Download From milliseconds to millions of years: guard cells and

421
From milliseconds to millions of years: guard cells and
environmental responses
Sarah M Assmann* and Xi-Qing Wang†
During the past year, significant advances have been made in
our understanding of stomatal development and its response to
climate change, and in our knowledge of how guard cell Ca2+
oscillations encode environmental signals. Recent studies on
(de)phosphorylation mechanisms have provided new information
on how guard cells respond to abscisic acid and blue light.
Addresses
Biology Department, Pennsylvania State University,
208 Mueller Laboratory, University Park, Pennsylvania 16802-5301, USA
*e-mail: [email protected]
† e-mail: [email protected]
Current Opinion in Plant Biology 2001, 4:421–428
1369-5266/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
AAPK
ABA-activated protein kinase
ABA
abscisic acid
abi1-1 ABA-insensitive1-1
cADPR cyclic ADP-ribose
HIC
HIGH CARBON DIOXIDE
inositol 1,4,5-trisphosphate
IP3
NAADP nicotinic acid adenine dinucleotide phosphate
PLC
phosphoinositide-specific phospholipase C
PLD
phospholipase D
S1P
sphingosine 1-phosphate
sdd1
stomatal density and distribution1
tmm
too many mouths
Introduction
Stomata, located in the plant epidermis, consist of a pair of
guard cells and the pore they enclose (Figure 1a,b).
Stomata open and close through turgor changes driven by
massive ion fluxes, which occur mainly through the guard
cell plasma membrane and tonoplast.
The guard cell has become a model system for the study
of signal transduction. Guard cell signaling is considered
to be intriguing, not only because stomates play an
important role in the plant’s response to its variable
environment but also because the guard cell is mechanically
separate from the surrounding cells, and because guard
cells have sensitive reversible reactions to internal and
external stimuli. A detailed analysis of guard cell signal
transduction may be useful for studies of other plant
signaling systems such as, pulvinar movement, root
gravitropism and pollen-tube growth.
Almost every kind of ion transporter known to function in
plants has been described in the guard cell (Figure 1c,d)
[1–3]. In the plasma membrane, H+-ATPases pump H+ out
of the cytosol. The plasma membrane is thus hyperpolarized,
which drives ion influx by other transporters. Inward K+
channels and a presumed Cl– transporter are responsible
for ion influx during stomatal opening. Sucrose uptake,
likely occurring via a H+–sucrose co-transport mechanism,
also appears to play a significant role in osmotic build-up
under some stomatal opening conditions [4–6]. Although the
guard cell inward K+ channel, KAT1, was identified several
years ago, sensitive reverse transcription polymerase chain
reaction techniques have also shown expression of three
other inwardly rectifying K+ channels — AKT1, AKT2/3,
and KAT2 — in guard cells. KAT1 may not play as
dominant a role in K+ uptake as previously believed [7•].
Outward K+ channels [8] and anion channels are the main
conduits for passive ion efflux from the cytosol to the
apoplast during stomatal closure. Evidence is accumulating
that the guard cell S-type anion channel may consist of, or
be regulated by, an ATP-binding cassette (ABC)-type transporter [9•]. In the tonoplast, anion channels, fast-vacuolar
channels (FV), vacuolar K+-selective channels (VK), slowvacuolar channels (SV), and vacuolar voltage-dependent
Ca2+ channels (VV) have been characterized (Figure 1) [2,3].
How so many ion transporters are organized in response to
various stimuli over both the short term and the long term
is a key question in stomatal physiology. This review will
provide an overview of some of the advances in this area
over the past 18 months. Other valuable recent reviews
covering various aspects of stomatal physiology are also
available [1,10••–12••,13].
Stomata are modified by environmental factors
The stomatal signaling system has been moulded by
environmental factors over both long (millions of years)
and short (hours, minutes, or even seconds) timescales.
Considerable progress has been made in the elucidation of
environmental perception and ion transport mechanisms
occurring over the short timescale. Our understanding
of what has taken place over the developmental and
evolutionary timescales remains limited, but several
papers have broken new ground. In 1987, Woodward [14]
pointed to evidence in the fossil record that plants have
responded to post-industrial increases in atmospheric CO2
levels with decreases in stomatal density. This response
may have been selected for because it allowed reduced
water loss, while photosynthesis was maintained due to the
increased availability of carbon dioxide. Conversely, an
increase in stomatal density can lead to increased water
loss, accompanied by increases in leaf cooling.
In 2001, it has been hypothesized that the evolution of the
broad lamina typical of most present-day leaves was made
possible by an increase in stomatal density that occurred
422
Cell signalling and gene regulation
Figure 1
(a)
(c)
(b)
H+
H+-ATPase
K+in
K+out
Anion (R-type)
Ca2+in
H+
Sucrose
Cl–
(d)
cells under high CO2 concentrations, and prevents neighboring cells from differentiating into guard cells, may be
absent in the hic mutants or may be immobilized as a result of
alterations in the composition of the cuticular layer [18••,19].
