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Light Regulation ofStomatal MovementKen-ichiro Shimazaki,1
Michio Doi,2
Sarah M. Assmann,3 and Toshinori Kinoshita1,41Department of
Biology, Faculty of Science, 2Research and Development Center
forHigher Education, Kyushu University, Ropponmatsu, Fukuoka,
810-8560, Japan;email: [email protected],
[email protected] of Biology, Pennsylvania
State University, University Park,Pennsylvania 16802-5301; email:
[email protected], Japan Science and Technology Agency, 4-1-8
Honcho Kawaguchi,Saitama, Japan; email:
[email protected]
Annu. Rev. Plant Biol. 2007. 58:219–47
The Annual Review of Plant Biology is online
atplant.annualreviews.org
This article’s doi:10.1146/annurev.arplant.57.032905.105434
Copyright c© 2007 by Annual Reviews.All rights reserved
First published online as a Review in Advance onJanuary 5,
2007
1543-5008/07/0602-0219$20.00
Key Words
blue light, chloroplasts, guard cells, phototropin,
plasmamembrane H+-ATPase, stomata
AbstractStomatal pores, each surrounded by a pair of guard
cells, regulateCO2 uptake and water loss from leaves. Stomatal
opening is drivenby the accumulation of K+ salts and sugars in
guard cells, which ismediated by electrogenic proton pumps in the
plasma membraneand/or metabolic activity. Opening responses are
achieved by coor-dination of light signaling, light-energy
conversion, membrane iontransport, and metabolic activity in guard
cells. In this review, wefocus on recent progress in blue- and
red-light-dependent stom-atal opening. Because the blue-light
response of stomata appearsto be strongly affected by red light, we
discuss underlying mecha-nisms in the interaction between
blue-light signaling and guard cellchloroplasts.
219
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Contents
INTRODUCTION. . . . . . . . . . . . . . . . . 220BLUE-LIGHT
RESPONSE
OF STOMATA . . . . . . . . . . . . . . . . . . 221Properties . .
. . . . . . . . . . . . . . . . . . . . . . 221Electrogenic H+ Pump
Drives K+
Uptake and StomatalOpening . . . . . . . . . . . . . . . . . . .
. . . 222
Entity of H+ Pump andRegulation . . . . . . . . . . . . . . . .
. . . . 224
Blue-Light Receptors . . . . . . . . . . . . . 225Biochemical
Evidence for
Phototropins as Blue-LightReceptors . . . . . . . . . . . . . .
. . . . . . . 226
Blue-Light Signaling in GuardCells . . . . . . . . . . . . . . .
. . . . . . . . . . . 227
Energy Source forBlue-Light-Dependent ProtonPumping . . . . . .
. . . . . . . . . . . . . . . . 229
Metabolism SupportingBlue-Light-DependentStomatal Opening . . .
. . . . . . . . . . 230
Role of Blue-Light-SpecificStomatal Opening . . . . . . . . . .
. . . 231
Interaction of Blue-Light Signalingwith Abscisic Acid . . . . .
. . . . . . . . 231
Other Photoreceptors . . . . . . . . . . . . 232RED-LIGHT
RESPONSE
OF STOMATA . . . . . . . . . . . . . . . . . . 233SYNERGISTIC
EFFECT OF BLUE
AND RED LIGHT INSTOMATAL OPENING . . . . . . . . 233
CONCLUDING REMARKS . . . . . . . 236
INTRODUCTION
Stomata regulate gas exchange between plantsand atmosphere,
optimize photosyntheticCO2 fixation, and minimize
transpirationalwater loss (8, 17, 144, 189, 193). Stomatamove
rapidly to adjust plants to the ever-changing environment. The
opening of stom-ata is driven by the accumulation of K+ salts(37,
42, 64, 191) and/or sugars (124, 174)in guard cells, which results
in a decrease
in water potential and subsequent water up-take. Turgor
elevation from water uptakeincreases guard cell volume, which
widensstomatal apertures because of the asymmet-ric positioning of
microfibrils in the cell wall.The volume increase requires an
increase insurface area of the guard cell plasma mem-brane, and
this needed area is provided bythe internal membranes of guard
cells (162).Guard cells possess a number of small vac-uoles in the
closed state of stomata (117). Suchsmall vacuoles fuse with each
other and gener-ate bigger vacuoles during stomatal opening(43).
Prior to these processes, a large num-ber of ions move from the
cytosol to the vac-uole via channels and pumps in the
tonoplast(186). Stomatal closure is caused by the re-lease and/or
removal of osmotica from guardcells under drought, darkness,
elevated CO2,or low humidity, resulting in the internaliza-tion of
the plasma membrane and the gener-ation of small vacuoles.
Stomatal opening is induced by light, in-cluding blue and red
light, and distinct mecha-nisms underly stomatal opening in
response tothese different wavelengths (193). Blue lightacts as a
signal and red light as both a sig-nal and an energy source. Blue
light acti-vates the plasma membrane H+-ATPase (21,76),
hyperpolarizing the membrane poten-tial with simultaneous apoplast
acidification,and drives K+ uptake through voltage-gatedK+
channels. Red light drives photosynthesisin mesophyll and guard
cell chloroplasts anddecreases the intercellular CO2 concentra-tion
(Ci ). Red-light-induced stomatal open-ing may result from a
combination of guardcell response to the reduction in Ci and a
di-rect response of the guard cell chloroplasts tored light (132,
151, 183). In the afternoon,sugars also accumulate in guard cells
as os-motica and maintain stomatal opening (174).The accumulation
of positively charged K+
ions in guard cells must be compensated byanions, mainly in the
form of the organic acidmalate2− (189). Malate forms in response
toweak blue light under a red-light background,and the formation
does not occur without
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red light (109, 110). Guard cell chloroplastsare responsible for
malate formation (151),and the chloroplasts also act as a
reservoirfor starch and catabolize it as a precursor ofmalate (183,
189). Guard cells also utilize Cl−
and NO3− as counterions for K+, althoughsuch utilization varies
with plant species andgrowth conditions (51).
In this review, we focus on recent progressconcerning
light-stimulated stomatal open-ing. On the basis of the highly
specializedmetabolism in guard cells, which facilitatesthe rapid
transport of ions across membranes,a synergistic effect between
blue and red lighton stomatal opening has been proposed. Forother
stomatal responses, particularly stom-atal closure responses
induced by the phyto-hormone abscisic acid (ABA) under
drought,please refer to other recent reviews on thissubject (11,
17, 35, 56, 132, 144).
BLUE-LIGHT RESPONSEOF STOMATA
Properties
Blue-light-specific stomatal opening is foundin a number of C3
and C4 plants understrong background red light, and in
facultativeCrassulacean acid metabolism plants func-tioning in the
C3, but not the CAM, mode(89).
In intact leaves, blue light, on a quantumbasis, is several to
20 times more effective thanred light in opening stomata (19, 59,
71, 151,152). The high sensitivity of this blue-lightresponse
becomes prominent in the presenceof background red light.
Simultaneous mea-surements of stomatal conductance, photo-synthetic
CO2 fixation, and intercellular CO2concentration (Ci ) in leaves of
Arabidopsisthaliana and rice (Oryza sativa L. cv, Taichung65)
plants in response to light are presentedin Figure 1. When a leaf
kept in the darkfor 1 h was illuminated with strong red light(600
μmol m−2 s−1), photosynthetic CO2 fix-ation occurred instantly with
a sharp drop inCi to 250–270 ppm, followed by a gradual in-
crease in photosynthetic rate for 20 min, untila steady state
was achieved. Stomata showed agradual increase in stomatal
conductance witha short lag time, and conductance reached amaximum
within 20 min, then showed smallfluctuations (Figure 1a). Weak blue
light (5μmol m−2 s−1), superimposed on the red lightfor 10 min,
elicited rapid stomatal opening,with a threefold faster rate than
that caused byred light (151). The opening responses wereobserved
repeatedly in response to blue light,and the magnitude of the
responses showeda slight decrease from morning to afternoon(28).
Stomata showed virtually no opening inresponse to the weak
blue-light stimulus inthe absence of red light (Figure 1b).
