutants (Figures 2, 3, S2, and S3), it is evident that
cell-fate decisions in the epidermis are not simply correlated
with changes in stomatal or epidermal cell density. This is
particularly evident when considering the phytochrome
mutants. For example, in the Col-0 accession, phyA mutants
show reduced cell densities (both of stomata and epidermal
cells) at both 175 and 50 mmol m22 s21, whereas this is not
the case in phyAD mutants in the Ws accession (compared
with Ws [phyD]) (Figures S2AS2D). Significantly, in both
Col-0 and Ws, phyA genotypes do not show differences in
stomatal index compared with their respective wild-type
controls. Also, both the pif4 and pif5 mutants show reductions
in cell density at both of the photon irradiances used in this
study, yet only pif4 mutants show a significantly reduced
stomatal index (at 175 mmol m22 s21). These data indicate
that the control of the proportion of cells in the epidermis
that are guard cells (stomatal index) may be distinguishable
in signaling terms from the pathway(s) that controls the final
number of cells that form per unit area (density), though further
experimentation will be required to examine this possibility.
The consequences, in terms of plant physiology and fitness,
remain to be determined.
Stomatal development is regulated at multiple levels [3, 4].
The initiation process and the spacing of divisions are
controlled by a presumed peptide-based signaling pathway involving the peptide product of the EPIDERMAL
PATTERNING FACTOR 1 (EPF1) gene [28] and an independent

pathway involving STOMATAL DISTRIBUTION AND DENSITY

1 (SDD1, encoding a putative subtilisin-like protease) [29].
Several members of the bHLH transcription factor family
have been shown to regulate stomatal development [3033].
The closely related bHLH transcription factors SPEECHLESS
(SPCH), MUTE, and FAMA have been shown to regulate
consecutive steps in the differentiation pathway of stomata
[3032]. Two more distantly related bHLH transcription
factors, ICE1/SCREAM and SCREAM2, are also required to
initiate the stomatal lineage and, through interactions with
SPCH, MUTE, and FAMA, are believed to modulate progression through the consecutive stages of stomatal development
[33]. Entry into the stomatal developmental pathway is, in part,
mediated by the leucine-rich repeat receptor-like protein TOO
MANY MOUTHS (TMM) [34], which is believed to form an
active complex at the cell surface with members of the
ERECTA family of LRR-RLK [14]. This putative complex is
then believed to signal through a MAP kinase signaling
pathway, headed by the putative MAP kinase kinase kinase
YODA [35], to negatively regulate stomatal development by
an as yet undetermined mechanism, potentially by targeting
the bHLH transcription factors that positively mediate the
steps in stomatal differentiation. Photoactivated phyB and
PIF4 are both nuclear factors [7, 19] and are, therefore, less
likely to interact with cell-surface receptors. In considering
possible mechanisms of action, it is interesting to note that
the bHLH transcription factors ICE1/SCM and SCM2 are able
to interact with SPCH, MUTE, and FAMA, interactions that
presumably influence the activity of these bHLH heterodimers
[33]. It is possible, therefore, that, as a member of the bHLH
family, PIF4 may influence the activity of the stomatal developmental pathway by interactions with these core factors, with
phyB influencing the stability of these interactions via its interactions with PIF4. It has been shown that the response to light
quantity, as well as to changes in carbon dioxide concentration, is mediated by mature leaves signaling to the developing
leaf primordia [10, 36]. Although this systemic signaling
response was not investigated in this study, it seems that
phyB is most likely to be necessary at the site of signal perceptionthe mature leavesand, therefore, may be a component
of the signaling pathway responsible for the generation of
a putative systemic signal, though a role for phyB locally
cannot be discounted. PIF4 may act either in the mature leaves
downstream of phyB in the generation of such a signal, in the
developing leaf primordia by potentially interacting with the
stomatal pathway, or both locally and systemically to regulate
the response to light quantity. It is unusual that, in this study,
phyB and PIF4 both display positive roles in regulating
stomatal development in response to light quantity, given
that previous research has shown that PIF4 negatively regulates phyB signaling [7, 23, 25, 26]. These studies have dealt
with responses that typically focus on cell elongation rather
than cellular differentiation as is the case here. Similar observations have been made for PIF3, which has been shown to
be both a positive and negative regulator of different aspects
of phytochrome signaling [24, 37, 38]. It is clear that an understanding of the mechanisms involved in this response will
require significant further experimentation.
To date, the only gene known to be involved in the control of
stomatal development in response to environmental signals is
the HIGH CARBON DIOXIDE (HIC) gene, which, by an unknown
mechanism, regulates stomatal development in plants grown
at elevated levels of carbon dioxide [39]. Here, we provide
evidence that a specific photoreceptor (phyB) and a putative

