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Research
Red light activates a chloroplast-dependent ion uptake
mechanism for stomatal opening under reduced CO2
concentrations in Vicia spp.
Blackwell Science Ltd
Rebecca L. Olsen1, R. Brandon Pratt1,2, Piper Gump1, Andrea Kemper1 and Gary Tallman1
1
Department of Biology, Willamette University, 900 State Street, Salem, OR 97301, USA; 2Natural Science Division, Pepperdine University, 24255 Pacific
Coast Highway, Malibu, CA 90265, USA
Summary
Author for correspondence:
Gary Tallman
Tel: +1 503 370 6611
Fax: +1 503 375 5425
Email: [email protected]
Received: 30 July 2001
Accepted: 22 October 2001
• Under red light in ambient CO2 guard cells of faba bean (Vicia faba) fix CO2 and
accumulate sucrose, causing stomata to open. We examined whether at [CO2] low
enough to limit guard cell photosynthesis stomata would open when illuminated
with red (R) or far-red (FR) light.
• After illumination with R or FR in buffered KCl solutions, net stomatal opening
was c. 3 µm (R and FR) in air containing 210–225 µl l–1 CO2 and was 5 µm (R) or
6.5 µm (FR) in air containing 40–50 µl l–1 CO2. Opening was fully inhibited by 3(3,4-dichlorophenyl)-1,1 dimethyl urea, the calmodulin antagonist W-7, the ser/thr
kinase inhibitor ML-9, and sodium orthovanadate, but not by dithiothreitol, which
inhibits formation of zeaxanthin, the blue light photoreceptor of guard cells.
• Stomatal opening was accompanied by K+ uptake and starch loss. Similar results
were obtained when leaves were exposed to conditions designed to lower intercellular leaf [CO2].
• These data suggest that the guard cell chloroplasts transduce reduced [CO2],
activating stomatal opening through an ion uptake mechanism that depends on
chloroplastic photosynthetic electron transport and that shares downstream
components of the blue light signal transduction cascade.
Key words: CO2, guard cell chloroplasts, guard cells, red light transduction,
stomata.
© New Phytologist (2002) 153: 497–508
Introduction
Two environmental signals trigger stomatal opening in
intact, healthy leaves of C3 and C4 plants: increased light intensity (Sharkey & Ogawa, 1987) and decreased intercellular
concentrations of leaf CO2 (c i; Mott, 1988, 1990; Morison,
1998; Assmann, 1999). Experiments with detached leaf
epidermis and guard cell protoplasts (GCPs) have shown that
the guard cells that flank each stoma have intrinsic mechanisms that equip them to perceive and transduce these
signals into stomatal opening. The cellular mechanism(s) by
which guard cells translate lowered ci into stomatal opening
is/are not well understood. According to Assmann (1999)
we do not know with certainty: (1) how many mechanisms
guard cells may have for perceiving changes in CO2 concentra-
© New Phytologist (2002) 153: 497– 508 www.newphytologist.com
tions; (2) where in the guard cell such mechanism(s) is/are
located; (3) the components of any mechanism for CO2
perception; (4) whether the mechanisms for stomatal opening
under reduced CO2 concentrations are the same as those that
result in stomatal closure at increased CO2 concentrations; or
(5) how CO2-sensing mechanisms may be integrated either
with each other or with guard cell signal transduction
mechanisms for responding to light, abscisic acid, etc.
Complicating the analysis of CO2 signaling of stomatal
opening is the possible interaction of the guard cell CO2
response with light signaling mechanisms for opening. Among
the best studied photosensory signal transduction cascades
of guard cells is the one activated by low fluences of blue
light (photosynthetic photon fluence rate [PPFR] = 0.5–
50 µmol m–2 s–1; Zeiger et al., 1987; Zeiger, 2000). Blue
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498 Research
light-induced stomatal opening is observed when blue light
is applied over a background of red light to whole leaves,
isolated guard cells, or GCPs (Zeiger et al., 1987). The blue
light photoreceptor is zeaxanthin (Z) (Frechilla et al., 1999),
a carotenoid pigment formed when the thylakoid membrane
of the chloroplast is energized by light (Zeiger, 2000). Lightinduced acidification of the thylakoid lumen activates
violaxanthin (V) de-epoxidase, which catalyses the conversion
of V to Z (Zeiger & Zhu, 1998; Zeiger, 2000). By some
unknown mechanism that may be initiated by blue lightactivated cis–trans isomerization of Z (Zeiger, 2000), a signal
transduction cascade is triggered that involves calmodulin and
a ser/thr kinase (Shimazaki et al., 1992; Shimazaki et al.,
1993; Assmann & Shimazaki, 1999). The kinase catalyses
phosphorylation of the C-terminal end of a P-type H+translocating ATPase (proton pump) in the guard cell plasma
membrane (Kinoshita & Shimazaki, 1999). Phosphorylation
promotes binding of a guard cell-specific 14-3-3 protein to
the C-terminal end of the pump, stabilizing pump activation
(Emi et al., 2001). The resulting proton extrusion causes the
guard cell plasma membrane to hyperpolarize to voltages that
regulate opening and closing of a variety of ion channels to
favor influx of K+ and water (Grabov & Blatt, 1998), guard
cell turgor production, and stomatal opening.
