Plant, Cell and Environment (1998) 21, 813–820
ORIGINAL ARTICLE
OA
220
EN
The stomatal response to CO2 is linked to changes in guard
cell zeaxanthin*
J. ZHU, L. D. TALBOTT, X. JIN & E. ZEIGER
Department of Biology, University of California, Los Angeles, CA 90095–1606, USA
ABSTRACT
The mechanisms mediating CO2 sensing and light–CO2
interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light
(500 µmol m–2 s–1) and temperature, guard cell zeaxanthin
content tracked ambient [CO2] and stomatal apertures.
Increases in [CO2] from 400 to 1200 cm3 m–3 decreased
zeaxanthin content from 180 to 80 mmol mol–1 Chl and
decreased stomatal apertures by 7·0 µm. Changes in zeaxanthin and aperture were reversed when [CO2] was lowered. Guard cell zeaxanthin content was linearly
correlated with stomatal apertures. In the dark, the CO2induced changes in stomatal aperture were much smaller,
and guard cell zeaxanthin content did not change with
chamber [CO2]. Guard cell zeaxanthin also tracked [CO2]
and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin
formation, eliminated CO2-induced zeaxanthin changes in
guard cells from illuminated epidermal peels and reduced
the stomatal CO2 response to the level observed in the
dark. These data suggest that CO2-dependent changes in
the zeaxanthin content of guard cells could modulate CO2dependent changes of stomatal apertures in the light while
a zeaxanthin-independent CO2 sensing mechanism would
modulate the CO2 response in the dark.
Key-words: Vicia faba; stomatal response to CO2; xanthophyll
cycle; zeaxanthin.
INTRODUCTION
Stomata have been shown to respond to changes in [CO2]
in over 50 species (Morison 1987). In general, increases in
intercellular [CO2] (Ci) reduce stomatal apertures and
decreases in Ci lead to stomatal opening. These responses
are optimally suited to couple photosynthetic activity in the
mesophyll and stomatal conductance in the epidermis
(Mott 1990).
Interactions between the stomatal response to CO2 and
other signals such as light, relative humidity, water stress,
Correspondence: Dr Eduardo Zeiger. Fax (310) 825–9433; e-mail:
[email protected]
*This work was supported by NSF #DCB 8904254 and DOE
#90ER20011.
© 1998 Blackwell Science Ltd
temperature, leaf age and nutrients have been extensively
studied (Morison 1987). In the intact leaf, the stomatal
responses to CO2 and light are functionally coupled and
hard to separate experimentally. It is well established, however, that guard cells have independent responses to light
and CO2 (Sharkey & Raschke 1981; Zeiger 1983), that
guard cells respond to CO2 in both the light and in darkness, and that stomatal sensitivity to CO2 increases with
incident radiation (Heath & Russell 1954; Morison &
Jarvis 1983; Wong et al. 1987). The mechanisms mediating
CO2 sensing in guard cells are not well understood
(Assmann 1993). Guard cells have two well-characterized
carboxylation reactions catalysed by Rubisco and phosphoenol pyruvate (PEP) carboxylase, which yield sugars
and malate, respectively. Both reactions have been invoked
as potential CO2 sensing mechanisms (Zeiger et al. 1987;
Raschke et al. 1988). The problem with these hypotheses,
however, is that higher [CO2] should increase the rate of
synthesis of sugars and malate and thus cause stomatal
opening, rather than closing. Results from a recent study
argue against CO2 sensing by osmoregulatory mechanisms
(Talbott et al. 1996). Pulses of CO2 applied to Vicia leaves
caused very similar stomatal responses irrespective of
whether guard cell osmoregulation was mediated by
sucrose or by potassium and its counterions.