Ca2+
K+
Anion
channel
Anion
H+
V-ATPase
Anion (S-type)
Anion
K+
(Stretch-activated)
VK
SV
VV
FV
K+
Ca2+
K+
Ca2+
Current Opinion in Plant Biology
Ion transport in guard cells. (a) Stomatal complex of Vicia faba. Scale
bar = 10 µm. (b) Stomatal complex of Arabidopsis thaliana. Scale bar
= 10 µm. (c) Ion channels and transporters discussed in this article
that reside at the guard cell plasma membrane. The Cl– transporter
and the sucrose transporter are inferred from physiological
experiments but have yet to be characterized by molecular or
electrophysiological techniques. (d) Ion channels and transporters
discussed in this article that reside at the guard cell tonoplast. FV,
fast-vacuolar channel; SV, slow-vacuolar channel; VK, vacuolar K+selective channel; VV, vacuolar voltage-dependent Ca2+ channel.
following a drastic decrease in atmospheric CO2 levels
that occurred in the late Paleozoic, 410–363 million years
ago. It is speculated that this increased stomatal density
promoted evaporative cooling, which in turn kept broader
undissected leaves with their attendant thicker boundary
layers from overheating [15•,16].
Over a much shorter developmental timescale, some plant
species also show an acclimatory response to atmospheric
CO2, with a decrease in stomatal density observed under
elevated CO2 conditions [17]. Recently, Gray et al. [18••]
demonstrated the expression in guard cells of the gene
HIC (HIGH CARBON DIOXIDE). In plants in which the
HIC gene was disrupted by insertion of the β-glucuronidase
gene, the stomatal index increased upon doubling of
experimental CO2 levels. The hic mutants thus exhibited a
reversal of the normal inverse correlation between stomatal
index and ambient CO2 concentration. On the basis of
nucleotide and derived peptide sequence homologies, HIC
encodes a putative 3-keto acyl coenzyme A synthase.
Enzymes of this type are involved in the synthesis of verylong-chain fatty acids. In plants, long-chain fatty acids are
found in waxes and cutins, which are constituents of the
cuticle, as well as in glycerolipids and sphingolipids. Some
substance that normally diffuses from pre-existing guard
Other wax mutants also lend support to the idea that disruption
of the extracellular matrix leads to a change in stomatal
index: eceriferum-1 and eceriferum-6 mutants of Arabidopsis
[20], both of which have alterations in wax composition on
the leaf surface, exhibit higher stomatal indices than do
wild-type plants.
The effect of the hic mutation differs from that of other
stomatal mutants such as too many mouths (tmm) [21] and
stomatal density and distribution1 (sdd1) [22•], in that the
stomata of the hic mutants show increased density but do
not show abnormal clustering. TMM has not yet been
cloned, but SDD1 encodes a subtilisin-like serine protease,
suggesting that the processing of a proteinaceous signal
may be involved in stomatal patterning. It is interesting to
speculate that TMM, SDD1, or other gene products
implicated in stomatal patterning may, like HIC, prove
sensitive to environmental factors that regulate stomatal
movements, such as light, drought, air pollutants, and
humidity. Some studies have already shown that long-term
drought stress and abscisic acid (ABA) treatment result in
increased stomatal density and reduced guard cell size
([23] and references therein). The ABA effect on cell size
indicates that not only stomatal patterning but also guard
cell development is influenced by the environment. Normal
differentiation of the stomatal complex involves cytoskeletal
reorganization, and some studies have implicated a sensitivity
of guard cell actin filament and microtubules arrays to
signals such as ABA and light [24,25].
The role of Ca2+ in guard cell responses to
ABA, cold, and oxidative stress
Guard cells can sense and rapidly respond to many signals,
including not only external stimuli but also internal stimuli
such as ABA and auxin [26,27]. This area of study is
advancing rapidly, and this past year has seen particular
advances in our understanding of the inter-relationships
between ABA and Ca2+ signaling.
Changes in cytosolic calcium are elicited by many of the
signals to which plants respond, and cytosolic calcium has
been proposed to be a universal second messenger. How is
the specificity and reliability of Ca2+ signaling achieved?
Perhaps a given external stimulus could produce a specific
type of Ca2+ response that amplifies the signal upstream of
Ca2+, such as signals from receptors and G proteins, and
then signals to Ca2+-regulated downstream molecules, for
example, calcium-dependent protein kinases [28], inward
K+ channels and anion channels.
Along these lines, recent studies have utilized proteinaceous Ca2+ reporters to measure Ca2+ oscillations in guard
cells, with particular reference to ABA signaling. Allen et al.
Guard cells and environmental responses Assmann and Wang
423
Table 1
Ca2+ release and re-uptake of resources in guard cells.
Stimulus
Mechanism
ABA, H2O2
Hyperpolarization-activated Ca2+ channel
Ca2+
Ca2+ destination
Location
Reference(s)
Cytosol
PM
[32•,33•]
ABA
Nonselective
Cytosol
PM
[65]
?
Inward K+ channel
Cytosol
PM
[66]
Stretch
Ca2+ channel
Cytosol
PM
[67]
ABA
IP3-induced Ca2+ release through PLC
Cytosol
*Tonoplast, *ER or *PM
[68]
ABA
cADPR-induced Ca2+ channel
Cytosol
Tonoplast, *ER
[39,69]
?
*NAADP
Cytosol
ER
[43]
channel
ABA
IP6
Cytosol
?
[37]
ABA
S1P-induced Ca2+ release
Cytosol
?
[40••]
[46]
Ca2+
Slow-vacuolar channel
Cytosol
Tonoplast
?
VV channel
Cytosol
Tonoplast
[70]
?
*Ca2+-ATPase
ER
ER
[71]
?
*Ca2+-ATPase
Apoplast
PM
[72,73]
*Identified in other plant cell types, not yet identified in guard cells. ER, endoplasmic reticulum; PM, plasma membrane.