Theseproperties are in accord with those shown inprevious reports
on other plant species (63,86, 151). When the rice leaf was
illuminatedwith red light, photosynthetic CO2 fixationincreased
instantly but remained at a low levelfor 10 min due to the low
stomatal conduc-tance, and then exhibited a large gradual in-crease
(Figure 1c). The gradual increase inphotosynthesis resulted from
the removal ofstomatal limitation, because the increase inCO2
fixation paralleled that in stomatal con-ductance of this phase.
Weak blue light super-imposed on the red light induced a very
rapidincrease in the stomatal aperture in this plantspecies (Figure
1c), as has also been shownfor other monocots, such as wheat (70,
71)and sugarcane (9).
Blue light acts as a signal, and even ashort period of light
(pulse, 30–60 s) inducesstomatal opening, which is sustained for
morethan 10 min after the pulse (63). Blue-light-specific stomatal
responses can be inducedby illuminating the leaves with blue light
su-perimposed on high-intensity red light, inwhich photosynthesis
was not further acti-vated by the blue-light stimulus (110,
148).The magnitude of the response was propor-tional to the photon
flux of blue light andreached saturation. Once a single saturat-ing
pulse was given to the leaf, the respon-siveness to a second pulse
was greatly re-duced when the second pulse was immediately
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a
b
c
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-2
0
2
4
6
0.04
0.06
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0.1
0.12
0.14
0.16
0.18
0 50 100 150
-2
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0 60 120 180
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0
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0 20 40 60 80 100
Ph
oto
syn
thet
ic R
ate
(µm
ol C
O2
m-2
s-1
) an
d C
i (10
2 p
pm
)
Time (min)
Sto
matal C
on
du
ctance (m
ol H
2 O m
-2 s-1)
Photosynthesis
Conductance
Ci
applied, and the responsiveness was graduallyrestored with a
half-life time (T1/2) of 9 minin Commelina communis (63). The
blue-lightreceptor was suggested to exist in two inter-convertible
forms: one physiologically activeand the other inactive (63). The
conversionfrom the inactive form to the active form was
alight-induced fast reaction and the active formgradually returned
to the inactive form via athermal reaction. These properties seem
to beattributable to the molecular properties of theresponsible
blue-light receptors, which werediscovered later (60, 72, 138).
Electrogenic H+ Pump Drives K+Uptake and Stomatal Opening
Medium acidification by guard cells inVicia epidermal peels was
reported, and the←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1Stomatal
conductance, photosynthetic CO2fixation, and intercellular CO2
concentration inresponse to light in leaves of Arabidopsis thaliana
(a,b) and Oryza sativa (c). Red upward arrows indicatecontinuous
irradiation of the leaf with a strong redlight (600 μmol s−1m−2).
Upward and downwardblue arrows indicate onset and termination
ofirradiation, respectively, with a weak blue light (5μmol s−1m−2).
(a) When an Arabidopsis leaf wasilluminated with red light,
stomatal conductancegradually increased and reached a steady
statewithin 20 min. Photosynthetic CO2 fixationshowed an initial
rapid increase with a subsequentgradual increase. This gradual
increase is probablydue to removal of stomatal limitation. When
weakblue light was superimposed on red light, thestomatal
conductance increased rapidly anddecreased after turning off the
blue light.Photosynthetic CO2 fixation was only slightlyenhanced by
blue light. (b) When an Arabidopsisleaf was illuminated with weak
blue light for 60min in the absence of red light, neither
stomatalconductance nor photosynthetic CO2 fixation wasincreased.
(c) When a rice leaf was illuminated withred light, stomatal
conductance did not change for10 min, whereas photosynthetic CO2
fixationshowed a rapid increase to a very low level. Uponthe
gradual increase in stomatal conductance,photosynthetic CO2
fixation showed a parallelincrease. When the leaf was irradiated
with weakblue light superimposed on red light, a very rapidincrease
in stomatal conductance was observed.
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acidification was required for K+ uptake andstomatal opening
(126). The H+ release fromguard cells was stimulated by light and
wasinhibited by vanadate (46). Zeiger & Hepler(197) found that
isolated guard cell proto-plasts of onion (Allium cepa) increased
theirvolume in response to blue light, which isindicative of
stomatal opening. This findingdemonstrated that all components
responsi-ble for blue-light-specific stomatal openingare localized
in guard cells. Using a large num-ber of guard cell protoplasts
from Vicia faba,it was found that guard cells extruded H+
inresponse to a pulse of blue light (157). Theproperties of H+
extrusion were very consis-tent with those of blue-light-induced
stomatalopening in intact leaves (63). The H+ extru-sion was
mediated by electrogenic pumping,and patch clamp experiments
demonstratedthat blue light induced a transient
membranehyperpolarization (12). These results indicatethat blue
light increases the inside-negativeelectrical potential across the
plasma mem-brane by activating the electrogenic protonpump (Figure
2). Because acidification ofthe external medium by epidermal strips
andswelling of guard cell protoplasts by blue lightwere inhibited
by vanadate, an inhibitor of theplasma membrane H+-ATPase (5, 46),
and theblue-light-stimulated electrogenic current re-quired
cytosolic ATP (12), the H+ pumpwas proposed to be the plasma
membraneH+-ATPase. In this initial report, the blue-light-induced
H+ current across the plasmamembrane as measured by whole-cell
patchclamp was not of the magnitude requiredfor sufficient K+
accumulation in guard cellsto drive stomatal opening. This may be
dueto loss of essential cytoplasmic componentsin the whole-cell
configuration, and a muchlarger current was obtained in a slow
whole-cell configuration (8, 143). A sufficientlylarge current was
also measured in whole-cellpatch clamp experiments (177) and in
intactVicia guard cells (135). A blue-light-sensitiveelectron
transport chain in the guard cellplasma membranes, which releases
H+ to themedium, was hypothesized as an alternative
H+
H+-ATPase
Kinase ?
Blue light
P14-3-3
Decrease inwater potential
H2O
P
Stomatal openingIncrease of
turgor pressure
K+
Inwardly-rectifyingK+ channel
ATPADPPi
ABA
Malate2-
Phototropins(phot1, phot2)
≤-100mv
14-3-3
Plasma membranehyperpolarization
H2O2
Figure 2Processes of stomatal opening in response to blue
light.
candidate mechanism (45, 125). Redox reg-ulation of the
blue-light-activated H+ pumpmay be possible (181, 183), although
manipu-lation of cytosolic NADH and NADPH con-centrations did not
alter the magnitude ofblue-light-stimulated pump current (177).
After discovery of the blue-light-inducedH+ pump, voltage-gated
K+ channels, acti-vated by membrane hyperpolarization,
werediscovered in the guard cell plasma membraneof the same plant
species (146). The channels(inward-rectifying K+ channels, K+in)
func-tion to take up K+ when guard cells are hy-perpolarized to
values more negative than theequilibrium potential for K+ (Figure
2). K+inchannels require ATP for the activation andmaintenance of
their activity (47, 190), andare sensitive to inhibition by Ca2+
(34, 145).The first genes for K+in channels to be clonedfrom
Arabidopsis were designated as KAT1 (6)and AKT1 (149). These genes
encoded K+inchannels, as determined by expressing thegenes in
Xenopus oocytes and evaluating the
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resultant currents (104, 141, 184). Subsequentresearch showed
that in addition to KAT1and AKT1, the Arabidopsis K+ channel
genesAKT2/3, AtKC1, and KAT2 are also expressedin guard cells (107,
170). KST1 and KPT1 arehighly expressed in guard cells of potato
andpoplar plants, respectively (84, 104, 123). In-troducing a
dominant-negative nonfunctionalKAT1 gene lowered the inward K+
currentby 75% in guard cells and reduced light-induced stomatal
opening by 40% (82), sug-gesting that the inward current largely
re-quires KAT1, although other K+in channelsare also functional
(170).
Entity of H+ Pump and Regulation
The pump was subsequently demonstrated tobe a plasma membrane
H+-ATPase that is ac-tivated by phosphorylation (76).