Findings showing that the xanthophyll zeaxanthin is
involved in signal transduction in guard cells (Srivastava &
Zeiger 1995a,b) suggested another possible CO2 sensing
mechanism. Studies on the xanthophyll cycle of mesophyll
chloroplasts have shown that the de-epoxidation reaction
that catalyses the conversion of violaxanthin into zeaxanthin is regulated by lumen pH (Hager 1980). Lumen pH
depends on the balance between light-driven electron
transport and carbon fixation rates, and recent studies have
shown that, under constant irradiation, decreases in ambient [CO2] increased the zeaxanthin content of chloroplasts
from cotton leaves (Gilmore & Björkman 1994). The
steady-state yield of chlorophyll a fluorescence from guard
cell chloroplasts is sensitive to changes in [CO2] (Melis &
Zeiger 1982; Cardon & Berry 1992), indicating that
changes in [CO2] alter the balance of ATP and NADPH in
guard cell chloroplasts and have the potential to affect
lumen pH. Therefore, changes in CO2 could cause changes
in the zeaxanthin content of guard cell chloroplasts.
In the present study, we used growth chamber-grown
Vicia faba leaves to test the hypothesis that the xanthophyll
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cycle of the guard cell chloroplast might be involved in
CO2 sensing. Stomata from growth chamber-grown Vicia
leaves have an enhanced CO2 response and thus provide a
useful experimental system for the study of CO2 sensing
(Talbott et al. 1996). Stomata in intact leaves and in
detached epidermis were used for parallel measurements
of stomatal apertures and guard cell zeaxanthin content, as
a function of varying ambient [CO2]. Obtained results
implicate the xanthophyll cycle of guard cells in the regulation of the stomatal response to CO2.
MATERIALS AND METHODS
Plant material
Plants were grown as described previously (Talbott et al.
1996). Briefly, seeds of Vicia faba L. cv. Windsor Long
Pod (Bountiful Gardens Seeds, Willits, CA, USA) were
planted in pots using commercial potting soil (Sunshine
Mix #1, American Horticulture Supply, Camarillo, CA,
USA). Plants were grown in a walk-in growth chamber
(PGV-36, Conviron, Asheville, NC, USA) at 85 ± 3% RH,
12 h day, 500 µmol m–2 s–1, 25 ± 0·5 °C/12 h night,
15 ± 0·5 °C. Plants were watered 4 times a day with an
automatic watering system and fertilized (20–10–20 mix,
Grow More Research and Manufacturing Co., Gardena,
CA, USA) once a week. Fully expanded, recently matured
and unshaded leaves from the third and fourth nodes of
4–5-week-old plants were used for experiments.
Winchester, KY, USA) or in darkness. The Petri dishes
were placed in a 25 °C constant temperature bath to prevent heating. The solution was aerated with 800 cm3 m–3
CO2-containing air during the first hour of incubation, and
then switched to CO2-free, 400 cm3 m–3 or 800 cm3 m–3
CO2-containing air for an additional hour.
At the end of each treatment, a few peels were used for
measurement of stomatal apertures (see below) and the rest
were sonicated on ice for 30 s in a Branson Sonifier (model
250, Branson Ultrasonics Corp., Danbury, CT, USA) at a
continuous duty cycle and a power setting of 7, to eliminate contamination from mesophyll chloroplasts. After
sonication, the peels were rinsed thoroughly in tap water
and stored at – 80 °C for pigment analysis by HPLC (Zhu
et al. 1995).
Measurement of stomatal apertures
Apertures were measured with an Olympus BH-2 microscope (Olympus Corp., Lake Success, NY, USA) connected to a digital camera (JE2362 A, Javelin Electronics,
Torrance, CA, USA). Abaxial epidermal strips from either
intact leaves or the epidermal peel experiments were
mounted on a slide for image analysis as described previously (Talbott et al. 1996). Apertures of 30 stomata from
three or more epidermal strips were determined at each
data point.
Pigment extraction and analysis
In vivo CO2 pulse experiments
Carbon dioxide levels in the growth chamber were
increased by addition of 100% CO2 into the fan compartment of the growth chamber using a mass flow controller,
as described previously (Talbott et al. 1996). Chamber
[CO2] was continuously monitored with an infrared gas
analyser (Model EGM-1, PP Systems, Hitchin Herts, UK).
At the onset of the light period, chamber [CO2] was around
340–440 cm3 m–3 depending on the ambient [CO2] of the
room in which the chamber was located. Chamber CO2
levels were raised from the initial level to 1200 cm3 m–3 in
200 cm3 m–3 increments and maintained for 1 h at each
concentration to allow for the attainment of steady-state
apertures at each [CO2]. To lower chamber [CO2], the flow
of CO2 was decreased, allowing photosynthetic carbon fixation in the canopy and leakage to remove CO2 until the
desired level was reached.