[29••] transformed a calcium reporting green fluorescent
protein construct or ‘cameleon’ into Arabidopsis, and
assessed guard cell Ca2+ responses to several distinct stimuli. They found that ABA caused both repetitive cytosolic
transients and more prolonged oscillations. Cold stimulated small, repetitive cytosolic Ca2+ transients, whereas
H2O2 induced one or two separate Ca2+ transients. Thus,
each stimulus led to a Ca2+ response with distinct features.
Even for a single signal, Ca2+ responses may vary in a manner that reflects signal strength. Staxen et al. [30] found
that different ABA concentrations administered to
Commelina communis epidermis led to guard cell Ca2+
oscillations of different periods, with higher concentrations
of the hormone eliciting lower frequency oscillations. This
phenomenon may arise from differential contributions of
Ca2+ uptake and internal Ca2+-release pathways at different
ABA concentrations [12••].
How are Ca2+ oscillations produced?
Ca2+ oscillations (periodic Ca2+ concentration changes over
time) and Ca2+ waves (spatial changes in calcium concentration) are produced through the integration of Ca2+ influx,
release of Ca2+ from the endomembrane system, Ca2+
efflux from the cytosol to the apoplast, and Ca2+ sequestration
into intracellular stores. Different stimuli presumably use
different Ca2+ sources or releasing pathways to establish
characteristic Ca2+ responses, and this hypothesis was
recently lent credence by two studies. Wood et al. [31•]
used aequorin to report Ca2+ responses to several stimuli in
the presence of Ca2+ channel blockers and the Ca2+ chelator
EGTA. They concluded that mechanical and ABA signaling
in guard cells rely heavily on Ca2+ release from intracellular
stores, whereas low-temperature signaling relies heavily
on Ca2+ uptake from the apoplast. Allen, Schroeder, and
colleagues [29••] studied Ca2+ oscillations in the Arabidopsis
det3 mutant. This mutant exhibits reduced expression of
the C-subunit of the V-type ATPase, accompanied by
reduced endomembrane energization. They found that
the det3 mutation changed the Ca2+ oscillations observed
when stomata were challenged with external Ca2+ and H2O2,
but they found no detectable changes in response to ABA
and cold stimuli [29••].
The result with cold treatment is consistent with the
results of Wood et al. [31•]. However, it is perhaps unexpected that the det3 mutation affected the H2O2 response
but not the ABA response, as Schroeder’s group has also
implicated H2O2 as a secondary messenger for ABA action
(see below) [32•]. The guard cell may utilize multiple
redundant pathways, thus ensuring adequacy of the vital
ABA response. In fact, this idea of redundancy is consistent
with the multiplicity of Ca2+ pathways implicated in guard
cells, as described below.
Ca2+ release and re-uptake pathways in
guard cells
One mechanism for increasing cytosolic Ca2+ levels is via
Ca2+ uptake from the apoplast. In 2000, two laboratories
characterized plasma membrane channels in Vicia faba and
Arabidopsis that mediate Ca2+ uptake and are activated by
ABA during hyperpolarization [32•,33•]. In Vicia faba,
these Ca2+ channels show some hyperpolarizationinduced activity in the absence of ABA. In Arabidopsis, it
was demonstrated that ABA activation of the channels
occurred via a H2O2 secondary messenger. It will be
interesting to ascertain the extent to which reactive oxygen
species regulate this channel type in Vicia faba and in
other plant species.
Endomembrane Ca2+ efflux in response to ABA has been
studied extensively and several secondary messengers
have been implicated in this response. Previous studies
have shown that ABA stimulates inositol 1,4,5-trisphosphate (IP3) production in guard cells and that IP3 induces
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Cell signalling and gene regulation
cytosolic Ca2+ elevation and partial stomatal closure [34–36].
IP3 may also serve as a precursor to IP6 (inositol hexakisphosphate), which has also been linked to Ca2+-dependent
ABA responses [37]. Staxen et al. [30] characterized ABAinduced Ca2+ oscillations in detail and found that these
oscillations and stomatal closure were both partially inhibited
by U-73122 (1-[6-{(17β-3-methoxxyestra-1,3,5(10)-trien17-yl)amino}hexyl]-1H-pyrrole-2,5-dione), an inhibitor of
phosphoinositide-specific phospholipase C (PLC).
PLC and another phospholipase, PLD, which cleaves the
phospholipid headgroup to yield phosphatidic acid, both
show transient increases in activity following ABA application.
PLD seems to function in the same pathway as PLC:
inhibitors of PLC and PLD each partially oppose ABAinduced stomatal closure, but simultaneous application of
both inhibitors does not result in enhanced attenuation of
ABA-induced closure [38•]. The relative positions of the two
phospholipases in their signaling chain remains unknown,
but it does seem that PLD activation is downstream of —
or independent of — Ca2+ release because the direct
product of PLD action, phosphatidic acid, does not elicit
cytosolic Ca2+ increases in guard cells.
The lack of complete reversal of ABA-induced stomatal
closure by combined PLC and PLD inhibitors implicates
other Ca2+ release pathways in the ABA response, and a
parallel pathway appears to be that mediated by cyclic
ADP-ribose (cADPR). In contrast to the situation with
PLC and PLD, simultaneous application of PLD and
cADPR antagonists does increase the extent to which
ABA-induced stomatal closure is reduced. For example,
simultaneous application of nicotinamide, an inhibitor of
cADPR action, and 1-butanol, an inhibitor of phosphatidic
acid production, results in an almost complete block of
ABA-induced stomatal closure in Vicia faba epidermal
peels [38•]. Patch clamping of isolated guard cell vacuoles
confirms that cADPR activates Ca2+ release from this
organelle via Ca2+-selective channels, and microjection of
cADPR into guard cells elicits cytosolic Ca2+ increases and
stomatal closure [39].