H+-ATPaseactivity in guard cell protoplasts was increasedby blue
light, and the activity was closely cor-related with the rate of H+
pumping. Lev-els of phosphorylation of the H+-ATPasewere increased
by blue light and the amountof increase was proportional to the
rate ofH+ pumping. The phosphorylation occurredexclusively on the C
terminus of the H+-ATPase, with subsequent binding of a 14-3-3
protein to the phosphorylated C terminus,which has been shown to
function as an au-toinhibitory domain of the H+-ATPase
(119).Phosphorylation of the Ser and Thr residueswithin this domain
promotes H+-ATPase ac-tivity (165), indicating that a
serine/threonineprotein kinase is involved in this
regulation(Figure 2).
Binding of a 14-3-3 protein to the H+-ATPase was absolutely
required for its acti-vation; phosphorylation alone was not
suffi-cient to activate the H+-ATPase (78). Fourisoforms of 14-3-3
proteins (Vf14-3-3a, b, c,and d) were detected in Vicia guard
cells, andmass analysis of the 14-3-3 protein that hadbound to the
H+-ATPase in vivo identifiedVf14-3-3a as a specific isoform binding
to theplasma membrane H+-ATPase (33). The iso-form Vf14-3-3a had a
higher binding affin-
ity to the H+-ATPase than Vf14-3-3b, andVf14-3-3a might more
effectively activate theH+-ATPase than Vf14-3-3b. The binding
siteof a 14-3-3 protein was determined to be thepenultimate Thr
residue in the C terminus bycompetition experiments with synthetic
phos-phopeptides (78). The site identified by thismethod was the
same as that determined bygenetic analysis of fusicoccin-dependent
acti-vation of the plasma membrane H+-ATPasein spinach leaves
(169).
Because the plasma membrane H+-ATPase shows primary and pivotal
functionsin many plant tissues by coupling with variouscarriers and
channels, the regulatory mecha-nisms of these enzymes have been
extensivelyinvestigated using the fungal toxin fusicoccin(119,
168). However, activation of the H+-ATPase by this toxin occurrs
irreversibly andsuch mechanisms might not be the same asthose
occurring in vivo. Guard cells are theideal systems to manipulate
the activity of thisenzyme in vivo, and the associated
regulatorymechanisms can be investigated under physi-ological
conditions (76).
There are several to 10 isoforms of theplasma membrane H+-ATPase
in plant cells,and 11 functional H+-ATPases are present
inArabidopsis (119). Tissue-specific expression ofthe H+-ATPase
isoforms has been observedin several organs, including AHA3 in
phloemcompanion cells (26), AHA9 in anther cells(58), and AHA4 in
roots (185). Therefore, itis possible that the H+-ATPase(s), which
isspecifically activated by blue light, is expressedin guard cells
in a cell-specific manner. InVicia guard cells, isoproteins of VHA1
(ViciaH+-ATPase1) and VHA2 were activated byblue light (76).
However, these isoproteinswere also expressed in roots, leaves,
stems,and flowers (55, 106). These observations sug-gest that guard
cells have an additional blue-light signaling system that is cell
specific,and the light signal is delivered to the usualH+-ATPase(s)
via this system. Interestingly,all 11 isogenes of AHA were
expressed inguard cells, whereas only 4 isogenes were ex-pressed in
mesophyll cells of Arabidopsis plants
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(180). In pulvinar motor cells of Phaseolus vul-garis, the
plasma membrane H+-ATPase ex-ists in a highly active and heavily
phosphory-lated state in the dark, and is inactivated
bydephoshorylation via phototropin-mediatedreactions (66),
suggesting that the blue-lightsignaling systems are flexible and
can be mod-ulated by cell-specific regulatory mechanisms.The
H+-ATPase inactivation caused waterloss from the motor cells and
resulted in leafmovement.
Blue-Light Receptors
Flavin and zeaxanthin, a component of thexanthophyll cycle
(108), are candidate chro-mophores for the blue-light receptor of
guardcells on the basis of action spectra for stom-atal opening in
intact leaves and malate syn-thesis in epidermal peels (71, 110,
199).The sensitivity of stomata to blue light in-creased with their
zeaxanthin content (198),and inhibition of zeaxanthin formation
sup-pressed blue-light-stimulated stomatal open-ing. The
zeaxanthin-less mutant of Arabidop-sis, npq1, failed to respond to
blue light (40).Strong green light suppressed the
blue-light-dependent stomatal opening in epidermalstrips of
Arabidopsis (39), and this effect wasalso found in isolated
epidermal peels fromlight-treated leaves (173). These results
sug-gest the involvement of zeaxanthin in theblue-light response of
stomata. However, fur-ther confirmation is needed because the
stom-atal aperture responses reported for the npq1mutant could not
be reproduced in leaves, epi-dermes, or guard cell protoplasts (29,
72, 180).
In 1997, a blue-light receptor for pho-totropism was identified
by the Briggs group(60). The photoreceptor has two Light, Oxy-gen,
Voltage (LOV) domains in the N ter-minus and a serine/threonine
protein kinasein the C terminus, and undergoes autophos-phorylation
in response to blue light (21,24). The LOV domains (LOV1 and
LOV2)function as binding sites for flavin mononu-cleotide and
absorb blue light (25). A ho-molog of this protein was found (67)
and
these proteins were initially called nonpho-totropic hypocotyl
(NPH1) and nonpho-totropic hypocotyl like (NPL1), and were
sub-sequently renamed phototropins 1 (phot1)and 2 (phot2),
respectively (20). Later, phot2was demonstrated to be the
photoreceptorthat mediates the chloroplast avoidance re-sponse to
high-intensity light (68, 69).
The absorption spectrum of the LOV2 do-main has peaks at 378 nm
in the UV-A re-gion, and 447 nm and 475 nm in the blueregion (138).
The action spectrum for stom-atal opening in wheat leaves closely
matchesthe absorption spectrum of the LOV domains(71). It was thus
plausible to hypothesize thatphototropins function as the
blue-light re-ceptors in guard cells (Figure 2). However,in the
Arabidopsis phot1 mutant, transpirationin the leaf still responded
to blue light (87).Thus, phot2 might act as a blue-light recep-tor
in guard cells, or phot1 and phot2 mightfunction redundantly in the
cells. To resolvethis issue, the stomatal response was
investi-gated using a single mutant of phot2, and thephot1 phot2
double mutant of Arabidopsis. Inthe epidermal peels of Arabidopsis,
stomata inthe phot1 phot2 double mutant did not openin response to
blue light, but stomata openedin the phot1, phot2, and npq1 single
mutants(72). Epidermal strips from the double mutantwere unable to
extrude H+ in response to bluelight. In this mutant, no change was
found inthe amount of plasma membrane H+-ATPase,and the mutant
could respond to fusicoccinby opening stomata. Thus, the lack of
stom-atal opening and H+ extrusion by blue lightis due to an
impairment in blue light percep-tion. Stomata in the phot2 mutant
retain moresensitivity to blue light than those of phot1 inthe
opening response.
Involvement of phototropins in stomatalresponse was confirmed by
the complemen-tation of the phot1 phot2 double mutant withthe PHOT1
gene (28): Stomatal conductancein leaves of the phot1 phot2 double
mutant didnot increase following blue-light irradiation,and the
response was restored in the transfor-mant. Chlamydomonas
reinhardtii phototropin
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(Crphot), which acts as a blue-light receptor inthe sexual life
cycle of algae (61), also restoredstomatal response when the phot1
phot2 doublemutant was transformed with CrPHOT (113).
Biochemical Evidence forPhototropins as Blue-LightReceptors
There are at least two Vicia faba phototropins,1a and 1b
(Vfphot1a and 1b), with molec-ular masses of 125 kDa, and both are
ex-pressed in guard cells. If phototropins func-tion upstream of
the H+-ATPase, then theyshould be phosphorylated more quickly
thanthe H+-ATPase. Simultaneous determina-tion of the
phosphorylation levels of pho-totropins and the H+-ATPase in Vicia
guardcells revealed that this was indeed the case(73). The maximum
phosphorylation levels ofVfphots and the H+-ATPase appeared around1
and 5 min, respectively, after the onsetof blue light. When
phototropin phospho-rylation was inhibited by diphenyleneiodo-nium
chloride (DPI), a flavoprotein inhibitor,or by protein kinase
inhibitors (K-252a andstaurosporine), the H+-ATPase
phosphory-lation was inhibited to the same degree asthat of
phototropins. Phosphorylation of pho-totropins and the H+-ATPase
showed similarphoton flux dependency. Phototropin 1 is as-sociated
with the plasma membrane in guardcells (137), where the H+-ATPase
is also lo-calized, and this is consistent with the idea thatphot 1
plays a role in blue-light-induced stom-atal opening. Thus,
biochemical and pharma-cological evidence indicate that
phototropinsact upstream of the plasma membrane H+-ATPase (Figure
2).