Epidermal peel experiments
Detached Vicia leaves were wrapped in wet paper towels
and kept for 2·5 h in the dark, except as indicated. Abaxial
epidermal peels were removed with forceps under dim
room light and incubated in Petri dishes containing 1·0 mol
m–3 KCl, 0·1 mol m–3 CaCl2, and 1·0 mol m–3 Mes/NaOH
(pH 6·8), either under 150 µmol m–2 s–1 white light
(Sylvania DAH 300 W projector bulb, GTE Products Co.,
In the intact leaf experiments, 5–6 leaves were immersed in
ice water immediately after the CO2 treatments to minimize xanthophyll cycle activity, and the abaxial epidermes
were detached and sonicated within 6 min. Mesophyll tissue was obtained from the same leaves used for epidermis
preparation.
Pigments were extracted under dim room light on ice.
Clean epidermal peels were ground thoroughly (20 min) to
ensure complete pigment extraction in a mortar in a mixture of 1 cm3 acetone and about 500 mg anhydrous
Na2SO4, and 0·5 cm3 NaHCO3-saturated hexane. KCl
(1·0 cm3, 0·2 kmol m–3) was then added and the mixture
centrifuged at 1500 g for 3 min. The upper solvent phase of
the extracts was collected and evaporated in vacuo. The
dried samples were either analysed immediately after
extraction or flushed with N2 and stored at – 80 °C.
Samples were chromatographed on a Beckman HPLC system (a 421 controller, a 165 detector and two 110 A pumps)
provided with a Spectra-Physics SP4270 integrator and an
Alltech Spherisorb ODS-1 5µ column, using the method
described by Gilmore & Yamamoto (1991). Pigments were
separated by elution with solvent A (acetonitrite:methanol:
0·1 kmol m–3 Tris-HCl buffer; 72:8:3, v/v; pH 8·0 adjusted
with 10 kmol m–3 KOH) for 4 min at a flow rate of
2 cm3 min–1, followed by a 2·5 min linear gradient of
0–100% solvent B (methanol:hexane; 4:1, v/v), and then a
7·5 min elution with 100% solvent B. Pigments were quantified by integrating the area under the 440 nm absorption
© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 813–820
Guard cell zeaxanthin and stomatal response to CO2
peak using empirically determined response factors.
Response factors used in the calculation were 0·360 for
violaxanthin, 0·346 for antheraxanthin, 0·501 for zeaxanthin, 0·321 for chlorophyll b, and 0·345 for chlorophyll a.
They were obtained by separating leaf pigment extracts by
HPLC and collecting fractions containing pigments of
interest, based upon published HPLC profiles (Gilmore &
Yamamoto 1991). The collected fractions were dried and
resuspended in hexane, acetone or ethanol. The identity of
the pigment was confirmed by its absorption spectra and its
concentration
determined
spectrophotometrically.
Response factors were determined from peak areas
obtained from different concentrations of the pigment (M.
Quiñones, unpublished results).
RESULTS
Changes in stomatal apertures and guard cell
zeaxanthin content in intact leaves as a function
of ambient [CO2]
Typical pigment profiles from growth chamber-grown
mesophyll and guard cells are shown in Fig. 1. Under con-
stant light and temperature, abaxial guard cells from intact,
growth chamber-grown Vicia leaves showed large changes
in zeaxanthin content as a function of changes in ambient
[CO2] (Figs 1 & 2a). Guard cell zeaxanthin decreased from
181 mmol mol–1 Chl to 79 mmol mol–1 Chl when chamber
[CO2] increased from 400 to 1200 cm3 m–3. As reported
previously (Talbott et al. 1996), stomatal apertures also
showed large changes as a function of ambient [CO2]
(Fig. 2a). A plot of stomatal apertures as a function of
guard cell zeaxanthin content showed that the two parameters were linearly related (Fig. 2b).