Control of Stomatal Development by phytochrome B

233

transcription factor (PIF4) known to be involved in phytochrome signaling are required in the increased photon irradiance-mediated control of epidermal cell-fate decisions in
stomatal development. Not only does this work establish
a region of the light spectrum responsible for controlling
stomatal development together with the photoreceptor and
a putative transcription factor involved in the pathway, but it
also provides a tractable system for investigating how pathways from environmental signals interact with the basal
pathway responsible for the control of stomatal development.
Experimental Procedures
Plant Material and Growth Conditions
phytochrome mutant alleles for the Col-0 accession of Arabidopsis thaliana
were phyA-211 and phyB-9. phyC, phyAC, and phyBC have been described
[40]. phytochrome mutants in the Ws accession have been previously
described [41]. pif4, pif5, pif4 pif5, and phyB pif4 mutants have been previously described [26], as has pif3-1 [24]. A T-DNA insertion in the first exon of
the PIF6 gene (SALK_090239c) was obtained from the Nottingham Arabidopsis Stock Centre, and homozygous plants were verified by PCR and
sequencing.
Arabidopsis plants were grown in growth chambers (Snijder Microclima
1000E, Snijder Scientific, The Netherlands) from seed in 3:1 mix of
compost-horticultural silver sand in short days (10 hr photoperiod, 70%
RH, 22 C) at photon irradiances of either 175 or 50 mmol m22 s21 supplied
by fluorescent light (Brite Gro and T5). For monochromatic red light experiments, densely packed light-emitting diodes provided light at lmax 665 nm
[42] at either 130 or 65mmol m22 s21, filtered through 20 mm water to remove
radiant heating (18 hr photoperiod, 22 C).
Stomatal Counts
Impressions of the abaxial surface of mature rosette leaves, principal
growth stage 5.10 [43], were made with dental resin (President Jet Light
Body, Colte`ne/Whaledent, Burgess Hill, UK). Clear nail varnish was applied
to the set impression after removal from the leaf, and the varnish impressions were viewed on a Zeiss Axiovert 200M inverted microscope and
imaged with Volocity software (Improvision Ltd, Coventry, UK). Stomatal
and epidermal cell counts were taken from three areas per leaf with three
leaves per plant from four separate plants, for a total of 36 measurements.
For the density data, the mean was calculated from the total number of
stomata or epidermal cells. The stomatal index was calculated for each
area individually, and the mean was then calculated from these data.
Stomatal index was calculated with the following formula: S.I. = [number
of stomata/(number of other epidermal cells + number of stomata)] 3 100.
For statistical analysis, an unpaired t test was performed on the data
following arcsine transformation, which was performed because stomatal
index is a proportion and not a direct measurement. Leaf area data are
provided in Figure S6.
Supplemental Data
Supplemental Data include six figures and can be found with this article online athttp://www.current-biology.com/supplemental/S0960-9822(09)00539-9.
Acknowledgments
This paper is dedicated to Garry Whitelam, who died during the course of
the final revisions to this manuscript. He was a generous and inspirational
colleague who has left a lasting impression on international photobiological
research. He will be greatly missed.
phyC, phyAC, and phyBC seed was kindly supplied by Peter Quail
(University of California, Berkeley). pif4, pif5, pif4 pif5, and phyB pif4 seed
was kindly supplied by Christian Fankhauser (University of Lausanne).
S.A.C., A.M.H., and C.S.G. acknowledge the support of the Leverhulme
Trust.
Received: October 15, 2008
Revised: December 10, 2008
Accepted: December 11, 2008
Published online: January 29, 2009