The binding of 14-3-3 proteins to targetproteins can modify
enzyme activity, changeintracellular localization, and generate a
scaf-fold for the interaction with other proteins(15, 36, 62, 78,
101). Phototropin binds re-versibly to a 14-3-3 protein upon its
autophos-phorylation in guard cells (73). The 14-3-3-phototropin
complex might confer a signal-ing state to phototropins, which then
could
transmit the light signal to downstream ele-ments (Figure 2).
Binding of a 14-3-3 proteinto phototropin was also seen in the
tissues ofboth etiolated seedlings and green leaves.
Binding of a 14-3-3 protein to target pro-teins was
phosphorylation-dependent in gen-eral. However, the amount of
14-3-3 pro-tein bound did not parallel the levels ofphototropin
phosphorylation. The maximumlevel of 14-3-3 binding in guard cells
wasreached faster than that of phosphorylation,and dissociation of
a 14-3-3 protein fromphototropin was also faster than the
dephos-phorylation of phototropin. This is proba-bly because
phototropins are phosphorylatedon multiple sites (139, 163), and
only someof these sites act as the binding sites for14-3-3 protein;
these sites appeared to bephosphorylated and dephosphorylated
fasterthan other sites. Because the binding sites of14-3-3 protein
had phospho-Ser consensusmotifs, including R/KXXpSXP, R/XXXpSXP,and
R/XXpS/T (1, 105), the binding siteswere determined by substituting
all candidateSer residues in phototropins. The bindingsites were
located in the hinge region betweenLOV1 and LOV2 in Vicia
phototropins (73).Two phosphorylation sites were determinedto be
localized in the N terminus and six inthe hinge region between LOV1
and LOV2in Avena sativa phot1 by combining in vitroand in vivo
phosphorylation using protein ki-nase A (139). The phosphorylation
sites haddifferent blue-light sensitivities.
Phototropin in etiolated seedlings under-went phosphorylation by
blue light and wasdephosphorylated gradually under darkness(118),
and the long-lived phosphorylated statecould act as the
desensitized state of the pho-toreceptor (163). The recovery of
sensitivityto blue light in phototropism occurred witha kinetics
similar to that for recovery fromthe phosphorylated state.
Essentially the samedesensitization process was found in the
blue-light response of stomata. The responsivenessof stomata to a
second blue-light pulse de-creased when guard cells were irradiated
im-mediately after a first pulse, and sensitivity was
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gradually restored during the dark interval(63, 73, 157).
Blue-Light Signaling in Guard Cells
The signaling mechanism from phototropinsto the plasma membrane
H+-ATPase islargely unknown. However, several signalingmolecules
appear to be present between pho-totropins and the H+-ATPase
(Figure 2).
14-3-3 proteins. The 14-3-3 proteins areinvolved in blue-light
signaling in stomataas regulators of both phototropins and
H+-ATPase. A 14-3-3 protein functions as an ac-tivator of the
plasma membrane H+-ATPasevia phosphorylation of the H+-ATPase in
re-sponse to blue light (33, 78). A 14-3-3 pro-tein also binds
phototropin upon its phospho-rylation, and the binding appears to
precedethe activation of the plasma membrane H+-ATPase in guard
cells (73). However, the func-tional significance of the
phototropin-14-3-3protein complex has not been determined.
RPT2. Root Phototropism 2 (RPT2), amember of a unique,
plant-specific familyof proteins (102), was suggested to act as
asignal transducer from phot1 to the down-stream components of the
signaling chainin phototropism and stomatal opening, butwas not
involved in chloroplast movement(65). RPT2 interacted genetically
and phys-ically with phot1 but not phot2, and the phot2rpt2 double
mutant lost the capacity for blue-light-induced stomatal opening in
the epider-mis (Figure 3). Because the determination ofstomatal
aperture in these experiments wasperformed only in the epidermis,
other sepa-rate lines of evidence will be needed to con-firm this
result.
VfPIP. A protein interacting with Viciaphot1a was isolated from
guard cells (32).The Vicia faba phot1a interacting protein (Vf-PIP)
bound to the N terminus of Vfphot1abut not to Vfphot1b. The VfPIP
transcriptwas predominantly expressed in guard cells.
VfPIP has sequence homology to dynein lightchain and localizes
to cortical microtubules.Vfphot1a seemed to exert its function
throughthe microtubules, because treating guard cellswith
microtubule depolymerizing compoundsresulted in partial inhibition
of blue-light-stimulated stomatal opening and H+ extru-sion. A
recent investigation indicated thatthe microtubules in guard cells
are orga-nized in parallel, straight, and dense arrays byblue light
and are not affected by red light(83). Reorganization of the
microtubules wasnot stimulated by activating the H+-ATPaseby
fusicoccin. VfPIP may accelerate stom-atal opening via
phototropin-mediated orga-nization and reorientation of
microtubules(Figure 3).
Ca2+. Ca2+ was suggested to be responsiblefor blue-light
signaling in guard cells by phar-macological tools. Calmodulin
antagonists in-hibited both blue-light-dependent H+ pump-ing and
stomatal opening (158). The pumpingwas inhibited by verapamil, a
Ca2+ channelblocker, albeit at high concentrations (160).The H+
pumping was inhibited reversibly bycaffeine, which releases Ca2+
from intracel-lular stores, and by inhibitors of endoplas-mic
reticulum Ca2+-ATPase. Although stim-ulation of blue-light-induced
acidification byexternal Ca2+ was reported in Arabidopsis
epi-dermis (134), neither Ca2+ channel blockersnor change in
external Ca2+ concentration af-fected H+ pumping in Vicia (154).
Therefore,the Ca2+ that is thought to be required forstomatal
opening might originate from in-tracellular Ca2+ stores, most
likely from theendoplasmic reticulum. In accord with
thispossibility, the anion channels that facilitateuptake of Cl−
and malate2− into the vacuole,as occurs during stomatal opening,
were ac-tivated by CDPK, a Ca2+-activated proteinkinase in guard
cells (122).
Recent investigations demonstrated thatphot1 activation elicited
an increase in cytoso-lic Ca2+ in Arabidopsis and tobacco
seedlings(16). The Ca2+ increase exhibited a transientchange with a
possible lag period of 3–6 s and
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phot1
phot2
Ca2+ channel
Ca2+*PLC
PPI*Protein kinase
(K-252a insensitive)H+-ATPase
Membranehyperpolarization K
+ uptake
Microtubule organization
RPT2
VfPIP
Blue lightspecific
Mesophyllchloroplast
ATPNADPH
Guard cellchloroplast
[Ca2+]cyt
ATPNADH
NADH
Oxaloacetic acidStarch
degradation
Malate synthesis
Ci decreaseAnion channel
inactivationMaintain membrane
hyperpolarization
Mesophyll cell
Guard cell
Sucrose
K+in channelactivation
Red light
Stomatalopening
NADH-MDH
ATP
Mitochondrion
CO2 fixation
CO2 fixation
Sucrose
Figure 3Cooperation of blue-light signaling and metabolism in
guard cells for stomatal opening. Blue light isperceived by phot1
and phot2. The light signals are transmitted and activate the
plasma membraneH+-ATPase, thereby inducing K+ uptake. Blue light
also stimulates starch degradation. These responsesare specific to
blue light under normal conditions. Red light is absorbed by guard
cells and mesophyllchloroplasts. Guard cell chloroplasts provide
ATP and NADH in the cytosol, and mesophyll chloroplastsfix CO2,
thereby resulting in Ci decrease. The Ci decrease activates
transport mechanisms that result inmembrane hyperpolarization.