Elevated CO2 reduces both guard cell Z content and lightinduced stomatal opening (Zhu et al., 1998); thus, it has been
suggested that guard cells use Z levels to integrate both blue
light and CO2 signals (Zeiger & Zhu, 1998; Zeiger, 2000). In
this model, Z levels would be expected to vary inversely with
rates of photosynthetic carbon fixation in guard cells. At lower
CO2 concentrations, photosynthetic carbon fixation would be
decreased, creating: (1) increased demand for Z to ‘de-energize’
the thylakoid membrane; (2) conditions in the thylakoid
lumen that would favor Z formation; and (3) increased guard
cell sensitivity to blue light (Zeiger, 2000). Elevated CO2 concentrations would favor increased photosynthetic carbon
fixation by guard cells, which would reduce acidification of
the thylakoid lumen, decrease conversion of V to Z and decrease
guard cell sensitivity to blue light (Zeiger, 2000). In this
model, CO2 would exert its action through the blue light
signal transduction pathway. An attractive feature of this
hypothesis is the idea that the chloroplast may be used to integrate light and CO2 responses of guard cells. It has long been
thought that guard cell chloroplasts are specialized for transducing the signals that trigger stomatal opening (Outlaw
et al., 1981), but other possible mechanisms involving Ca2+
(Webb et al., 1996) or malate (Hedrich & Marten, 1993)
have been proposed to explain the effects of CO2 on stomata
(for a review, see Assmann, 1999), and even stomatal opening
in darkness in response to reduced CO2 concentrations has
been documented (Dale, 1961; Mansfield & Heath, 1961).
One way to separate other effects of CO2 on stomatal
opening from those mediated by its interaction with the blue
light signal transduction mechanism would be to examine
CO2 responses under red light. In gas exchange studies by
Assmann (1988) in which intact leaves of Commelina communis
were illuminated with red light prior to administration of
low-fluence blue-light pulses, stomatal conductance was
c. 150 µmol m–2 s–1 at a red light fluence of only 263 µmol m–2 s–1.
This initial stomatal response to red light prior to blue light
treatment could be interpreted as a guard cell response to
reduced c i due to photosynthesis. Based on these studies, we
hypothesized that in epidermis detached from leaves of Vicia
faba, stomata illuminated with red light would open more
than those of dark controls under reduced concentrations of
CO2. Even though under red light GCPs can fix CO2 (Gotow
et al., 1988) and isolated guard cells can accumulate sucrose
(Poffenroth et al., 1992; Talbott & Zeiger, 1993), we reasoned
that any red light-induced opening at low concentrations of
CO2 would be unrelated to guard cell photosynthetic carbon
fixation and/or Z formation, because: (1) low CO2 concentrations would be expected to reduce photosynthetic rates; and
(2) Z is specific for blue light photoreception. Because chlorophyll is the only known red light photoreceptor of guard cells,
we hypothesized that any mechanism for opening observed at
reduced concentrations of CO2 under red light would be
located in the guard cell chloroplast and that it might be
sensitive to inhibitors of photosynthetic electron transport
such as 3-(3,4-dichlorophenyl)-1, dimethyl urea (DCMU).
Finally, we hypothesized that any such response should be
insensitive to dithiothreitol (DTT), a known inhibitor of V
de-epoxidase and Z formation (Srivastava & Zeiger, 1995). The
results of our studies indicate that the guard cell chloroplast
harbors at least one thylakoid-dependent, but Z-independent,
mechanism for sensing reduced concentrations of CO2.
Materials and Methods
Plants
Vicia faba L. (Loretta) was grown from seed ( Johnny’s Seed,
Albion, ME, USA) in a greenhouse on the campus of
Willamette University in Salem, OR, USA. Seeds (two per
6.3-l pot) were germinated and plants were grown in HP
Premier Pro-mix potting soil (Premier Horticulture Ltee,
Rivere-du-Loup, Quebec, Canada). Upon germination,
plants were watered every other day until the soil was
saturated. Plants were grown under natural daylight
supplemented with light from sodium vapor lamps (PPFR
of lamps = 100 ± 15 µmol m–2 s at the top of the canopy;
mean ± SE, n = 30) to give a 20-h daylength. Experiments
were conducted between May 15 and September 1 in 1997–
2000. Greenhouse temperature, PPFR and relative humidity
were measured daily at 12 : 00 h Pacific Standard Time (PST)
using a steady-state porometer (Model LI-1600, Li-Cor Inc.,
Lincoln, NB, USA) held at canopy height. Noon glasshouse
temperatures ranged from 19 to 26°C, relative humidity from
26 to 58%, and PPFR from 230 to 1780 µmol m–2 s–1.
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0.2
Epidermis
Experimental
In experiments with detached epidermis, peels were incubated
under red or far-red (FR) light or in darkness for up to 4 h in
solutions containing KCl bubbled with air containing various
concentrations of CO2. Potential antagonists or inhibitors of
stomatal opening were added to solutions. Peels were removed
from solutions periodically for measurement of stomatal
apertures and for histological analysis for K+ and starch in
guard cells. Dishes containing peels were incubated under
inverted plastic cups (4 cm diameter, 4 cm deep) that were
covered with foil and clamped down onto light filters or the
black surface of the laboratory bench. In light experiments,
light was delivered through filters to the bottom of each dish
by a 150 W halogen photo optic lamp (model EKE, Osram
Corp., Winchester, KY, USA) from a fiber optic illuminator
(Model 170 D, Dolan-Jenner Industries, Lawrence, MA,
USA). Although chlorophyll is the only known red light
photoreceptor of guard cells, because both chlorophylls and
phytochromes are red-light photoreceptors, experiments were
performed with light of near-red (R) wavelengths or with light
of FR wavelengths (Fig. 1). Red light (620–900 nm with
c. 50% of energy output between 650 nm and 680 nm; Fig. 1a)
was produced with a Kodak 1 A safelight filter. Far-red light
(700–900 nm with 98.5% of energy output between these
wavelengths; Fig. 1b) was produced using black Plexiglas
(Rohm & Haas, Philadelphia, PA, USA). Most experiments with
red light were performed at PPFRs of 125–150 µmol m–2 s–1.