References
1. Hetherington, A.M., and Woodward, F.I. (2003). The role of stomata in
sensing and driving environmental change. Nature 424, 901908.
2. Roelfsema, M.R., and Hedrich, R. (2005). In the light of stomatal
opening: New insights into the Watergate. New Phytol. 167, 665691.
3. Casson, S., and Gray, J.E. (2008). Influence of environmental factors on
stomatal development. New Phytol. 178, 923.
4. Bergmann, D.C., and Sack, F.D. (2007). Stomatal development. Annu.
Rev. Plant Biol. 58, 163181.
5. Salisbury, E.J. (1928). On the causes and ecological significance of
stomatal frequency, with special reference to the woodland flora. Philosophical Transactions of the Royal Society of London. 216B, 165.
6. Franklin, K.A., and Whitelam, G.C. (2005). Phytochromes and shadeavoidance responses in plants. Ann. Bot. (Lond.) 96, 169175.
7. Huq, E., and Quail, P.H. (2002). PIF4, a phytochrome-interacting bHLH
factor, functions as a negative regulator of phytochrome B signaling
in Arabidopsis. EMBO J. 21, 24412450.
8. Chen, M., Chory, J., and Fankhauser, C. (2004). Light signal transduction
in higher plants. Annu. Rev. Genet. 38, 87117.
9. Ticha, I. (1982). Photosynthetic characteristics during ontogenesis of
leaves. 7. Stomata density and sizes. Photosynthetica 16, 375471.
10. Lake, J.A., Quick, W.P., Beerling, D.J., and Woodward, F.I. (2001). Plant
development. Signals from mature to new leaves. Nature 411, 154.
11. Franklin, K.A., Larner, V.S., and Whitelam, G.C. (2005). The signal transducing photoreceptors of plants. Int. J. Dev. Biol. 49, 653664.
12. Clack, T., Mathews, S., and Sharrock, R.A. (1994). The phytochrome
apoprotein family in Arabidopsis is encoded by five genes: The
sequences and expression of PHYD and PHYE. Plant Mol. Biol. 25,
413427.
13. Devlin, P.F., Patel, S.R., and Whitelam, G.C. (1998). Phytochrome E influences internode elongation and flowering time in Arabidopsis. Plant Cell
10, 14791487.
14. Shpak, E.D., McAbee, J.M., Pillitteri, L.J., and Torii, K.U. (2005).
Stomatal patterning and differentiation by synergistic interactions of
receptor kinases. Science 309, 290293.
15. Masle, J., Gilmore, S.R., and Farquhar, G.D. (2005). The ERECTA gene
regulates plant transpiration efficiency in Arabidopsis. Nature 436,
866870.
16. Aukerman, M.J., Hirschfeld, M., Wester, L., Weaver, M., Clack, T.,
Amasino, R.M., and Sharrock, R.A. (1997). A deletion in the PHYD
gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing. Plant Cell 9, 13171326.
17. Sakamoto, K., and Nagatani, A. (1996). Nuclear localization activity of
phytochrome B. Plant J. 10, 859868.
18. Yamaguchi, R., Nakamura, M., Mochizuki, N., Kay, S.A., and Nagatani,
A. (1999). Light-dependent translocation of a phytochrome B-GFP
fusion protein to the nucleus in transgenic Arabidopsis. J. Cell Biol.
145, 437445.
19. Huq, E., Al-Sady, B., and Quail, P.H. (2003). Nuclear translocation of the
photoreceptor phytochrome B is necessary for its biological function in
seedling photomorphogenesis. Plant J. 35, 660664.
20. Monte, E., Al-Sady, B., Leivar, P., and Quail, P.H. (2007). Out of the dark:
How the PIFs are unmasking a dual temporal mechanism of phytochrome signalling. J. Exp. Bot. 58, 31253133.
21. Al-Sady, B., Kikis, E.A., Monte, E., and Quail, P.H. (2008). Mechanistic
duality of transcription factor function in phytochrome signaling. Proc.
Natl. Acad. Sci. USA 105, 22322237.
22. Al-Sady, B., Ni, W., Kircher, S., Schafer, E., and Quail, P.H. (2006). Photoactivated phytochrome induces rapid PIF3 phosphorylation prior to
proteasome-mediated degradation. Mol. Cell 23, 439446.
23. Leivar, P., Monte, E., Al-Sady, B., Carle, C., Storer, A., Alonso, J.M.,
Ecker, J.R., and Quail, P.H. (2008). The Arabidopsis phytochrome-interacting factor PIF7, together with PIF3 and PIF4, regulates responses to
prolonged red light by modulating phyB levels. Plant Cell 20, 337352.
24. Monte, E., Tepperman, J.M., Al-Sady, B., Kaczorowski, K.A., Alonso,
J.M., Ecker, J.R., Li, X., Zhang, Y., and Quail, P.H. (2004). The phytochrome-interacting transcription factor, PIF3, acts early, selectively,
and positively in light-induced chloroplast development. Proc. Natl.
Acad. Sci. USA 101, 1609116098.
25. de Lucas, M., Daviere, J.M., Rodriguez-Falcon, M., Pontin, M., IglesiasPedraz, J.M., Lorrain, S., Fankhauser, C., Blazquez, M.A., Titarenko, E.,
and Prat, S. (2008). A molecular framework for light and gibberellin
control of cell elongation. Nature 451, 480484.