These reactions are not specific to red light. Blue light is also
absorbed bychloroplasts and has the same effect as red light, but
the role of blue light through the chloroplasts is notdepicted in
this figure.
∗We placed Ca2+ upstream of protein phosphatase in this
signaling pathway, but
the order is speculative. K-252a, a broad inhibitor of Ser/Thr
protein kinases; NADH-MDH,NADH-malate dehydrogenase; phot1,
phototropin 1; phot2, phototropin 2; PLC, phospholipase C;RPT2,
ROOT PHOTOTROPISM 2; VfPIP, Vicia faba phototropin interacting
protein; PP1, type 1protein phosphatase.
was sustained for 80 s after the pulse of bluelight. The
responsiveness to blue light was de-sensitized after the first
pulse and graduallyrestored within 3–4 h. Based on mutant
anal-ysis, cry1 and cry2 do not participate in thesignal
transduction chain triggering this Ca2+
increase, whereas both phot1 and phot2 areinvolved and appear to
mediate the cytoso-lic Ca2+ increase via calcium-permeable
chan-nels in the plasma membrane of mesophyllcells (16, 54, 167).
phot2 may also function viaphospholipase C-mediated Ca2+ release
fromintracellular stores (Figure 3), as inferredfrom assays with
pharmacological inhibitors
of phospholipase C (PLC) (54). Phototropin-mediated Ca2+
increase was likely to be re-quired for rapid inhibition of
hypocotyl elon-gation (38), chloroplast movement (178),
andphototropism (14, 16), and the rapid growthinhibition was
phot1-dependent. However,note that an increase in cytosolic Ca2+ by
bluelight has not yet been reported in guard cells.
It has been well documented that Ca2+ is asecond messenger for
ABA-induced stomatalclosure (17, 144). A high concentration of
cy-tosolic Ca2+ caused stomatal closure or inhib-ited stomatal
opening (42, 96, 147) throughactivation of anion channels, and
inactivation
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of the plasma membrane H+-ATPase andK+in channels (74, 145).
These results appearto conflict with the notion that Ca2+ is
re-quired for stomatal opening. ABA and a highconcentration of
external Ca2+ induced theoscillation of cytosolic concentration of
Ca2+
with a definite period, and such oscillation wasabsolutely
required for the maintenance ofstomatal closure (3, 95). However,
if cytosolicCa2+ increased continuously without oscilla-tion, or
the intervals between oscillations weretoo short or too long (3),
the Ca2+ responsedid not cause sustained stomatal closure (3,
4,140). We note that, in other tissues, the Ca2+
increase elicited by blue light via phototropinsdid not show
oscillations (16, 54, 167), andsuch a nonoscillatory increase might
encodeinformation opposite to that for stomatalclosure.
Protein kinase. A serine/threonine proteinkinase that directly
phosphorylates the plasmamembrane H+-ATPase is present in
guardcells, and this kinase may be activated by bluelight (76)
(Figure 2). This kinase is unlikelyto be phototropin itself,
because the kinasewas almost completely insensitive to kinase
in-hibitors that are effective against phototropinkinase activity
(73, 77, 158, 167). A proteinkinase activity that phosphorylates
the H+-ATPase has been demonstrated in the plasmamembrane of
spinach leaves in vitro (169)(Figure 3).
Protein phosphatase. Okadaic acid and ca-lyculin A inhibit both
blue-light-dependentH+ pumping and stomatal opening (75).
Thisresult implies that type 1 or type 2A pro-tein phosphatase(s)
mediate the signaling be-tween phototropins and the plasma
mem-brane H+-ATPase in guard cells. However,it has been unclear
which type of proteinphosphatase is involved in this signaling,
be-cause okadaic acid and calyculin A inhibitboth phosphatases.
Recent work indicates thattype 1 protein phosphatase is responsible
forthis signaling (172) (Figure 3). When thedominant-negative form
of type 1 protein
phosphatase or inhibitor-2, a proteinaceous-specific inhibitor
of type 1 protein phos-phatase, was expressed in Vicia guard
cellsvia particle bombardment, stomatal open-ing by blue light was
inhibited. Tautomycin,a preferential inhibitor of type 1
proteinphosphatase in vivo, suppressed blue-light-induced
phosphorylation of the H+-ATPasebut did not affect the
autophosphorylation ofphototropins.
Energy Source forBlue-Light-Dependent ProtonPumping
Protons are pumped out at the expenseof ATP by the plasma
membrane H+-ATPase. It is likely that the energy
forblue-light-dependent proton pumping is pro-vided mainly by
mitochondria and partly bychloroplasts (Figure 3), although the
energysource may vary with the prevailing envi-ronment (13, 120,
189). Several chloroplastsand numerous mitochondria exist in
guardcells, which exhibit high rates of respira-tion (2, 156, 182).
Inhibited respiration re-duced ATP levels drastically in guard
cells,but the reduction was not severe in mes-ophyll cells (44,
156, 181). Stomatal open-ing specific to blue light was inhibited
bypotassium cyanide (KCN) and anoxia andpartially affected by
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor
ofphotosystem II (PSII) in photosynthetic elec-tron transport
(148). Oligomycin, KCN, andlow oxygen tension largely reduced
blue-light-dependent H+ pumping, and DCMUpartially inhibited these
responses (93).
Guard cell chloroplasts synthesize ATPvia cyclic and noncyclic
photophosphoryla-tion (161, 195) (Figure 3) and can be the
mainenergy source when mitochondrial activityis reduced. In the
presence of oligomycin,fusicoccin-induced H+ pumping in guardcells
was greatly reduced, whereas the addi-tion of red light enhanced
both H+ pumpingand stomatal opening, and this enhancementwas
eliminated by DCMU (179).
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Metabolism SupportingBlue-Light-Dependent StomatalOpening
Malate. The main events of blue-light-dependent stomatal opening
are an activationof the plasma membrane H+-ATPase and K+
uptake from the medium. To compensate thepositively charged K+
accumulated in guardcells, malate2− is synthesized, and Cl− andNO3−
are taken up from the medium. Mostplant species (except onion,
which does notaccumulate starch in guard cells) accumulatemalate2−
preferentially over other anions. Aclose correlation has been
obtained betweenstomatal opening and malate accumulationin guard
cells (183, 189). Malate2− can beproduced through the degradation
of starchin guard cells under blue light (Figure 3).The opening of
stomata by blue light wasseverely impaired in Arabidopsis
phosphoglu-comutase mutant plants, which did not ac-cumulate starch
in guard cell chloroplasts(88). However, this blue-light response
wasrestored in the presence of high concentra-tions of Cl−,
possibly because Cl− replacedmalate2− as a necessary counterion for
K+.The mechanisms mediating uptake of anions,including Cl− and
NO3−, across the plasmamembranes in guard cells are largely
un-known, but it is likely that a cotransporter ofH+/Cl− (NO3−)
acts as the anion uptake car-rier in guard cells.
Recent work indicated that AtSTP1 (theH+-monosaccharide
symporter gene) expres-sion was quickly and largely upregulated
inguard cells in darkness (166). The AtSTP1transporter may function
in the guard cell im-port of apoplastic glucose derived from
mes-ophyll cells under darkness and contribute tothe accumulation
of starch. The products ofstarch breakdown could be exported to
thecytosol from guard cell chloroplasts and actas osmotica under
the light (114, 116, 129),i.e., in the opposite direction as in
mesophyllchloroplasts, where starch is broken down indarkness
(164). Exported compounds mightbe converted to phosphoenolpyruvate
(PEP)
in the cytosol, and PEP catalyzed by PEP car-boxylase to produce
OAA, which is then re-duced to malate (189) (Figure 3). High
ac-tivities of PEP carboxylase and NAD+ malatedehydrogenase,
required for these reactions,exist in guard cells.
Malate could be produced through light-activated NADP+ malate
dehydrogenase inguard cell chloroplasts (49), but the malateformed
by this pathway marginally con-tributes to stomatal opening,
because the ac-tivity of NADP+ malate dehydrogenase ismuch lower
than that of NAD+- malate dehy-drogenase in guard cells (142, 189).