One of the most recent calcium-mobilizing molecules to
be identified is sphingosine 1-phosphate (S1P). In
Commelina communis, ABA-induced stomatal closure is
opposed by a competitive inhibitor of sphingosine kinase,
the enzyme responsible for producing S1P from sphingosine.
Application of S1P to epidermal peels induces Ca2+
oscillations in guard cells and stomatal closure. An increase
in S1P levels occurs in whole leaves following drought
stress, and it will be important to ascertain whether or not
such responses also occur in guard cells [40••]. S1P is
unusual in that it can serve as an intracellular signal or, if
located extracellularly, interact with outwardly facing
receptors [41]. The idea of a mesophyll messenger that
coordinates photosynthetic and stomatal functions has
intrigued stomatal physiologists for years [42]. Could S1P
play such a role?
Yet another Ca2+ mobilization pathway has yet to be
assayed in guard cells, although it has been detected in
other types of plant cells. Navazio et al. [43] demonstrated
that endoplasmic reticulum vesicles from cauliflower
respond to nicotinic acid adenine dinucleotide phosphate
(NAADP), a Ca2+-releasing molecule recently described in
both vertebrates and invertebrates. In contrast to the IP3
and cADPR pathways, NAADP did not elicit Ca2+ release
from tonoplast vesicles. The results with NAADP thus
highlight the importance of the endoplasmic reticulum as
a Ca2+ store in plants. Given the plethora of Ca2+ release
pathways described in guard cells to date, one would
predict that NAADP will also be found to mobilize Ca2+ in
this cell type.
Ca2+-independent pathways
The existence of multiple Ca2+ mobilization pathways
and Ca2+ release sites undoubtedly contributes to the
generation of stimulus-specific Ca2+ signals. It should be
kept in mind, however, that Ca2+-independent pathways
also exist in the guard cell. ABA can inhibit K+ channels
involved in stomatal opening and in some circumstances
elicit stomatal closure without detectable calcium increases
in guard cells [31•,39,44]. Protons have been implicated as
one of the other secondary messengers in the guard cell
ABA response. First, ABA also induces pH increases prior
to stomatal closure; and second, elevated pH activates the
plasma membrane outward K+ channel through which K+
efflux occurs during stomatal closure. This channel is
Ca2+-insensitive [45]. The activities of FV and SV channels,
which release K+ and Ca2+ to the cytosol (Figure 1) [2] are
also stimulated by alkaline cytosolic pH, although the VK
current is inhibited under these conditions [3,46]. These
data imply that changes in pH function alongside changes
in pCa to modulate stomatal movement.
Nonionic signaling in guard cell reponses
to stress
The guard cell not only uses Ca2+ oscillations and pH
changes to establish specific responses but also employs
nonionic signaling molecules. Evidence that guard cell
pathways have novel features not found universally in all
plant cells has been provided recently by two studies.
Sutton et al. [47] found that co-injection of oocytes with
guard cell protoplast mRNA along with cRNA for KAT1
resulted in the expression of an inward K+ channel that was
inhibited upon ABA application to the oocyte. However,
oocytes co-injected with mesophyll mRNA and KAT1
cRNA produced inward K+ currents that were not inhibited
by ABA [47]. These results show that the mesophyll ABA
signaling pathway is distinct from, and cannot substitute
for, the guard cell ABA signaling pathway.
One guard-cell-specific component may be AAPK (ABAactivated protein kinase), a serine-threonine protein
kinase whose biochemical activity has only been detected
in guard cells to date [48]. On the basis of sequencing of
two peptides from AAPK using tandem mass spectrometry,
Guard cells and environmental responses Assmann and Wang
Li et al. [49••] cloned the AAPK cDNA from Vicia faba
guard cells. Mutating Lys43 in AAPK to an alanine
residue resulted in a kinase with apparent dominantnegative effects, and guard cells transformed with AAPK
K43A no longer closed in response to ABA application to
epidermal peels. Guard cells expressing the mutant
version of the kinase also failed to activate plasma membrane
anion channels, which play a central role in ABA-induced
stomatal closure.
Protein phosphorylation is reversed by protein phosphatases.
abi1-1 and abi2-1 are two ABA-insensitive dominant
mutant alleles of the protein phosphatase 2C (PP2C) genes
ABI1 and ABI2. Both of these mutations confer a wilty
phenotype and ABA insensitivity of stomatal closure.
Recently, Gosti et al. [50••] isolated seven recessive alleles
of ABI1 as intragenic revertants of the abi1-1 mutation.
These revertants show hypersensitive responses to ABA in
terms of the inhibition of seed germination and seedling
root growth. When water is withheld, the revertants also
show enhanced drought tolerance. The recessive nature of
the revertants suggests that a loss of function in ABI1 is
responsible for the ABA-hypersensitive phenotype.
Consistent with the above hypothesis, recombinant proteins
derived from the intragenic revertant alleles do not exhibit
detectable PP2C activity. In the same assay, the PP2C
encoded by the dominant, possibly gain-of-function,
abi1-1 allele exhibits residual phosphatase activity [50••].