Figure 3 shows the changes in violaxanthin, antheraxanthin and zeaxanthin – the three components of the xanthophyll cycle – of mesophyll and guard cell chloroplasts
as a function of changes in [CO2]. The xanthophyll cycle
of mesophyll chloroplasts was insensitive to the changes
in [CO2] under the experimental conditions used, and
showed no detectable zeaxanthin under all tested [CO2]
(Fig. 3, inset). On the other hand, the xanthophyll cycle
from guard cell chloroplasts showed a very high CO2
sensitivity (Fig. 3).
The reversibility of these responses to ambient [CO2]
was tested in experiments in which chamber [CO2] was
Figure 1. Typical HPLC chromatography profiles of photosynthetic pigments from guard cells under 400 and 900 cm3 m–3 CO2, and
mesophyll cells under 400 cm3 m–3 CO2 from growth chamber-grown Vicia faba. Light fluence rate at the adaxial leaf surface was
500 µmol m–2 s–1 in the chamber. Individual pigment is indicated above the corresponding peak. Neo, neoxanthin; Vio, violaxanthin; Ant,
antheraxanthin; Lut, lutein; Zea, zeaxanthin; Chl b, chlorophyll b; Chl a, chlorophyll a; β-car, β-carotene.
© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 813–820
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J. Zhu et al.
The effect of the de-epoxidase inhibitor
dithiothreitol (DTT) on CO2-dependent
zeaxanthin formation and stomatal movements in
the light and in the dark
The results of experiments with intact leaves showing that
guard cell zeaxanthin content tracks CO2-dependent stomatal movements in the light but not in the dark (Fig. 2) suggest that the metabolic processes associated with the
stomatal responses to CO2 in the dark might differ from
those in the light. Experiments with epidermal peels made
it possible to use the inhibitor of violaxanthin de-epoxidase, DTT, to test whether inhibition of zeaxanthin formation altered the stomatal response to CO2. Stomata from
abaxial epidermis detached from dark-adapted (2·5 h)
leaves were incubated for 1 h in a solution aerated with
800 cm3 m–3 CO2 either in 150 µmol m–2 s–1 white light or
in darkness, and then treated with 400 cm3 m–3 CO2 or
CO2-free air for an additional hour in the presence or
absence of 3 mol m–3 DTT (Srivastava & Zeiger 1995a).
Table 1 shows the net changes in apertures after the second
hour of incubation. In the dark, stomata transferred from
800 to 400 cm3 m–3 CO2 and to CO2-free air opened about
0·6 and 1·4 µm, respectively, and these responses were
insensitive to 3 mol m–3 DTT. In the light, the stomatal
responses to CO2 were larger than in the dark, and 3 mol
m–3 DTT reduced the response by ≈ 50% (Table 1). The
DTT-insensitive component of the stomatal response to
Figure 2. (a) CO2-dependent changes in stomatal apertures and
guard cell zeaxanthin (Z) in leaves of growth chamber-grown
Vicia faba under constant light (open symbols) and in the dark
(closed symbols); (b) Relationship between guard cell
zeaxanthin and stomatal apertures in leaves of growth chambergrown Vicia faba as a function of ambient [CO2] under constant
light. Light fluence rate at the adaxial leaf surface was
500 µmol m–2 s–1 in the chamber. Chamber CO2 levels were
increased from about 400 to 1200 cm3 m–3 in 200 cm3 m–3
increments. Each [CO2] was maintained for 1 h prior to
measurements. Each value represents an average of four
experiments ± SE.
raised from 400 to 1000 cm3 m–3 and then lowered to
400 cm3 m–3 in a stepwise fashion. Both guard cell zeaxanthin content and stomatal apertures tracked the changes in
[CO2] in both directions (Figs 4a & b).
In the dark, average stomatal apertures at ambient [CO2]
were about 7 µm (vs. nearly 14 µm in the light) and apertures decreased by about 3·3 µm over the 400–1000 cm3
m–3 range, compared to about 7 µm in the light. Guard cell
zeaxanthin remained constant in the 400–800 cm3 m–3
range and decreased only slightly at 1000 cm3 m–3
(Fig. 2a). There was no significant correlation between
stomatal apertures in the dark and the zeaxanthin content in
guard cells (r = 0·21).