Becauseguard cell chloroplasts are likely to overreduceelectron
acceptors due to their low Rubiscoactivity and resulting low CO2
fixation, thelight-activated NADP+-MDH may functionas a malate
valve to prevent damage to guardcells under strong sunlight
(41).
Sucrose. Early investigations indicated thatstarch breakdown
produced sugars that actedas principal osmotica for stomatal
opening.This starch-sugar hypothesis had been re-placed by the
theory of guard cell osmoreg-ulation via K+ and its counterions
(37, 42,189). However, some studies have reporteda parallel
relationship between stomatal aper-tures and sucrose content in
epidermal peels(128). Recent investigations demonstrate thatsucrose
accumulates in guard cells and re-places K+ in the afternoon,
thereby serv-ing as a dominant osmoticum to maintainstomatal
apertures (176). Sugar accumulatesin guard cells under the light;
however, themechanism by which the sucrose accumu-lation occurs is
largely unknown (183). Be-cause guard cells are not connected
withneighboring cells via plasmodesmata (188),two other pathways
must operate in the su-crose accumulation: either uptake from
theapoplast across the plasma membrane and/orproduction in these
symplastically isolatedcells. It is unlikely that photosynthetic
CO2fixation in guard cell chloroplasts providesall of the sucrose,
because the chlorophyll
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content in guard cells is less than 2% of that ofmesophyll cells
on a cell basis and Rubisco ac-tivity is low on a chlorophyll basis
(50, 115,127, 153, 156). It is possible that sugars areproduced in
guard cells, but fast starch degra-dation is required for
sufficient and continu-ous provision of sugars (112, 176) (Figure
3).Import of sucrose/hexose from the apoplastwill likely occur
(183). It is very likely thatblue light stimulates sucrose/hexose
uptake bycoupling with a H+/sucrose or glucose sym-porter across
the plasma membrane of guardcells through acidification of the
apoplastby the H+-ATPase (Figure 3). In supportof this idea,
fusicoccin-induced activationof the H+-ATPase enhanced sucrose
up-take in guard cell protoplasts (128, 130). Su-crose uptake by
guard cells was reported inepidermal peels when they were
overlayedonto mesophyll tissue (27). Because apoplas-tic sucrose
reaches a high concentration inthe daytime (91, 183), the
H+/sucrose andH+/monosaccharide symporters could func-tion coupled
with the H+-ATPase. Transientupregulation of AtSTP1
H+/monosaccharidesymporter gene in the midday might con-tribute to
guard cell-specific accumulation ofsucrose (166). Expression of the
sucrose trans-porter AtSUC3 was demonstrated in guardcells of
Arabidopsis (100).
Role of Blue-Light-Specific StomatalOpening
The opening of stomata in response to bluelight is fast and
sensitive (151). Photosyn-thetic CO2 fixation starts upon
illuminationand reaches maximum levels within severalminutes. In
general, stomatal opening occursmore slowly than photosynthesis,
requiringmore than 20 min to reach the maximum aper-ture in many
plant species. At dawn, sunlightis rich in blue light, and the
consequent rapidstomatal opening would enhance CO2 uptakefor
photosynthesis (194).
Blue-light-induced stomatal opening ismediated via phototropins
and enhancesphotosynthetic CO2 fixation by removing
the stomatal limitation to CO2 entry. Pho-totropins also mediate
phototropic bending,chloroplast accumulation, leaf expansion,
andleaf movement (21), and these responses maxi-mize photosynthetic
electron transport by im-proving the efficiency of light capture.
Thus,the blue-light response of stomata optimizesphotosynthesis in
increasing the CO2 provi-sion for stromal reactions in coordination
withthese phototropin-mediated thylakoid reac-tions. Such functions
of phototropins weredemonstrated by a dramatic growth enhance-ment
of Arabidopsis under low and moderatelight environments (171).
A fast response of stomata to blue light issuggested to decrease
stomatal limitation ofCO2 uptake and facilitate CO2 fixation
undersun flecks in the canopy (79). More impor-tantly, the
sustained nature of stomatal open-ing after stimulation by a short
period of (blue)light, such as a sun fleck (63), will
enhancelight-energy capture by providing CO2 whenthe leaf
encounters successive sun flecks.
Interaction of Blue-Light Signalingwith Abscisic Acid
When plants are under drought stress,stomata close in the
daytime to minimizewater loss by transpiration. ABA
inducesanion-channel activation with simultaneousmembrane
depolarization and a subsequentactivation of K+out channels (11,
144). Theactivated anion and K+out channels allowsustained K+ salt
efflux from guard cells,ultimately resulting in stomatal closure.
ABAalso stimulates stomatal closure by inhibitingH+ pumping (48,
157) (Figure 2). Thepump inhibition is important to
maintainmembrane depolarization and to reduce ATPconsumption by the
H+-ATPase. ABA in-hibits blue-light-dependent phosphorylationof the
H+-ATPase, and the inhibition maybe mediated by H2O2 in guard cells
(202).ABA produces H2O2 in guard cells throughNADPH oxidases in the
plasma membrane(81, 121, 201, 202), resulting in NO pro-duction
that stimulates stomatal closure
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(22). H2O2 also induces an increase throughactivation of
hyperpolarization-activatedCa2+-permeable channels (52, 121). It
ispossible that the increase in cytosolic Ca2+
via H2O2 inhibits the H+-ATPase, althoughthis seems
contradictory if Ca2+ is requiredfor blue-light signaling, as
mentioned above(74, 90). ABA inhibited blue-light-inducedapoplastic
acidification in the epidermis by theH+-ATPase, and the inhibition
was not foundin abi1 and abi2 mutants of Arabidopsis
(134).Phototropins could be the target of H2O2,because phototropins
have cysteine residuesthat are essential for their activities
(138).
Other Photoreceptors
Phytochrome might be involved in the stom-atal movements of
several plant species, butthe response was quite small (136).
Phy-tochrome was reported as a modulator ofthe blue-light response
of stomata in Phase-olus vulgaris seedlings (57). The time
requiredto initiate stomatal opening was shortenedby R and
lengthened by FR, and the re-sponse showed R/FR reversibility. In
the or-chid Paphiopedilum, stomata opened in re-sponse to low
photon flux density of red lightand this opening was reversed by
far-red light,suggesting phytochrome involvement (175).Negative
results were also reported on phy-tochrome involvement, e.g., in
wheat (71)and Vicia (112). Recently, a triple mutationof gene
families of phytochrome kinase sub-strates (PKS1, PKS2, and PKS4)
was reportedto impair phototropism (85). PKS proteinsinteracted
with phot1 in vivo and in vitro,and PKS proteins were suggested to
representa molecular link between phytochrome andphototropin
signaling. However, no data wereobtained on the involvement of PKS
proteinsin the blue-light response of stomata.
The action spectrum of stomatal openingin the UV region was
obtained using Vicia epi-dermis (31). The spectrum showed a
majorpeak at 280 nm and a minor peak at 360 nm.The response at 280
nm was three times largerthan that at 459 nm. The
UV-B-dependent
response was antagonized by green light (30).It is suggested
that the response to UV ismediated by a blue-light receptor, and
thatthe energy is directly transferred to the signalmolecule from
the protein-pigment complexupon absorption of UV. However,
Arabidop-sis mutants of both npq1 and phot1 phot2 re-sponded to
UV-B (287 nm), suggesting thepresence of a separate, unidentified
UV-Bphotoreceptor.