Taken together, the evidence suggests that wild-type ABI1
phosphatase negatively regulates ABA responses, and it is
tempting to speculate that it reverses the action of the
ABA-activated kinase, AAPK.
Guard cells respond not only to drought stress but also to
stresses such as salinity and air pollution [51]. In addition
to their previously implicated roles in cell division and
organ development, polyamines have been found to accumulate upon plant exposure to such stresses [52]. Liu et al.
thus addressed the hypothesis that polyamines are
involved in stomatal action and, indeed, found that application of all of the natural polyamines inhibited stomatal
opening and induced stomatal closure. Polyamines also
inhibited whole-cell inward K+ currents, plausibly acting
on the KAT1 inward K+ channel. This inhibition of inward
K+ channels was not observed in isolated membrane patches,
suggesting that the response also requires cytoplasmic
factors [53]. These data suggest that polyamines may be
components of stress-induced signal transduction in guard
cells, and the next step will be to document the extent to
which guard cell polyamine concentrations change in
response to stress signals.
Blue light and stomatal opening
Stomatal closure in response to ABA and stomatal opening
in response to light comprise two major areas of investigation by stomatal physiologists. Stomatal opening is
triggered by both red light — acting through chlorophyll
425
— and blue light. Low-intensity blue light is much more
effective in stimulating stomatal opening than comparable
fluence rates of red light, pointing to a sensitive, nonchlorophyllous photoreceptor as the starting point for this
photoresponse. Identification of this photoreceptor is still
under investigation. Zeiger and colleagues have provided
photobiological evidence that zeaxanthin, a carotenoid
intermediate in the xanthophyll cycle, is the specific bluelight photoreceptor [54]. Guard cells from the npq1 mutant
of Arabidopsis, which lacks zeaxanthin, show reduced stomatal
opening in response to blue light when assayed in isolated
epidermal peels [54]. Gas exchange experiments measuring
blue-light-induced transpiration from whole leaves of the
npq1 mutant, however, show a wild-type stomatal response
to blue light. The same is true for all other blue-light
photoreceptor mutants assayed to date (i.e. nph1, cry1, and
cry2) [55,56].
Moreover, in Vicia faba at least, the ultraviolet action spectrum
for stomatal opening does not match the absorption spectrum
of bulk (trans) zeaxanthin, and is instead consistent with
that of either a cis-carotenoid (including cis-zeaxanthin) or
a flavin [57]. If zeaxanthin is indeed the guard cell bluelight photoreceptor, then the whole-leaf experiments with
npq1 imply that there must be a mesophyll blue-light
photoreceptor that senses and reports the presence of blue
light to the zeaxanthin-less guard cells of npq1.
Although the early stages of the blue-light photoresponse
remain mysterious, the ultimate targets of that signal
transduction chain are becoming clearer. Recently, two
studies [58••,59] have lent support to the idea that it is an
H+-ATPase, rather than a redox pathway, that mediates the
proton pumping that hyperpolarizes the plasma-membrane
to drive K+ uptake and stomatal opening in response to
blue light [60]. Shimazaki’s group used biochemical
techniques to demonstrate that blue-light activation of the
H+-ATPase involves blue-light-dependent phosphorylation
of the carboxyl terminus of the H+-ATPase [58••]. This
phosphorylation increases the affinity of the H+-ATPase
for a 14-3-3-protein (14-3-3 proteins are positive regulators
of H+-ATPases). Three 14-3-3 cDNAs were identified in a
Vicia faba guard cell cDNA library, and analysis of the
native 14-3-3 protein co-immunoprecipitated with the
H+-ATPase indicates that just one of the 14-3-3 isomers,
14-3-3a, specifically associates with the H+-ATPase following
blue-light irradiation [61]. These elegant experiments have
thus identified 14-3-3a as a key player in the stomatal
response to blue light.
Conclusions
Massive progress has been made since the first successful
patch clamp recordings from guard cells in 1984. A number
of important signaling elements have been identified that
function in guard cells on the timescale of milliseconds to
minutes, although the ordering and interactions of these
signaling molecules remain unknown in many cases. A few
key genes have also been identified that play a role in
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Cell signalling and gene regulation
guard cell development. However, on the intermediate
timescale of the life-time of a single mature guard cell or a
single leaf, we still know very little about environmental
effects on guard cell physiological function and gene
expression [62,63].
4.
Zhao R, Dielen V, Kinet JM, Boutry M: Cosuppression of a plasma
membrane H+-ATPase isoform impairs sucrose translocation,
stomatal opening, plant growth, and male fertility. Plant Cell 2000,
12:535-546.
5.
Ritte G, Rosenfeld J, Rohrig K, Raschke K: Rates of sugar uptake by
guard cell protoplasts of Pisum sativum L. related to the solute
requirement for stomatal opening. Plant Physiol 1999, 121:647-656.
Many of the most telling experiments carried out over
the past few years have been conducted on isolated epidermal peels or guard cell protoplasts, or on whole leaves
monitored with gas-exchange techniques. It has been
demonstrated unequivocally, however, that in response to
signals such as ABA and atmospheric humidity, not all of
the guard cells in a leaf act in unison. There exist domains
or ‘patches’ within the leaf that show differential responses
to an identical stimulus; furthermore, the boundaries and
behavior of such patches fluctuate over time. Such results
indicate that stomata in the intact leaf do not function
independently but rather are coupled, perhaps hydraulically,
within a given patch. This ‘emergent collective behavior’
[64••] adds one more layer of complexity to stomatal function
under real-world conditions.