Current Biology Vol 19 No 3

234

26. Lorrain, S., Allen, T., Duek, P.D., Whitelam, G.C., and Fankhauser, C.
(2008). Phytochrome-mediated inhibition of shade avoidance involves
degradation of growth-promoting bHLH transcription factors. Plant J.
53, 312323.
27. Feng, S., Martinez, C., Gusmaroli, G., Wang, Y., Zhou, J., Wang, F.,
Chen, L., Yu, L., Iglesias-Pedraz, J.M., Kircher, S., et al. (2008). Coordinated regulation of Arabidopsis thaliana development by light and
gibberellins. Nature 451, 475479.
28. Hara, K., Kajita, R., Torii, K.U., Bergmann, D.C., and Kakimoto, T. (2007).
The secretory peptide gene EPF1 enforces the stomatal one-cellspacing rule. Genes Dev. 21, 17201725.
29. Berger, D., and Altmann, T. (2000). A subtilisin-like serine protease
involved in the regulation of stomatal density and distribution in Arabidopsis thaliana. Genes Dev. 14, 11191131.
30. Ohashi-Ito, K., and Bergmann, D.C. (2006). Arabidopsis FAMA controls
the final proliferation/differentiation switch during stomatal development. Plant Cell 18, 24932505.
31. MacAlister, C.A., Ohashi-Ito, K., and Bergmann, D.C. (2007). Transcription factor control of asymmetric cell divisions that establish the
stomatal lineage. Nature 445, 537540.
32. Pillitteri, L.J., Sloan, D.B., Bogenschutz, N.L., and Torii, K.U. (2007).
Termination of asymmetric cell division and differentiation of stomata.
Nature 445, 501505.
33. Kanaoka, M.M., Pillitteri, L.J., Fujii, H., Yoshida, Y., Bogenschutz, N.L.,
Takabayashi, J., Zhu, J.K., and Torii, K.U. (2008). SCREAM/ICE1 and
SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20, 17751785.
34. Nadeau, J.A., and Sack, F.D. (2002). Control of stomatal distribution on
the Arabidopsis leaf surface. Science 296, 16971700.
35. Bergmann, D.C., Lukowitz, W., and Somerville, C.R. (2004). Stomatal
development and pattern controlled by a MAPKK kinase. Science 304,
14941497.
36. Coupe, S.A., Palmer, B.G., Lake, J.A., Overy, S.A., Oxborough, K.,
Woodward, F.I., Gray, J.E., and Quick, W.P. (2006). Systemic signalling
of environmental cues in Arabidopsis leaves. J. Exp. Bot. 57, 329341.
37. Bauer, D., Viczian, A., Kircher, S., Nobis, T., Nitschke, R., Kunkel, T.,
Panigrahi, K.C., Adam, E., Fejes, E., Schafer, E., et al. (2004). Constitutive photomorphogenesis 1 and multiple photoreceptors control degradation of phytochrome interacting factor 3, a transcription factor
required for light signaling in Arabidopsis. Plant Cell 16, 14331445.
38. Kim, J., Yi, H., Choi, G., Shin, B., Song, P.S., and Choi, G. (2003). Functional characterization of phytochrome interacting factor 3 in phytochrome-mediated light signal transduction. Plant Cell 15, 23992407.
39. Gray, J.E., Holroyd, G.H., van der Lee, F.M., Bahrami, A.R., Sijmons,
P.C., Woodward, F.I., Schuch, W., and Hetherington, A.M. (2000). The
HIC signalling pathway links CO2 perception to stomatal development.
Nature 408, 713716.
40. Monte, E., Alonso, J.M., Ecker, J.R., Zhang, Y., Li, X., Young, J., AustinPhillips, S., and Quail, P.H. (2003). Isolation and characterization of phyC
mutants in Arabidopsis reveals complex crosstalk between phytochrome signaling pathways. Plant Cell 15, 19621980.
41. Franklin, K.A., Davis, S.J., Stoddart, W.M., Vierstra, R.D., and Whitelam,
G.C. (2003). Mutant analyses define multiple roles for phytochrome C in
Arabidopsis photomorphogenesis. Plant Cell 15, 19811989.
42. Franklin, K.A., Allen, T., and Whitelam, G.C. (2007). Phytochrome A is an
irradiance-dependent red light sensor. Plant J. 50, 108117.
43. Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E.,
Davis, K.R., and Gorlach, J. (2001). Growth stage-based phenotypic
analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell 13, 14991510.