In all experiments, PPFR was measured with a LiCor
quantum/radiometer/photometer (LiCor, Lincoln, NB,
USA). To achieve similar photon fluence rates in FR as in R,
spectral energy transmittance profiles of R and FR filters were
measured with a spectroradiometer (Optronic Laboratories
model OL 754, Orlando, FL, USA) calibrated to a standard
bulb (Model 752–10, National Institute of Standards and
Technology, Optronics Laboratories) using the lamp
described at the same PPFR for each measurement. Because
© New Phytologist (2002) 153: 497– 508 www.newphytologist.com
0.15
0.1
Energy (W m–2 nm–1)
At 17 : 00 h on the day prior to each experiment, plants were
watered until the soil was saturated and then placed in a
darkened cabinet. At 10 : 00 h the next day, leaves from 3- to
5-wk-old plants were removed from insertion levels 3 or 4
above the soil surface and were transported to the laboratory
in moist towels in a plastic bag. Epidermis was detached
(‘peeled’) from between major veins on abaxial leaf surfaces
using forceps. To minimize contamination with mesophyll
tissue and prevent dehydration, peeling was performed under
water at a 180° angle to the leaf surface (Weyers & Travis,
1981). Peels were trimmed to c. 0.5 × 0.5 cm squares and
transferred to 10 ml glass dishes (20–25 per dish) containing
5 ml of 5 mM KCl, 5 mM (2-[N-morpholino] ethanesolfonic
acid (Mes), pH 6.5.
(a)
0.05
0
0.04
(b)
0.03
0.02
0.01
0
400
500
600
700
800
Wavelength (nm)
Fig. 1 Spectral profiles of filters used to illuminate stomata in
epidermis detached from leaves of Vicia faba L. and subjected to
reduced concentrations of CO2, as described in the Materials and
Methods section. (a) Full data for both filters (open circles, red;
crosses, far red); (b) reduced scale to shown fine features of the
spectral profile of the far-red filter.
the quantum sensor measured wavelengths between 400
and 700 nm only, equivalent fluence rates for R and FR
wavelengths were calculated by dividing the integrated area
between 653 nm and 680 nm on the FR profile into the
integrated area between 712 nm and 740 nm on the same
profile. Using the inverse of this ratio (67.5) as a multiplier, a
PPFR of 2 µmol m–2 s–1 through the FR filter was calculated to
be equivalent to 135 µmol photons m–2 s–1 in the 712–740 nm
waveband. Air containing reduced concentrations of CO2
(c. 30–50 µl l–1) was produced by bubbling line air (400–
470 µl l–1 CO2) through two flasks connected in series, each
containing 400 ml of saturated NaOH. To produce air with
210–225 µl l–1 CO2, line air and air coming from flasks
was mixed in a downstream 1-l flask before it was delivered
to incubation cups. Air was bubbled through incubation
solutions at 6.7 ml min–1 using hypodermic needles (26
gauge) inserted through the tops of the cups and connected to
air lines. To prevent positive pressure in cups, two 5-mm holes
were drilled at 180° to each other, centered 1 cm beneath the
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500 Research
rim at the open end of each cup. The same holes were used
to withdraw air during experiments for monitoring CO2
concentrations. Each day, CO2 concentrations were recorded
every minute in one of four available chambers using an
EGM-1 Environmental Gas Monitor (PP Systems, Hitchin,
Herts, UK). Measurements of CO2 concentration were
rotated daily among the four chambers. Epidermal peels were
maintained on the surface of the liquid by the aeration
protocol and thus were not submerged in solutions for any
significant length of time (< 1 s).
Stomatal apertures were measured periodically with a
microscope equipped with a digital video camera and monitor
(Tallman & Zeiger, 1988). Light from the microscope illuminator was filtered through a green filter to minimize changes in
aperture that might be caused by illumination during
measurements. Means and standard errors of sample means
were calculated using STATVIEW 4.01 (Abacus Concepts,
Berkeley, CA, USA).
All experimental solutions were made by adding compounds to the incubation solution described above. All chemicals were from Sigma (St Louis, MO, USA). Potassium ion
uptake by guard cells was evaluated with sodium cobaltinnitrite staining as described by Green et al. (1990). The starch
content in guard cell chloroplasts was evaluated with iodine–
potassium iodide (IKI) stain (Tallman & Zeiger, 1988). To
determine whether stomatal opening observed at lower CO2
concentrations in epidermis illuminated with red or FR light
was dependent on photosynthetic electron transport in guard
cell chloroplasts, peels were preincubated in darkness for 1 h
in solutions containing or lacking 10 µM DCMU before light
treatments were administered. To evaluate whether Z was
required for the response to lowered CO2 concentration, peels
were incubated in solutions containing or lacking 5 mM DTT.
To test whether stomatal opening at lower CO2 concentrations required activation of the guard cell plasma membrane
proton pump, peels were illuminated in solutions containing or
lacking 10 mM sodium orthovanadate (Schwartz et al., 1991).