Figure 3. Antheraxanthin (A, ◆), violaxanthin (V, ●), and
zeaxanthin (■) content in abaxial guard cell chloroplasts from
leaves of growth chamber-grown Vicia, expressed as the
percentage of the total xanthophyll cycle pool, as a function of
ambient [CO2] under constant light. Inset: A, V, and Z content in
Vicia mesophyll chloroplasts under the same conditions. Light
fluence rate and ambient [CO2] were as described in Fig. 2. Each
value represents an average of four experiments ± SE.
© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 813–820
Guard cell zeaxanthin and stomatal response to CO2
817
CO2 in the light was of the same magnitude as the total
response to CO2 in the dark.
Figure 5(a) shows the zeaxanthin content in guard cells
from epidermal strips incubated in 150 µmol m–2 s–1 white
light under three different [CO2], in the presence or absence
of 3 mol m–3 DTT. Zeaxanthin content in guard cells incubated in 800 cm3 m–3 CO2 increased in parallel with stomatal apertures when transferred to 400 cm3 m–3 or CO2-free
air, and the increase in zeaxanthin was inhibited by DTT. In
the absence of DTT, stomatal apertures were closely correlated with the zeaxanthin content of guard cells (Fig. 5b).
DISCUSSION
Figure 4. Reversibility of the CO2-dependent changes in stomatal
apertures (a) and guard cell zeaxanthin (b) in growth chambergrown Vicia leaves kept under constant light. Light fluence rate at
the adaxial leaf surface was 500 µmol m–2 s–1. Chamber CO2
levels were increased from about 400 to 1000 cm3 m–3 in 200 cm3
m–3 increments, and then decreased to 400 cm3 m–3 in a stepwise
fashion. Each [CO2] was maintained for 1 h prior to measurements.
Arrows indicate the sequence of the [CO2] treatments. Each value
represents an average of three experiments ± SE.
Under the constant, relatively low light levels of the
growth chamber environment, mesophyll from Vicia
leaves had no detectable zeaxanthin, as shown previously
for growth chamber-grown cotton (Brugnoli & Björkman
1992). In contrast, guard cells had high zeaxanthin content
at ambient [CO2], comprising about 45% of the xanthophyll cycle pool (Fig. 3). These high levels of guard cell
zeaxanthin are comparable to those found at midday in
greenhouse-grown guard cells (Srivastava & Zeiger
1995b). The xanthophyll cycle of guard cell chloroplasts
was very sensitive to changes in [CO2], and guard cell
zeaxanthin decreased to about 20% of the xanthophyll
cycle pool when [CO2] increased from 400 to 1000 cm3
m–3. Concomitant increases in violaxanthin content indicated that the xanthophyll cycle was modulated by the
changes in ambient [CO2]. There were no detectable
changes in the xanthophyll cycle pigments of mesophyll
chloroplasts in response to these CO2 manipulations.
Studies of the xanthophyll cycle of mesophyll chloroplasts have shown that, at high irradiances, a decrease in Ci
stimulates violaxanthin de-epoxidation and causes an
increase in zeaxanthin content (Gilmore & Björkman
1994), and in non-photochemical fluorescence quenching
(Demmig-Adams & Adams 1992). A similar mechanism
Increase in stomatal aperture (µm)
light
dark
CO2 (cm3 m–3)
–DTT
+DTT
–DTT
+DTT
400
0
1·22 ± 0·15a
3·16 ± 0·25b
0·63 ± 0·05a*
1·55 ± 0·09b**
0·61 ± 0·09*
1·37 ± 0·20**
0·56 ± 0·07*
1·45 ± 0·32**
a
Significantly different (one-way ANOVA; F = 13·850, P = 0·020; at the 0·05 level).
Significantly different (one-way ANOVA; F = 36·472, P = 0·004; at the 0·05 level).
*Not significantly different (one-way ANOVA; F = 0·231, P = 0·799; at the 0·05 level).
**Not significantly different (one-way ANOVA; F = 0·134, P = 0·876; at the 0·05 level).
b
© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 813–820
Table 1. Stomatal response to CO2 in abaxial
epidermal peels incubated in the light or in
darkness in the presence or absence of 3 mol
m–3 DTT. Epidermal peels from dark-adapted
(2·5 h) leaves were incubated in a medium
(1·0 mol m–3 KCl, 0·1 mol m–3 CaCl2, and
1·0 mol m–3 Mes/NaOH pH 6·8) aerated with
800 cm3 m–3 CO2-containing air in either
150 µmol m–2 s–1 white light or in darkness for
1 h and then transferred to a medium in
equilibrium with 800, 400 and 0 cm3 m–3
CO2-containing air for an additional 1 hr.