6.
Talbott LD, Zeiger E: The role of sucrose in guard cell
osmoregulation. J Exp Botany 1998, 49:329-337.
Update
Heterotrimeric G proteins are one of the most important
components of animal signaling pathways. Recent studies
have shown that the Arabidopsis G protein α subunit,
GPA1, is also a vital regulator of plant growth and development [74••,75••], including guard cell signaling. Wang et al.
[75••] used plants harbouring T-DNA insertions in GPA1 to
demonstrate G protein involvement in ABA-inhibition of
stomatal opening and inward K+ channels, and in pH-independent ABA-activation of slow anion channels. gpa1
mutant plants exhibit greater rates of water loss than their
wild-type counterparts [75••]. These results place G proteins
in the guard cell ABA signaling network.
Calcium is a universal signaling element. Allen et al. [76••]
present elegant data showing that calcium oscillation
numbers frequency, and duration all regulate stomatal aperture
[76••]. A detailed analysis is presented in Hetherington’s
article on pp 415–420 of this issue.
Acknowledgements
Research on guard cell signal transduction in the authors’ laboratory is
supported by grants from the National Science Foundation (MCB 98-74438
and MCB 00-86315) and the US Department of Agriculture (00-35100-9420
and 01-35304-09916).
7.
•
Szyroki A, Ivashikina N, Dietrich P, Roelfsema MRG, Ache P,
Reintanz B, Deeken R, Godde M, Felle H, Steinmeyer R et al.: KAT1 is
not essential for stomatal opening. Proc Natl Acad Sci USA 2001,
98:2917-2921.
Genetic disruption of the inward K+ channel KAT1 has no effect on stomatal
behavior in the assays reported in this paper. Other inward K+ channels are
also expressed in the guard cell and may compensate for the loss of
functional KAT1.
8.
9.
•
Gaedeke N, Klein M, Kolukisaoglu U, Forestier C, Müller A,
Ansorge M, Becker D, Mamnun Y, Kuchler K, Schulz B et al.: The
Arabidopsis thaliana ABC transporter AtMRP5 controls root
development and stomata movement. EMBO J 2001,
20:1875-1887.
ABC (ATP-binding cassette) transporters constitute a large protein family in
Arabidopsis. This paper shows that the ABC transporter, AtMRP5, is
expressed in guard cells and is involved in stomatal opening.
10. Schroeder JI, Kwak JM, Allen GJ: Guard cell abscisic acid signalling
•• and engineering drought hardiness in plants. Nature 2001,
410:327-330.
This excellent review updates current knowledge of guard cell abscisic acid
(ABA) responses, summarizing most of the components that have been
shown to be involved in this signaling pathway.
11. Blatt MR: Cellular signaling and volume control in stomatal
•• movements in plants. Annu Rev Cell Dev Biol 2000, 16:221-241.
This review describes some of the recent developments in stomatal physiology,
with particular emphasis on membrane trafficking.
12. MacRobbie EAC: ABA activates multiple Ca2+ fluxes in stomatal
•• guard cells, triggering vacuolar K+ (Rb+) release. Proc Natl Acad
Sci USA 2000, 97:12361-12368.
This article provides a comprehensive overview of Ca2+ influx pathways across
the plasma membrane and Ca2+-release pathways from endomembranes.
13. McAinsh MR, Brownlee C, Hetherington AM: Calcium ions as
second messengers in guard cell signal transduction. Physiol
Plant 1997, 100:16-29.
14. Woodward FI: Stomatal numbers are sensitive to CO2 increases
from pre-industrial levels. Nature 1987, 327:617-618.
15. Beerling DJ, Osborne CP, Chaloner WG: Evolution of leaf-form in
•
land plants linked to atmospheric CO2 decline in the late
Palaeozoic era. Nature 2001, 410:352-354.
Atmospheric CO2 concentration influences on stomatal density and evaporative
cooling may have been key elements in the evolution of the broad leaf lamina
found in many present day land plants.
16. McElwain JC, Beerling DJ, Woodward FI: Fossil plants and global
warming at the Triassic–Jurassic boundary. Science 1999,
285:1386-1390.
17.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Assmann SM: Signal transduction in guard cells. Annu Rev Cell
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2.
Ward JM, Pei ZM, Schroeder JI: Roles of ion channels in initiation
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Allen GJ, Amtmann A, Sanders D: Calcium-dependent and calciumindependent K+ mobilization channels in Vicia faba guard cell
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Ache P, Becker D, Ivashikina N, Dietrich P, Roelfsema MRG,
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Woodward FI, Kelly CK: The influence of CO2 concentration on
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18. Gray JE, Holroyd GH, van der Lee FM, Bahrami AR, Sijmons PC,
•• Woodward FI, Schuch W, Hetherington AM: The HIC signalling
pathway links CO2 perception to stomatal development. Nature
2000, 408:713-716.
The HIC gene identified in this report may be crucial for stomatal response
to global climate change. hic mutants show an unusual increase in stomatal
index under elevated CO2 concentrations. HIC is hypothesized to encode
an enzyme involved in the synthesis of the leaf cuticle.
19. Serna L, Fenoll C: Plant biology. Coping with human CO2
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20. Jenks MA, Tuttle HA, Eigenbrode SD, Feldmann KA: Leaf epicuticular
waxes of the eceriferum mutants in Arabidopsis. Plant Physiol
1995, 108:369-377.