Calmodulin and a ser/thr kinase have been implicated as
downstream components of the blue light signal transduction
pathway (Zeiger, 2000). To assess whether these proteins
might be involved in a stomatal response to lower CO2 concentrations under red light, peels were incubated in solutions
containing: (1) 1 or 10 µM W-7 (N-(6-aminohexyl)-5-chloro1-napthalenesulfonamide), an antagonist of calmodulin and
calmodulin-dependent kinases; (b) 200 or 500 µM H-7 (1-(5isoquinolinesulfonyl)-2-methyl-piperazine), an inhibitor of
protein kinase C; or (3) 50 or 100 µM ML-9 (1-(5-chloronapthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine), a specific
inhibitor of mammalian myosin light chain kinases and, at
higher concentrations, protein kinases A and C.
To determine whether a stomatal CO2 response detected in
detached epidermis under red light might also occur in intact
leaves, individual leaflets were detached from dark-adapted
plants, submerged in 60 ml of deionized water and illumin-
ated from the adaxial surface for 1.5 h with 440 µmol m–2 s–1
of red light. This protocol is thought to reduce c i to low levels
as leaf photosynthesis is limited by (1) the low concentration
of CO2 dissolved in water relative to that required to saturate
photosynthesis; and (2) the low diffusibility of CO2 in water
vs air. Similar methods have been described elsewhere
(Weyers & Meidner, 1990). The time employed and PPFR
were sufficient to achieve maximum stomatal opening on the
abaxial leaf surface (not shown). After incubation, abaxial
epidermis was detached from leaves, apertures were examined
and guard cells were stained for K+, as described earlier in this
section.
Results
The time course of stomatal opening under R or FR light at
three different concentrations of CO2 is summarized in
Fig. 2. Over 4 h at CO2 concentrations of 400–450 µl l–1
(Fig. 2a,b), stomata did not open under R or FR light.
Stomata apertures reached their peak after 2 h of illumination
with R or FR light in air containing CO2 concentrations of
210–225 µl l–1 (Fig. 2c,d); net opening was c. 3 µm (Fig. 2c).
Under R light, net opening was c. 5 µm after 4 h at CO2
concentrations of 40–50 µl l–1 (Fig. 2e,f ); net opening under
FR after 4 h was 6.5 µm (Fig. 2e,f). Small increases in
aperture (c. 0.5–1 µm) were detected after 4 h in darkness in
air containing 210–225 µl l–1 CO2 (Fig. 2c) and 40–50 µl l–1
CO2 (Fig. 2e). No uptake of K+ by guard cells was detected
in epidermis incubated for 4 h under R light in air containing
450 µl l–1 CO2 (Fig. 3A). Substantial K+ uptake was observed
after 4 h of illumination with R light in air containing
260 µl l–1 CO2 (Fig. 3b). The greatest amounts of K+
accumulated after 4 h of illumination with R light in air
containing 20 µl l–1 CO2 (Fig. 3c). Small amounts of K+ were
detected in guard cells in epidermis incubated for 4 h in
darkness in air containing 20 µl l–1 CO2 (Fig. 3d). Results
under FR were similar (not shown). After 3 h of illumination
with R light in air containing 50 µl l–1 CO2, starch was
reduced in guard cell chloroplasts (Fig. 4b) over that of guard
cell chloroplasts of unincubated guard cells (Fig. 4a) or guard
cells treated with air containing 50 µl l–1 CO2 for 3 h in
darkness (Fig. 4c).
After 4 h of illumination under R light in air containing
50 µl l–1 CO2, mean net stomatal opening reached 4.5 µm
and 4.7 µm at PPFRs of 150 µmol m–2 s–1 and 200 µmol m–2 s–1,
respectively (Fig. 5). In air containing 30–50 µl l–1 CO2,
stomatal opening under 150 µmol m–2 s–1 of R or FR light was
fully inhibited by 10 µM DCMU (Fig. 6). Under similar
conditions, neither R- or FR-induced opening was inhibited
by 5 mM DTT (Fig. 7).
When epidermis was illuminated with R light
(150 µmol m–2 s–1) in air containing 30–50 µl l–1 CO2, stomatal opening was not inhibited by H-7 in concentrations as
high as 500 µM (Fig. 8a). Opening was fully inhibited by
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Stomatal
Aperture (µ m)
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10 (a)
9
8
7
6
5
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(c)
(e)
500 (b)
(d)
(f)
400
300
200
100
0
0
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120
180
240 0
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120
180
Time (min)
240 0
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120
180
240
Fig. 2 Mean stomatal apertures (a,c,e) in epidermis detached from leaves of Vicia faba L. incubated for up to 4 h in darkness (filled squares)
or under 125 µmol m–2 s–1 of light enriched in red (open circles) or far-red (closed triangles) wavelengths. Epidermis was incubated in 5 m M KCl,
5 mM Mes, pH 6.5. Each value is the mean of 90 measurements, 30 on each of three separate days from 10 measurements on each of three
epidermal peels each day. Values are means ± SE of the sample mean. If no error bar is visible it is contained within the width of the symbol.
Below each panel is a corresponding panel (b,d,f) that contains three digital records of CO2 concentrations in air surrounding epidermal peels
over the 4-h time-course of each experiment. On each of the three separate days (closed squares, day 1, dark; closed circles, day 2, red; open
circles, day 3, far red) that apertures were measured, an infrared gas analyser was used to measure and record CO 2 concentrations at 1-min
intervals in one of the three experimental chambers in which peels were being incubated.