Stomata from the cry1 cry 2 double mu-tant exhibited a normal
response to blue light,which was enhanced by red light in a man-ner
similar to the response in the wild type,and this result excluded
the involvement ofcryptochromes in these stomatal responsesto light
(87). By contrast, a recent detailedinvestigation indicated that
stomata of thecry1 cry2 double mutant showed a reducedblue-light
response, whereas those of CRY1-overexpressing plants revealed a
hypersensi-tive response (92), suggesting the involve-ment of
cryptochromes. In agreement withthese findings, stomata in
epidermal peelsfrom the phot1 phot2 double mutant openedslightly in
response to relatively strong bluelight. Note that in these
experiments theblue light stimulus used might have
activatedchlorophyll-dependent stomatal opening inthe epidermis of
the phot1 phot2 double mu-tant, because the background red light
in-tensity employed would not have sufficed tosaturate guard cell
photosynthesis. In othercryptochrome-mediated responses, COP1
isimplicated as a downstream element. There-fore, in an analysis of
stomatal responses ineither plants overexpressing COP1 or
plantslacking functional cop1, the COP1 protein wasimplicated as a
negative regulator of stom-atal opening that functions downstream
ofboth cryptochromes and phototropins. How-ever, stomata in CRY1
overexpressing plantsand in cop1 mutant plants are barely
closed(widely open) in darkness. Taken together,these results
suggest that cry signaling islikely related to the closure system
in stom-ata, and that COP1 activates the closure sys-tem, but is
not directly related to phototropin
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signaling. This conclusion is also consistentwith other reports
showing that neither cry1nor cry2 participates in stomatal opening
inresponse to blue light (111). Crys thus seem tofunction in a
blue-light-independent mannerto inhibit stomatal closure and
thereby pro-mote stomatal opening.
RED-LIGHT RESPONSEOF STOMATA
The opening response of stomata to red lightrequires a high
light intensity (see Figure 1).The red-light response also requires
contin-uous illumination in most plant species, withZea mays being
a notable exception (10). Thered-light response may be driven
partly by ac-cumulation of K+ salts (59) and partly by
sugaraccumulation (112, 174, 176). Potassium ac-cumulation may be
driven by the membranepotential, generated by a
red-light-activatedH+ pump in the plasma membrane (112, 150).The
pump activation might be brought aboutby the increase in ATP
concentration in thecytosol resulting from photophosphorylationin
guard cell chloroplasts (161, 179). How-ever, the pump activation
by red light reportedin initial patch clamp experiments (150)
wasnot reproduced (135, 177), and confirmationof this finding is
needed. Sugars might be pro-duced in guard cells from a combination
ofstarch degradation, photosynthesis, and im-port from the apoplast
(114).
A localized beam of red light applied to in-dividual guard cells
did not induce stomatalopening, giving rise to the hypothesis that
thered-light response is actually a response to thedecrease in Ci
(Figure 1) resulting from red-light-driven mesophyll photosynthesis
(131).This result is supported by the recent obser-vation that
stomata in albino leaf portions ofvariegated leaves of Chlorophytum
comosum donot open by red light (133). The reductionof Ci brought
about by mesophyll photosyn-thesis is one of the triggers that
cause stom-atal opening (Figure 3), and it is generallyaccepted
that guard cells sense Ci rather thanCa (ambient CO2 concentration)
(103). CO2
modulates cytosolic Ca2+ transients in guardcells, thereby
stimulating Ca2+-induced stom-atal closure in a process analogous
to that trig-gered by ABA (144, 187, 192). In addition,guard cells
were hyperpolarized in CO2-freeair and depolarized under high CO2,
probablydue to the activation of anion channels (18, 53,131), which
results in stomatal closure.
Several lines of evidence indicate that thered-light response is
not just an indirect re-sponse, mediated by the effects of red
light onmesophyll photosynthesis and Ci, but also re-sults from a
direct response of the guard cellsto this light stimulus. First, it
is well estab-lished that red light can induce stomatal open-ing in
the isolated epidermis. This responsedepends on guard cell
chloroplasts: The re-sponse is suppressed by DCMU (112, 148),
aninhibitor of PSII, and is not observed in theepidermis of the
orchid, Paphiopedilum, whichhas guard cells lacking chlorophyll
(193). Ex-periments in intact leaves also demonstratethat the
red-light response is not solely a re-sponse to Ci: Red light
stimulates stomatalopening even when the intracellular
concen-tration of CO2 is held constant via gas ex-change
methodology (99).
SYNERGISTIC EFFECT OF BLUEAND RED LIGHT IN STOMATALOPENING
Stomata in Arabidopsis opened rapidly in re-sponse to a weak
blue light under a back-ground of strong red light, but barely
openedwithout red light (Figure 1). Stomatal con-ductance in leaves
illuminated with both redand blue light is typically larger than
thesum of the conductances under blue and redlight alone,
suggesting a synergistic action ofstomatal opening in intact
leaves. This phe-nomenon is illustrated in Figure 1 and hasbeen
observed in numerous species (7, 63, 70,110, 151, 194).
It is likely that both Ci and guard cellchloroplasts play roles
in the synergistic ef-fect of blue and red light on stomatal
opening.That the synergistic effects of blue and red
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light could occur in part via a stomatal re-sponse to the
reduction of Ci induced by mes-ophyll photosynthesis is suggested
by the ob-servation that manipulation of Ci by gas ex-change
techniques affects the magnitude ofthe blue-light response.
Reducing Ci to a lowlevel increases the magnitude of the
blue-lightresponse in several plant species (7, 70, 86).In
addition, red-light enhancement of stom-atal conductance in
response to blue light wasfound in leaves of the orchid
Paphiopedilum,which has a chlorophyllous mesophyll
butachlorophyllous guard cells, whereas Paphio-pedilum stomata in
isolated epidermal peelsshowed no red-light response (196). Taken
to-gether, these results suggest that mesophyllphotosynthesis can
indirectly stimulate theblue-light response.
That guard cell chloroplasts also play arole in the synergistic
effect of red and bluelight is indicated by the fact that this
syner-gism is also found in the isolated epidermisfor both stomatal
opening (148) and malateformation (109, 110). In addition, in
wheat,red light enhanced the magnitude of the blue-light-induced
conductance response, even inthe presence of CO2-free air (70).
Such resultsindicate that there are also Ci-independent as-pects to
this synergism. We suggest that theseCi-independent aspects could
be accountedfor by cooperation of blue-light signaling andguard
cell chloroplasts with respect to malate
formation. We propose that guard cell chloro-plasts are primed
to translocate NADH andATP into the cytosol under red light.
Thesecompounds are required for malate synthe-sis and H+ pumping,
respectively, when bluelight is applied (Figure 4). Malate is
formedby the reduction of oxaloacetate (OAA) byNAD-malate
dehydrogenase at the expense ofNADH. The large amount of NADH
neces-sary for the reaction can be produced by re-verse reactions
of NAD+-glyceraldehyde 3-phosphate dehydrogenase (NAD+-GAPD) inthe
cytosol using DHAP/GAP as a reductant,which can be supplied by
guard cell chloro-plasts via the triose phosphate translocator(TPT)
under light (Figure 4). DHAP/GAPis oxidized to 1,3-bisphosphate
glyceric acid(1,3-BPGA), and the ATP is generated by thereverse
reaction via phosphoglycerate kinase(PGAK) from 1,3-BPGA, with
simultaneousproduction of 3-PGA (Figure 4).
Carbon skeletons for malate formationare derived from starch in
the chloroplasts.Blue light induces starch degradation
andultimately provides Triose-P (DHAP/GAP)in the cytosol, and
Triose-P is finally con-verted to PEP. OAA is then produced
throughPEP carboxylase at the expense of PEP andHCO3− (189). It is
interesting to note that,in potato, transcript levels of
NAD+-GAPD,H+-ATPase, PEP carboxylase, and inward-rectifying K+
channel (KST1), all of which are
−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure
4Hypothetical scheme of provision of ATP and NADH from guard cell
chloroplasts forblue-light-dependent H+ pumping in guard cells.
Bold black arrows indicate high enzyme activitiesdemonstrated in
guard cells. When guard cells are illuminated with blue light, the
plasma membraneH+-ATPase is activated via phototropin signaling.
The activated H+-ATPase would consume ATP andinduce K+ uptake
through voltage-gated K+ channels. The accumulated K+ would promote
malatesynthesis to compensate the positive charges at the expense
of NADH, resulting in the regeneration ofATP and NADH. The
resultant 3-PGA would be transported from the cytosol to the
chloroplasts, thenphosphorylated and reduced to produce DHAP/GAP.