Guard cells and environmental responses Assmann and Wang
427
21. Geisler M, Nadeau J, Sack FD: Oriented asymmetric divisions that
generate the stomatal spacing pattern in Arabidopsis are
disrupted by the too many mouths mutation. Plant Cell 2000,
12:2075-2086.
37.
22. Berger D, Altmann T: A subtilisin-like serine protease involved in
•
the regulation of stomatal density and distribution in Arabidopsis
thaliana. Genes Dev 2000, 14:1119-1131.
Understanding of stomatal development at the molecular level remains
limited. The authors of this paper used genetic techniques to identify a role for a
subtilisin-like serine protease in the regulation of stomatal density and distribution.
38. Jacob T, Ritchie S, Assmann SM, Gilroy S: Abscisic acid signal
•
transduction in guard cells is mediated by phospholipase D
activity. Proc Natl Acad Sci USA 1999, 96:12192-12197.
Phospholipase D (PLD) activity following ABA application to guard cells was
assessed by direct biochemical assays and use of pharmacological
inhibitors. PLD appears to function in a pathway that is distinct from the
cADPR pathway to mediate stomatal responses to ABA.
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39. Leckie CP, McAinsh MR, Allen GJ, Sanders D, Hetherington AM:
Abscisic acid-induced stomatal closure mediated by cyclic ADPribose. Proc Natl Acad Sci USA 1998, 95:15837-15842.
25. Hwang JU, Lee Y: Abscisic acid-induced actin reorganization in
guard cells of dayflower is mediated by cytosolic calcium levels
and by protein kinase and protein phosphatase activities. Plant
Physiol 2001, 125:2120-2128.
40. Ng CKY, Carr K, MacAinsh MR, Powell B, Hetherington AM: Drought•• induced guard cell signal transduction involves sphingosine-1phosphate. Nature 2001, 410:596-599.
Sphingosine-1-phosphate appears to be a novel calcium-mobilizing agent in
guard cells. Sphingosine-1-phosphate could induce Ca2+ oscillations and
stomatal closure. Disruption of sphingosine-1-phosphate production led to
the partial inhibition of ABA-induced stomatal closure.
26. Merritt F, Kemper A, Tallman G: Inhibitors of ethylene synthesis
inhibit auxin-induced stomatal opening in epidermis detached
from leaves of Vicia faba L. Plant Cell Physiol 2001, 42:223-230.
41. Young KW, Nahorski SR: Intracellular sphingosine 1-phosphate
production: a novel pathway for Ca2+ release. Semin Cell Dev Biol
2001, 12:19-25.
27.
42. Wong SC, Cowan IR, Farquhar GD: Stomatal conductance
correlates with photosynthetic capacity. Nature 1979,
282:424-426.
Bauly JM, Sealy IM, Macdonald H, Brearley J, Dröge S, Hillmer S,
Robinson DG, Venis MA, Blatt MR, Lazarus CM, Napier RM:
Overexpression of auxin-binding protein enhances the sensitivity
of guard cells to auxin. Plant Physiol 2000, 124:1229-1238.
28. Berkowitz G, Zhang X, Mercie R, Leng Q, Lawton M: Co-expression
of calcium-dependent protein kinase with the inward rectified
guard cell K+ channel KAT1 alters current parameters in Xenopus
laevis oocytes. Plant Cell Physiol 2000, 41:785-790.
29. Allen GJ, Chu SP, Schumacher K, Shimazaki CT, Vafeados D,
•• Kemper A, Hawke SD, Tallman G, Tsien RY, Harper JF et al.:
Alteration of stimulus-specific guard cell calcium oscillations and
stomatal closing in Arabidopsis det3 mutant. Science 2000,
289:2338-2342.
This article provides evidence that different stimuli elicit distinct Ca2+ oscillations, and that different stimuli use different Ca2+ resources in the production
of those oscillations. det3 is an Arabidopsis mutant with a 60% reduction in
the V-type H+-ATPase. det3 exhibits altered Ca2+ oscillations in response to
Ca2+ and H2O2, but ABA and cold-treatment-induced Ca2+ oscillations
retain the wild-type phenotype.
30. Staxen I, Pical C, Montgomery LT, Gray JE, Hetherington AM,
McAinsh MR: Abscisic acid induces oscillations in guard-cell
cytosolic free calcium that involve phosphoinositide-specific
phospholipase C. Proc Natl Acad Sci USA 1999, 96:1779-1784.
31. Wood NT, Allan AC, Haley A, Viry-Moussaïd M, Trewavas AJ: The
•
characterization of differential calcium signalling in tobacco guard
cells. Plant J 2000, 24:335-344.
The authors of this paper identify differential use of Ca2+ resources in guard
cell responses to various environmental stimuli.
32. Pei ZM, Murata Y, Benning G, Thomine S, Klüsener B, Allen GJ,
•
Grill E, Schroeder JI: Calcium channels activated by hydrogen
peroxide mediate abscisic acid signalling in guard cells. Nature
2000, 406:731-734.
This report characterizes a guard cell Ca2+ channel from the model plant
species, Arabidopsis, and presents evidence that reactive oxygen species
mediate ABA signaling in guard cells.
33. Hamilton DWA, Hills A, Köhler B, Blatt MR: Ca2+ channels at the
•
plasma membrane of stomatal guard cells are activated by
hyperpolarization and abscisic acid. Proc Natl Acad Sci USA 2000,
97:4967-4972.