Fig. 3 Potassium ion uptake by guard cells in
epidermis detached from leaves of Vicia faba
L. and incubated for 4 h in 5 mM KCl, 5 mM
Mes, pH 6.5, under 125 µmol m–2 s–1 of red
light in air containing (a) 450 µl l–1 CO2,
(b) 260 µl l–1 CO2 or (c) 20 µl l–1 CO2. A dark
control treated for 4 h in 20 µl l–1 CO2 is
shown in (d). Potassium ion was detected with
sodium hexanitrocobaltate III, which closes
stomata through its effects as a fixative and
an osmoticum. Original magnification, ×200;
bar (a), 100 µm.
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Fig. 4 Starch in chloroplasts of guard cells in epidermis detached from leaves of Vicia faba L. and not treated (a) or incubated for 3 h in 5 mM
KCl, 5 mM Mes, pH 6.5, under 125 µmol m–2 s–1 of red light (b) or in darkness (c) in air containing 50 µl l–1 CO2. Starch was stained with iodine–
potassium iodide, which is toxic to guard cells and causes them to slowly lose turgor. Original magnification ×600; bar (a), 50 µm.
5
Discussion
Mean net stomatal
opening (µ m)
4
3
2
1
0
0
50
100
150
200
250
PPFR (µ mol m–2 s–1)
Fig. 5 Mean net stomatal opening (difference between mean
stomatal aperture in light and mean stomatal aperture in darkness) in
epidermis detached from leaves of Vicia faba L. and incubated for
3 h in 5 mM KCl, 5 mM Mes, pH 6.5 at photosynthetic photon fluence
rates (PPFRs) of red light ranging from 10 to 200 µmol m–2 s–1.
Means for subtraction were obtained from 90 measurements, 30 on
each of three separate days from 10 measurements on each of three
epidermal peels each day; y = 2.962 log(x) – 2.652, r = 0.890.
concentrations of ML-9 as low as 50 µM (Fig. 8B) and by
concentrations of W-7 as low as 10 µM (Fig. 8C). Under
similar conditions, opening was also inhibited by 10 mM
sodium orthovanadate (Fig. 9).
When intact leaves were submerged in water and illuminated from the adaxial surface for 1.5 h with R light (PPFR
= 440 µmol m–2 s–1), guard cells on the abaxial leaf surface
accumulated K+ (Fig. 10a) and stomata opened (Fig. 10b).
The data reported here indicate that in addition to activating
the Calvin cycle (Gotow et al., 1988), sucrose accumulation
(Poffenroth et al., 1992; Talbott & Zeiger, 1993) and Z
formation in guard cells (Zeiger, 2000), red light can also
activate a K+ uptake mechanism for stomatal opening under
reduced concentrations of CO2 (Figs 2 and 3). Like the blue
light response of guard cells, the red light-activated CO2
response also triggers starch degradation in guard cell chloroplasts
(Tallman & Zeiger, 1988) (Fig. 4). Stomatal opening and K+
uptake occur in inverse proportion to CO2 concentration
(Figs 2 and 3). The response saturates at a PPFR of 150–
200 µmol m–2 s–1 (Fig. 5), a light level similar to that on the
abaxial surface of a leaf in full sun (Yera et al., 1986). The
observation that the response is similar in R and FR light
(Fig. 2) and that it is inhibited equally in R and FR light by
pretreatment of guard cells with DCMU (Fig. 6) indicates
that: (1) the sensory transduction mechanism for the response
is localized in the guard cell chloroplast; (2) the response is
dependent on chloroplast electron transport; and (3) the
photoreceptor is chlorophyll. The ratio of FR to R through
the R filter was 1.1 : 1 (Fig. 1a) and the ratio of FR to R ratio
through the FR filter was 67.5 : 1 (Fig. 1b). Despite the large
differences in FR to R ratios between the two filters, opening
responses in R and FR were similar (Figs 2, 6, 7 and 9),
suggesting that phytochrome does not act as a photoreceptor
in the response. The PPFR of wavelengths between 680 nm
and 700 nm was only 2–3 µmol m–2 s–1 through the FR filter
at a FR PPFR of 125–150 µmol m–2 s–1 (Fig. 1b). The observation
that opening responses were similar under FR and R treatments
(Figs 2, 6, 7 and 9) may suggest (1) that a primary signaling
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Stomatal
aperture (µ m)
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10
9
8
7
6
5
4
3
2
1
0
500
400
300
200
100
0
0
60
120
180
240 0
60
120
180
240
Time (min)
Fig. 6 Effects of 10 µM DCMU on mean stomatal apertures in epidermis detached from leaves of Vicia faba L. Epidermal peels were incubated
for 1 h in darkness and then up to 4 h in darkness or under 125 µmol m–2 s–1 of light enriched in red (left panels) or far-red (right panels)
wavelengths in air containing 30– 50 µl l–1 CO2. Epidermis was incubated in 5 mM KCl, 5 mM Mes, pH 6.5, ± DCMU (crossed circle,
light + DCMU; open circle, light – DCMU; crossed square, dark + DCMU; filled square, dark – DCMU). Each value is the mean of 90
measurements, 30 on each of three separate days from 10 measurements on each of three epidermal peels each day. Values are means ± SE of
the sample mean. If no error bar is visible it is contained within the width of the symbol. Below each of the top panels is a corresponding panel
that contains three digital records of CO2 concentrations in air surrounding epidermal peels over the 4-h time-course of each experiment. Left
panel: open circle, day 1; filled square, day 2; open circle, day 3. Right panel: filled circle, day 1; crossed square, day 2; crossed circle, day 3. On
each of the three separate days that apertures were measured, an infrared gas analyser was used to measure and record CO 2 concentrations
at 1-min intervals in one of the four experimental chambers in which peels were being incubated.
event in photosystem II (PSII) is highly amplified and /or (2)
that photosystem I (PSI) and its light-harvesting complex play
a significant role in the signal transduction cascade. If PSI
plays such a role, it is probably not through cyclic electron flow,
since the response under FR is inhibited by DCMU (Fig. 6).