DTT was added to the medium at the end of
the first hour of incubation. The values shown
are the net aperture changes measured under
0 or 400 cm3 m–3 CO2 in comparison with the
aperture under 800 cm3 m–3 CO2 at the end of
the 2 h incubation. Each value represents an
average of three experiments ± SE
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J. Zhu et al.
Figure 5. (a) Effect of DTT on CO2-dependent changes in guard
cell zeaxanthin in the light; (b) relationship between guard cell
zeaxanthin and stomatal apertures in abaxial epidermal peels as a
function of [CO2] in the light in the absence of DTT. Conditions
were as described in Table 1. The DTT concentration was 3 mol
m–3. Each value represents an average of four experiments ± SE.
appears to operate at low irradiance in chloroplasts of
abaxial guard cells (Figs 2 & 3).
The large changes in guard cell zeaxanthin content in
response to changes in ambient [CO2] suggest correspondingly large, CO2-mediated effects on lumen pH (Hager
1980). Changes in [CO2] could alter lumen pH via changes
in the rate of consumption of ATP and NADPH by the carboxylation reaction catalysed by Rubisco (Gotow et al.
1988; Cardon & Berry 1992). A second CO2-sensitive sink
for reducing equivalents from the guard cell chloroplast
could be provided by PEP carboxylase activity in the
cytosol. A 3-phosphoglycerate/dihydroxyacetone phosphate shuttle has been suggested to export ATP and reducing equivalents from the chloroplast to the cytosol
(Shimazaki et al. 1989) and would alter the energy status
of the thylakoid membrane in a [CO2]-dependent manner.
Under constant light, temperature and relative humidity,
changes in stomatal apertures were linearly related to
changes in guard cell zeaxanthin content over the
400–1000 cm3 m–3 range of [CO2] in intact leaves (Fig. 2b)
and under incubation in 0, 400, and 800 cm3 m–3 CO2 in
detached epidermis (Fig. 5b). In intact leaves, the changes
were reversible (Fig. 4) and both aperture and zeaxanthin
changes saturated in the same range of [CO2] (Figs 2 and
4). This response pattern argues for a causal relationship
between the two parameters.
Stomatal apertures in intact leaves also tracked ambient
[CO2] in the dark, in the absence of changes in guard cell
zeaxanthin content (Fig. 2a). As reported in previous studies, the stomatal response to [CO2] in the dark was smaller
than in the light (Heath & Russell 1954; Morison & Jarvis
1983; Wong et al. 1987).
In epidermal peels kept under constant irradiation, the
net increase in stomatal aperture in response to a decrease
in [CO2] from 800 to 0 cm3 m–3 CO2 was about twice as
large as that measured in the dark (Table 1). Decreases in
[CO2] caused an increase in guard cell zeaxanthin content
in the light (Fig. 5a) but not in the dark (Fig. 2a). The
inhibitor of violaxanthin de-epoxidase, DTT, prevented the
CO2-dependent zeaxanthin increases in the light (Fig. 5a)
and inhibited net aperture increases to the levels observed
in the dark controls (Table 1). Thus, both in the intact leaf
and in epidermal peels, net changes in apertures in
response to CO2 were larger in the light than in the dark,
and were accompanied by changes in guard cell zeaxanthin
content only in the light.
The tight coupling between CO2-dependent aperture and
zeaxanthin changes in the light, the co-directionality of the
aperture and zeaxanthin changes, and the inhibition by
DTT of the CO2-dependent zeaxanthin increase and of the
aperture increases in the light to the level observed in the
dark, suggest that zeaxanthin could play a role in the sensory transduction of CO2 signals in guard cells. Zeaxanthin
would transduce prevailing [CO2] at the guard cell chloroplast into modulated stomatal apertures in the light, while
the stomatal response to CO2 in darkness would be dependent on a second, zeaxanthin-independent sensory transduction pathway (Hedrich & Marten 1993).