A plasma membrane Ca2+ channel in Vicia faba guard cells that is activated
by hyperpolarization and ABA is described.
34. Lee YS, Choi YB, Suh S, Lee J, Assmann SM, Joe CO, Kelleher JF,
Crain RC: Abscisic acid-induced phosphoinositide turnover in
guard cell protoplasts of Vicia faba. Plant Physiol 1996,
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43. Navazio L, Bewell MA, Siddiqua A, Dickinson GD, Galione A,
Sanders D: Calcium release from the endoplasmic reticulum of
higher plants elicited by the NADP metabolite nicotinic acid
adenine dinucleotide phosphate. Proc Natl Acad Sci USA 2000,
97:8693-8698.
44. Romano LA, Jacob T, Gilroy S, Assmann SM: Increases in cytosolic
Ca2+ are not required for abscisic acid-inhibition of inward K+
currents in guard cells of Vicia faba L. Planta 2000, 211:209-217.
45. Blatt MR, Armstrong F: K+ channels of stomatal guard cells:
abscisic-acid-evoked control of the outward rectifer mediated by
cytoplasmic pH. Planta 1993, 191:330-341.
46. Ward JM, Schroeder JI: Calcium-activated K+ channels and
calcium-induced calcium release by slow vacuolar ion channels in
guard cell vacuoles implicated in the control of stomatal closure.
Plant Cell 1994, 6:669-683.
47.
Sutton F, Paul SS, Wang XQ, Assmann SM: Distinct abscisic acid
signaling pathways for modulation of guard cell versus
mesophyll cell potassium channels revealed by expression
studies in Xenopus laevis oocytes. Plant Physiol 2000,
124:223-230.
48. Li J, Assmann SM: An abscisic acid-activated and calciumindependent protein kinase from guard cells of fava bean. Plant
Cell 1996, 8:2359-2368.
49. Li J, Wang XQ, Watson MB, Assmann SM: Regulation of abscisic
•• acid-induced stomatal closure and anion channels by guard cell
AAPK kinase. Science 2000, 287:300-303.
The authors of this paper report cloning of a guard-cell-specific protein
kinase, AAPK. In guard cells expressing a dominant loss-of-function AAPK
allele, ABA failed to activate anion channels or induce stomatal closure.
50. Gosti F, Beaudoin N, Serizet C, Webb AAR, Vartanian N, Giraudat J:
•• ABI1 protein phosphatase 2C is a negative regulator of abscisic
acid signaling. Plant Cell 1999, 11:1897-1909.
The abi1-1 mutant is a wilty, ABA-resistant Arabidopsis mutant that has
been heavily used in stomatal physiology. The authors of this paper describe
the whole-plant phenotype and phosphatase activity of intragenic revertants
of abi1-1. The results indicate that the wild-type ABI1 phosphatase is a
negative regulator of ABA responses.
51. Torsethaugen G, Pell EJ, Assmann SM: Ozone inhibits guard cell K+
channels implicated in stomatal opening. Proc Natl Acad Sci USA
1999, 96:13577-13582.
35. Gilroy S, Read ND, Trewavas AJ: Elevation of cytoplasmic calcium
by caged calcium or caged inositol trisphosphate initiates
stomatal closure. Nature 1990, 346:769-771.
52. Evans PT, Malmberg RL: Do polyamines have roles in plant
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36. Blatt MR, Thiel G, Trentham DR: Reversible inactivation of K+
channels of Vicia stomatal guard cells following the photolysis of
caged inositol 1,4,5-trisphosphate. Nature 1990, 346:766-769.
53. Liu K, Fu H, Bei Q, Luan S: Inward potassium channel in guard
cells as a target for polyamine regulation of stomatal movements.
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428
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58. Kinoshita T, Shimazaki KI: Blue light activates the plasma
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stomatal guard cells. EMBO J 1999, 18:5548-5558.
The H+-ATPase is a key component in guard cell signaling. This paper
elegantly shows that blue light can stimulate phosphorylation of the carboxyterminus of the H+-ATPase, and induce H+-ATPase activity. 14-3-3 protein
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59. Taylor AR, Assmann SM: Apparent absence of a redox requirement
for blue light activation of pump current in broad bean guard cells.
Plant Physiol 2001, 125:329-338.
60. Assmann SM, Simoncini L, Schroeder JI: Blue light activates
electrogenic ion pumping in guard cell protoplasts of Vicia faba.
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61. Emi T, Kinoshita T, Shimazaki KI: Specific binding of vf14-3-3a
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62. Plesch G, Kamann E, Mueller-Roeber B: Cloning of regulatory
sequences mediating guard-cell-specific gene expression. Gene
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63. Smart LB, Cameron KD, Bennett AB: Isolation of genes
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Nicotiana glauca. Plant Mol Biol 2000, 42:857-869.
64. Mott KA, Buckley TN: Patchy stomatal conductance: emergent
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This insightful review summarizes knowledge and hypotheses concerning
patchy stomatal behavior in the intact leaf.
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71. Hwang I, Sze H, Harper JF: A calcium-dependent protein kinase
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74. Ullah H, Chen JG, Young JC, Im KH, Sussman MR, Jones AM:
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See ‘Update’.
75. Wang XQ, Ullah H, Jones AM, Assmann SM: G protein regulation of
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See ‘Update’.
76. Allen GJ, Chu SP, Harrington CL, Schumacher K, Hoffmann T,
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See ‘Update’.