Unlike the blue light response of guard cells, the red lightactivated CO2 response is not dependent on Z formation in
guard cell chloroplasts, as shown by failure of 5 mM DTT to
inhibit opening ( Fig. 7). The observation that DTT inhibits
blue light-induced opening but not the red light-induced
CO2 response, even though a number of components of the
signal transduction pathway are shared with those of the blue
light response pathway (see below), supports the arguments
that: (1) DTT is a very specific inhibitor of Z formation in
guard cells (Srivastava & Zeiger, 1995); and (2) that Z is the
blue light photoreceptor of guard cells responsible for
© New Phytologist (2002) 153: 497– 508 www.newphytologist.com
stomatal opening (Frechilla et al., 1999). As with the blue
light response, the red light-activated CO2 response depends
on activation of the guard cell plasma membrane proton
pump (Fig. 9). In patch clamp studies using GCPs of Vicia
faba, Serrano et al. (1988) reported that red light activated the
plasma membrane proton pump. It seems plausible that in
that study, experimental conditions may have elicited a
response similar to the one observed in our experiments. A
recent study with red light suggests that guard cell chloroplasts
can supply ATP to the plasma membrane H+ pump in guard
cells in which the pump has been activated by fusicoccin
(Tominaga et al., 2001). However, when patch-clamped
GCPs of Vicia faba were treated with DCMU to inhibit red
light-induced proton pumping, pumping was not restored by
addition of ATP to the patch pipette (Serrano et al., 1988). In
our experiments with FR filters, the pump was activated at
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Fig. 7 Effects of 5 mM dithiothreitol (DTT) on mean stomatal apertures in epidermis detached from leaves of Vicia faba L. Epidermal peels were
incubated for up to 4 h in darkness or under 125 µmol m–2 s–1 of light enriched in red (left panels) or far-red (right panels) wavelengths in air
containing 30– 50 µl l–1 CO2. Epidermis was incubated in 5 mM KCl, 5 mM Mes, pH 6.5, ± DT T (crossed circle, light + DT T; open circle, light –
DT T; crossed square, dark + DTT; filled square, dark – DT T). Each value is the mean of 90 measurements, 30 on each of three separate days
from 10 measurements on each of three epidermal peels each day. Values are means ± SE of the sample mean. If no error bar is visible it is
contained within the width of the symbol. Below each of the top panels is a corresponding panel that contains three digital records of CO 2
concentrations in air surrounding epidermal peels over the 4-h time-course of each experiment. Left panel: open circle, day 1; filled square, day
2; open circle, day 3. Right panel: filled square, day 1; open circle, day 2; filled square, day 3. On each of the three separate days that apertures
were measured, an infrared gas analyser was used to measure and record CO2 concentrations at 1-min intervals in one of the four experimental
chambers in which peels were being incubated.
PPFR of red light too low to drive large amounts of ATP synthesis (2–3 µmol m–2 s–1), suggesting that amplification of
primary light/CO2 signals through a signal transduction cascade
is required for the response. Indeed, the red light-activated
CO2 response appears to share some downstream components
of the blue light signal transduction pathway that lie between
the chloroplast and the plasma membrane. Similar to the blue
light response, the red light-activated CO2 response was not
inhibited by H-7 (Fig. 8a), an inhibitor of protein kinase C
(Assman & Shimazaki, 1999); but was inhibited by ML-9
(Fig. 8b), which in the relatively high concentrations used,
inhibits ser/thr kinases (Assmann & Shimazaki, 1999), and
by W-7 (Fig. 8c), a calmodulin antagonist and inhibitor of
calmodulin-dependent protein kinases (Shimazaki et al., 1992;
Shimazaki et al., 1993; Assmann & Shimazaki, 1999). Results
of experiments with whole leaflets submerged in water and
illuminated with red light to reduce the CO2 concentrations
around guard cells (Fig. 10) suggest that guard cells possess
this mechanism in intact leaves and that it is not an artifact
created by detachment of epidermis. Such a mechanism could
explain why in intact leaves of Commelina communis, Assmann
(1988) observed stomatal conductances of up to 150 mmol
m–2 s–1 at relatively low red light fluences.
It should be noted that a small amount of stomatal opening
was observed in darkness at lower CO2 concentrations
(Fig. 2c,e) and K+ uptake was detected in guard cells incubated in darkness at very low CO2 concentrations (Fig. 3d).
Whether this represents (1) a light-independent CO2
response; or (2) an effect of lower CO2 concentrations operating unamplified through the components of the red lightactivated signal transduction pathway described earlier in this
discussion awaits investigation.