Pharmacological, action spectroscopy, and genetic
studies have recently implicated zeaxanthin as a blue light
sensor in guard cells (Srivastava & Zeiger 1995a;
Quiñones et al. 1996; Zeiger & Zhu 1998). Interactions
between the stomatal responses to blue light and CO2 have
been reported (Assmann 1988; Lascève et al. 1993), and
measurements of guard cell zeaxanthin content over a daily
course of stomatal movements in a greenhouse have shown
that zeaxanthin content in guard cells tracked incident radiation to the leaf and was closely correlated with stomatal
apertures (Srivastava & Zeiger 1995b).
Available data indicate that the xanthophyll cycle of guard
cells has the same biochemical properties as its mesophyll
counterpart (Karlsson et al. 1992; Masamoto et al. 1993;
Srivastava & Zeiger 1995b) but it is regulated in a quite different way. On a chlorophyll basis, guard cell chloroplasts
have a nearly 4-fold higher capacity for electron transport
and oxygen evolution, substantially lower rates of carbon
fixation, and a larger LHCII unit size (Zeiger et al. 1981;
Shimazaki & Zeiger 1987; Mawson 1993; Zhu et al. 1997)
than mesophyll chloroplasts. High rates of electron transport
and low rates of carbon fixation can be expected to favour
lumen acidification and zeaxanthin formation at low photon
© 1998 Blackwell Science Ltd, Plant, Cell and Environment, 21, 813–820
Guard cell zeaxanthin and stomatal response to CO2
flux densities, as observed (Srivastava & Zeiger 1995b).
Stomata from greenhouse-grown leaves have an enhanced
light sensitivity (Talbott et al. 1996) and the xanthophyll
cycle of these guard cells is more sensitive to light than its
mesophyll counterpart (Srivastava & Zeiger 1995b).
Stomata from growth chamber-grown leaves have an
enhanced CO2 sensitivity, and the xanthophyll cycle of these
guard cells is dramatically more sensitive to CO2 than that of
mesophyll (Fig. 3). These contrasting properties underscore
the regulatory differences of the xanthophyll cycle from
mesophyll and guard cell chloroplasts, which is presumably
associated with the distinct function of the two chloroplast types: carbon fixation in the mesophyll, and sensory
transduction in the guard cell.
Modulation of the xanthophyll cycle of guard cells by
both light and ambient [CO2] would integrate light and CO2
sensing at the guard cell chloroplast into a single mechanism mediating light–CO2 interactions on stomatal apertures (Morison 1987). In such a mechanism, the modulation
of guard cell zeaxanthin content by lumen pH could sense
environmental and metabolic signals regulating stomatal
apertures via their effect on lumen pH. An empirically verifiable implication of this hypothesis is that recently characterized acclimations of the stomatal responses to blue light
and to CO2 (Assmann 1992; Frechilla et al. 1997) could
underlie changes in the sensitivity of the xanthophyll cycle
of guard cells to light and CO2.
A major question emerging from the zeaxanthin hypothesis is the nature of the mechanism transducing zeaxanthin-mediated signal processing within the chloroplast to
extrachloroplastic targets elsewhere in the guard cell. Light
sensing at the guard cell chloroplast has been shown to
mediate red light-stimulated outward electrical currents
from guard cell protoplasts (Serrano et al. 1988), and the
chloroplast appears to be the site of perception of blue light
in blue light-stimulated stomatal opening (Srivastava &
Zeiger 1995a). A metabolite of guard cell photosynthesis
(Serrano et al. 1988; Cardon & Berry 1992) and calcium
fluxes across the chloroplast envelope (Kinoshita et al.
1995) have been proposed as possible second messengers.
Recent studies have shown that cytosolic free calcium
appears to be a component of the CO2 signal transduction
pathway in stomatal guard cells (Webb et al. 1996). Further
studies on the role of zeaxanthin on signal transduction in
guard cells could provide insight on this important question.
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development on the morphology and stomatal physiology of
Commelina communis. Oecologia 92, 188–195.
Assmann S.M. (1993) Signal transduction in guard cells. Annual
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Received 6 October 1997; received in revised form 27 February
1998; accepted for publication 2 April 1998
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