DHAP/GAP could be shuttled out fromchloroplasts to the cytosol in
guard cells, and the phosphorylation and reduction reactions in
guard cellchloroplasts would be facilitated under a strong red
light. 1,3-BPGA, 1,3-Bisphosphoglycerate; DHAP,Dihydroxyacetone
phosphate; FBP, Fructose1,6-bisphosphate; G-1-P, glucose
1-phosphate; GAP,Glyceraldehyde 3-phosphate; GAPD,
Glyceraldehyde-3-phosphate dehydrogenase; MDH, Malatedehydrogenase;
OAA, Oxaloacetate; PEP, phosphoenolpyruvate; PEPC,
phosphoenolpyruvatecarboxylase; PGAK, Phosphoglycerate kinase;
3-PGA, 3-phosphoglycerate; Rubisco,Ribulose-1,5-bisphosphate
carboxylase/oxygenase; TPI, Triose-phosphate isomerase.
234 Shimazaki et al.
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involved in stomatal opening, were downreg-ulated simultaneously
in a guard cell–specificmanner under drought (80).
Guard cell chloroplasts have suitable prop-erties to meet this
hypothesis (Figure 4). (a)A considerable proportion of reducing
equiv-
alents and ATP are available for reactionsother than
photosynthetic CO2 fixation (153);for example, only 8% of reducing
equivalentswere used for CO2 fixation in Vicia guard cells(50). (b)
The chloroplasts have enzyme activ-ities that favor the
accumulation of DHAP;
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activities of PGAK, NADP+-GAPD, and TPIin guard cell
chloroplasts were several to morethan 10 times higher than those of
mesophyllchloroplasts, but the activity of FBPase inguard cell
chloroplasts was roughly half of thatin mesophyll chloroplasts.
Such accumulationfavors the export of DHAP to the cytosol viathe
phosphate translocator (116, 159). (c) Veryhigh activities of
NAD+-GAPD and PGAKwere found in the guard cell cytosol (159).These
enzymes utilize DHAP and catalyze theproduction of NADH and ATP. (d
) Indirectexport of ATP, which was generated in guardcell
chloroplasts, to the cytosol was suggestedby an increase in H+ pump
activity under redlight (179). The H+ pump activation was
sen-sitive to DCMU. The ATP production canindicate simultaneous
production of NADHin the cytosol.
Therefore, when guard cells are illumi-nated with blue light
under background redlight, the activated plasma membrane H+-ATPase
utilizes ATP for H+ pumping, withsubsequent K+ uptake. This
situation requiresmalate formation, and results in the pro-duction
of NAD+, ADP, 3-PGA, and Pi inthe cytosol, and thereby stimulates
the ex-port of DHAP/GAP from the chloroplasts(Figure 4). A low but
significant Rubisco ac-tivity in guard cell chloroplasts (23, 127,
153,200) would generate 3-PGA under red light,and the 3-PGA would
act as an intermedi-ate for DHAP production. The enhancementof
malate formation is brought about by theactivation of the H+-ATPase
in guard cells(Figure 4). In accord with this idea, suppres-sion of
PEPC activity through inhibition ofH+-ATPase was reported (97).
Because blue light likely stimulates thetransfer of metabolites
between chloroplastsand cytosol (Figure 4), it may also affect
theenergy state of thylakoid membranes. In ac-cord with this idea,
two distinct energy statesof thylakoid membranes of guard cells
werereported. The chlorophyll fluorescence tran-sient in guard
cells evoked by blue light hadno prominent M peak (98, 155). Such
uniquekinetics of the fluorescence was transformed
into the standard kinetics of mesophyll cells,with a prominent M
peak, when guard cellswere excited by green light, which did not
ac-tivate blue-light signaling (94).
Under CO2-free conditions, where red-light enhancement of the
blue-light responseis still observed (70) despite the absence ofCO2
as a substrate for Rubisco, perhaps redlight enhances the
blue-light stimulation ofstarch breakdown. Red light promotes
starchbreakdown under low CO2 conditions (112),although whether
this red-light effect also op-erates in the presence of blue light
has yet tobe evaluated.
CONCLUDING REMARKS
Stomatal pores surrounded by a pair of guardcells are important
structures and allow up-take of CO2 for photosynthesis by
opening,and reduce the water loss from plants byclosing in response
to ever-changing envi-ronments, thereby extending the land
plantgrowth area. Guard cells are machineries thattransduce
external and internal signals intothe transport of various ions and
metabo-lites across the membranes, and adjust thepore size of
stomata. Thus, guard cells areexcellent model systems to
investigate themechanisms of perception and transduction ofsignals,
such as light, phytohormones, chem-icals, temperature, and
humidity, into stom-atal movement. In this review, we focusedon the
light signaling in guard cells and de-scribed the properties of the
plasma mem-brane H+-ATPase and those of blue-light re-ceptor
phototropins. The plasma membraneH+-ATPases are distributed in most
planttissues and drive the secondary transport ofnumerous kinds of
inorganic ions, organicacids, and sugars by coupling with tissue-or
organ-specific transporters. Because theplasma membrane H+-ATPase
is activated byblue light in guard cells, the regulatory mech-anism
of the H+-ATPase is elucidated underphysiological conditions, which
has been dif-ficult in other tissues. Phototropins (phot1phot2),
light receptor-type Ser/Thr protein
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kinases, are identified as blue-light recep-tors, and both phot1
and phot2 function forstomatal opening. Phototropins also
regulatedivergent responses, including phototropism,chloroplast
movement, leaf movement, andleaf flattening. Because the signaling
fromphototropins to immediate downstream com-ponent(s) remains
unknown in any of these re-sponses, identification of the kinase
substrateor the signaling component in guard cells willprovide
valuable information. Type 1 proteinphosphatase mediates the
signaling betweenphototropins and the plasma membrane H+-ATPase in
guard cells. Although type 1 pro-tein phosphatase regulates many
fundamentalprocesses in animal cells, the role of this phos-
phatase has not been demonstrated in plantcells. Because the
type 1 protein phosphataseworks together with a number of
regulatorysubunits, identification of the subunit func-tioning in
blue-light responses is importantand will provide new insight into
the role ofthe phosphatase in plant cells. Finally, stom-atal
opening is supported by metabolic activityunique to guard cells,
and malate2− formationis an important process required for the
open-ing. Reducing equivalents and carbon skele-tons needed for the
malate2− formation arelikely provided from guard cell
chloroplasts.Guard cell chloroplasts seem to adapt theirrole to an
organ-specific function of stomatalmovement.
SUMMARY POINTS
1. Stomatal opening is induced by light and is mediated by two
distinct photosystems:blue-light photosystems and chloroplasts.
Stomata open in response to a weak bluelight and the opening is
enhanced by background red light.
2. Blue light activates the plasma membrane H+-ATPase via the
phosphorylation of theC terminus and increases the inside-negative
electrical potential across the plasmamembrane in guard cells. The
potential drives K+ uptake through voltage-gatedK+ channels, and
the accumulated positive K+ charges are compensated mainly
bymalate2− formed in guard cells.
3. Red light induces stomatal opening at high intensity. Red
light likely mediates stomatalopening via reduction of the
intercellular concentration of CO2 (Ci ) by
mesophyllphotosynthesis, but the role of guard cell chloroplasts in
the response could not beexcluded.
4. Phototropins (phot1 phot2), light-responsive serine/threonine
protein kinases, areidentified as blue-light receptors for stomatal
opening, and possess flavin mononu-cleotides as chromophores.
Involvement of other photoreceptors in the light-inducedopening
response of stomata is reported.
5. The signaling between phototropins to the plasma membrane
H+-ATPase is largelyunknown. Results indicate that type 1 protein
phosphatase mediates this signaling.
6. Abscisic acid inhibits phototropin-mediated phosphorylation
of the plasma membraneH+-ATPase via H2O2, thereby suppressing
stomatal opening.
7. Enhancement of the blue-light response of stomata by red
light is brought aboutby guard cell chloroplast activity. The
chloroplasts likely provide ATP for the H+-ATPase and NADH for
malate2− formation in the cytosol through the translocationof
triose phosphate (DHAP/GAP) across the chloroplast envelope.
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ACKNOWLEDGMENTS
This work was supported by Grants-in-Aid for Scientific Research
(on Priority Areas, no.17084005 and A, no. 16207003) to K. S., and
by grants from the National Science Foundation(MCB02-09694) and the
US Department of Agriculture (2006-35100-17254) to S.M.A.
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