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(b)
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Fig. 8 Effects of various concentrations of H-7 (a), ML-9 (b), or W-7
(c) on mean stomatal apertures in epidermis detached from leaves of
Vicia faba L. Epidermal peels were incubated for up to 4 h in darkness
or under 125 µmol m–2 s–1 of light enriched in red wavelengths in air
containing 30– 50 µl l–1 CO2. Epidermis was incubated in 5 mM KCl,
5 mM Mes, pH 6.5, ± H-7 (a: open circle, no treatment; crossed circle,
200 µM H-7; crossed diamond, 500 µM H-7; filled square, dark),
ML-9 (b: open circle, no treatment; crossed circle, 50 µM ML-9;
crossed diamond, 100 µM ML-9; filled square, dark) or W-7 (c: open
circle, no treatment; crossed circle, 1 µM W-7; crossed diamond,
10 µM W-7; filled square, dark). Each value is the mean of 90
measurements, 30 on each of three separate days from 10
measurements on each of three epidermal peels each day. Values are
means ± SE of the sample mean. If no error bar is visible it is contained
within the width of the symbol. On each of the three separate days
that apertures were measured, an infrared gas analyser was used to
measure and record CO2 concentrations at 1-min intervals in one of
the four experimental chambers in which peels were being incubated
(not shown).
© New Phytologist (2002) 153: 497– 508 www.newphytologist.com
To our knowledge, this is the first direct evidence that red
light can trigger K+ uptake by guard cells. The response
requires reduced CO2 concentrations and localizes to the
guard cell chloroplast. It is striking that all mechanisms
described to date for light- or CO2-induced stomatal opening
are harbored in the guard cell chloroplast.
Results of experiments with GCPs from Vicia faba
illuminated with red light demonstrate that guard cells can fix
CO2 into intermediates of the Calvin cycle, including phosphoglyceric acid, sugar monophosphates, sugar diphosphates
and triose phosphates (Gotow et al., 1988). Neither the
amount of Rubisco activity in guard cell extracts of Pisum
(Reckmann et al., 1990) nor in intact leaves of Vicia faba
grown at lower relative humidity (60%; Lu et al., 1997) were
sufficient to produce photosynthetic intermediates at rates
likely to support the magnitude of stomatal opening reported
for those species. Nevertheless, an immunohistochemical
assay of guard cells of 41 species showed that a majority contained Rubisco (Madhavan & Smith, 1982). Based on recent
results, Outlaw and De Vlieghere-He (2001) proposed that
accumulation in the guard cell apoplast of sucrose produced
by the mesophyll under conditions of high vapor pressure
differences between leaves and the air (VPD) (and hence, high
rates of transpiration) may downregulate transcription of
guard cell genes for Rubisco and other enzymes involved in
carbon fixation and metabolism. Were this hypothesis to be
supported experimentally, it could explain: (1) the wide
variability in the amount of guard cell Rubisco observed
among species in immunohistochemical studies (Madhavan
& Smith, 1982); and (2) why some investigators detected
Calvin cycle activity in guard cells of Vicia faba using plants
grown under conditions that would be expected to produce
lower VPD (Gotow et al., 1988), while others using plants
grown under conditions expected to produce higher VPD did
not (Lu et al., 1997). This hypothesis is also consistent with
the suggestion of Zeiger that Calvin cycle activity in guard
cells may modulate the blue light response (Zeiger & Zhu,
1998; Zeiger, 2000). When greenhouse-grown plants (higher
VPD) were moved to an environmental chamber (lower
VPD) the sensitivity of their stomata to decreased concentrations of CO2 increased dramatically (Talbott et al., 1996). If
low VPD = high guard cell Rubisco activity (Outlaw & De
Vlieghere-He, 2001), as proposed by Zeiger and colleagues
(Zeiger & Zhu, 1998; Zeiger, 2000) and observed by Talbott
et al. (1996), illuminated guard cells of chamber-grown plants
would be expected to accumulate more Z and become more
sensitive to blue light than those of greenhouse-grown plants
at the same PPFR. The sucrose regulation hypothesis could
also explain why, in the experiments reported here, stomata
illuminated with red light at higher CO2 concentrations did
not open (Fig. 2a) by producing sugars photosynthetically,
since temperature and humidity in the summer months in
the greenhouse would have been expected to produce higher
VPD.
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Fig. 9 Effects of 10 mM sodium orthovanadate on mean stomatal apertures in epidermis detached from leaves of Vicia faba L. Epidermal peels
were incubated for up to 4 h in darkness or under 125 µmol m–2 s–1 of light enriched in red (left panels) or far-red (right panels) wavelengths
in air containing 30– 50 µl l–1 CO2. Epidermis was incubated in 5 mM KCl, 5 mM Mes, pH 6.5, ± orthovanadate (crossed circle, light + vanadate;
open circle, light – vanadate; crossed square, dark + vanadate; filled square, dark – vanadate). Each value is the mean of 90 measurements, 30
on each of three separate days from 10 measurements on each of three epidermal peels each day. Values are means ± SE of the sample mean.
If no error bar is visible it is contained within the width of the symbol. Below each of the top panels is a corresponding panel that contains three
digital records of CO2 concentrations in air surrounding epidermal peels over the 4-h time-course of each experiment. Left panel: open circle,
day 1; filled square, day 2; open circle, day 3. Right panel: open circle, day 1; filled square, day 2; filled circle, day 3. On each of the three separate
days that apertures were measured, an infrared gas analyser was used to measure and record CO 2 concentrations at 1-min intervals in one of
the four experimental chambers in which peels were being incubated.
Another chloroplast mechanism for stomatal opening is
accumulation of sucrose in guard cells of V. faba under red
light by a DCMU-sensitive mechanism (Poffenroth et al.,
1992; Talbott & Zeiger, 1993). Calvin cycle activity is one
potential source of that sucrose. Sucrose could also accumulate from red light-induced starch breakdown in guard cell
chloroplasts, but no starch breakdown (judged by IKI staining) was evident during red light-induced stomatal opening
in air containing ambient concentrations of CO2, while starch
breakdown was detected in concurrent experiments with blue
light (Tallman & Zeiger, 1988). It is, of course, possible that
a concurrent Calvin cycle activity and starch degradation
would be futile insofar as it would lead to no net change in the
amount of starch in guard cell chloroplasts illuminated with
red light. Another possible source of sucrose in intact leaves
is the guard cell apoplast (Lu et al., 1997). The GCPs of
P. sativum do have sufficient sucrose transport capacity to
support stomatal opening at rates described in the literature
(Ritte et al., 1999), but no direct measurements of transport
of photosynthetically produced sucrose from guard cell apoplast to symplast have been made in any species.
Finally, the guard cell chloroplast is the site of production
of Z, the only pigment identified as a blue light photoreceptor
for triggering the blue light photosensory signal transduction
cascade of guard cells. In early experiments with low fluences
of blue light (Zeiger et al., 1987; Assmann, 1988), red light
was used to saturate any photosynthetic responses of guard
cells so that the blue light response could be evaluated independently of its potential effects on guard cell photosynthesis
(Zeiger et al., 1987). It was discovered that pre-illumination
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Fig. 10 Accumulation of potassium ions in guard cells in intact leaves
of Vicia faba L. submerged in water and illuminated from the adaxial
surface with 440 µmol m–2 s–1 of red light for 1.5 h. At the end of the
1.5-h illumination, epidermis was peeled from the abaxial leaf surface
and stained with sodium hexanitrocobaltate III (a) or examined with
a microscope for stomatal opening (b). The hexanitrocobaltate stain
closes stomata through its effects as a fixative and an osmoticum.
Magnification, ×300; bars, 50 µm. Results are representative of those
from three separate experiments.
with red light was required before a blue light response could
be measured. It has since been determined that the photosynthetic electron transport that results from red light illumination drives formation of Z as light-driven acidification of the
thylakoid lumen activates the de-epoxidase enzyme that
converts V to Z (Zeiger & Zhu, 1998).
In most species, the presence of chloroplasts in guard cells
is the most visible feature that differentiates them from neighboring epidermal cells. A wealth of published data now point
to the central role of the guard cell chloroplast and to the
importance of photosynthetic electron transport in regulating
stomatal responses to light and CO2. Their importance to
guard cell signal transduction may explain why guard cell
chloroplasts fail to senesce even when mesophyll cells in the
same leaf have yellowed completely (Zeiger & Schwartz,
1982; Ozuna et al., 1985). In addition to its role as an environmental sensor (Outlaw et al., 1981), emerging data point
to the guard cell chloroplast as an evolutionary repository specialized for integrated signal transduction to regulate stomatal
aperture in a variety of developmental, environmental and
physiological circumstances (Zhu et al., 1995; Quinones
et al., 1996).
While it is only possible to speculate as to why evolution
has preserved a multiplicity of mechanisms for triggering
and/or regulating stomatal opening, it is obvious that guard cells
are subject both to the developmental programs of the plants
in which they exist and to an environment that fluctuates
dramatically and that has the potential to regulate stomatal
development (Lake et al., 2001). As plants germinate, grow to
maturity, and then flower, fruit and set seed, guard cells move
through their own developmental programs, first as compo-
© New Phytologist (2002) 153: 497– 508 www.newphytologist.com
nents of young leaves, next as elements of expanding leaves
and then as constituents of senescing leaves. They may be
located near or far from a vein, on the top or the bottom
surface of a leaf, near the top or the bottom of the canopy, near
to or far from other various organs on the plant. Their proximity to light, temperature, nutritional, hormonal and water
potential gradients change with plant growth, leaf age and
position in the canopy and with fluctuations in the environment that include length of the photoperiod, humidity, soil
moisture, temperature, etc. Whether, at any point in its developmental history, each and every guard cell or population of
guard cells possesses each and every mechanism for opening
that has been discovered to date is unknown, but published
data suggest otherwise. Diurnal changes in guard cell physiology
(Dodge et al., 1992; Talbott & Zeiger, 1998), variations in
responses related to growth conditions (Talbott et al., 1996;
Outlaw & De Vlieghere-He, 2001), species differences in
guard cell biochemistry (Madhavan & Smith, 1982), and seasonal variations in guard cell responses (Lohse & Hedrich,
1992) are now well documented. Indeed, the experiments
described here were performed over 4 years from mid-May
to 1 September of each year because in other months of the
year, using greenhouse-grown plants, large dark-opening of
stomata was observed at lower concentrations of CO2 (not
shown). Staining revealed that K+ uptake was not the basis for
dark movements and guard cells illuminated with red light at
lower concentrations of CO2 still accumulated K+ in large
amounts. These types of variations reinforce the notion that
guard cells are dynamic by nature and that they can change
character dramatically over even relatively short periods of time.
If so, then any individual experiment may simply provide a
picture of how a guard cell conditioned by its environmental
history responds to experimental protocols that are imposed on
that history. In future, many careful studies under rigorously
controlled conditions will probably be needed if we are to define
how the developmental history of the guard cell shapes it
response capacity for various signals, and how it is shaped for
the future by the conditions to which it is currently exposed.
Acknowledgements
We thank E. Zeiger and L. Talbott for helpful comments
on the manuscript and S. D. Davis and Jeffrey Warren for
assistance with spectroradiometry. Supported in part by NSF
MCB-9900525 to G.T.
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