Department of Biological Sciences, University of Essex, Colchester,
CO4 3SQ United Kingdom
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INTRODUCTION |
Stomata are the main routes for leaf
gas exchange, controlling CO2 uptake and
transpiration. Stomatal movements are regulated by both internal and
external factors. Opening of stomata is stimulated by low
CO2 concentrations, blue light, and other
photosynthetically active wavelengths, whereas stomatal closure occurs
in response to a number of environmental cues, most notably darkness,
low air humidity, and high temperature (for reviews, see Assmann, 1993
;
Willmer and Fricker, 1996). Stomatal movements are brought about
through changes in turgor within guard cells and accessory cells. This
involves the active (energy requiring) net accumulation or loss of
K+ ions and the parallel loss or accumulation of
organic solutes, such as malate and Suc (e.g. Outlaw, 1996; Willmer and
Fricker, 1996; Asai et al., 2000).
Chloroplasts are a characteristic feature of guard cells and are
present in most (but not all) species, and they show photosynthetic electron transport (Willmer and Fricker, 1996; Tsionsky et al., 1997).
Although guard cell chloroplasts are generally smaller, less numerous,
and have fewer grana than mesophyll chloroplasts (Sack, 1987; Willmer
and Fricker, 1996), photophosphorylation, on a chlorophyll basis, has
been reported to be as high as 80% of that in the mesophyll cells
(Shimazaki and Zeiger, 1985). Although guard cells have much lower
chlorophyll contents than mesophyll cells (25- to 100-fold lower), they
are also considerably smaller (approximately 10-fold smaller; Table
III.1 in Willmer and Fricker, 1996), so their chloroplasts could
represent a significant energy source for guard cell ion transport and
other processes (Wu and Assman, 1993). The role of guard cell
chloroplasts in CO2 fixation is still a matter of
debate (e.g. Lee and Bowling, 1995; Willmer and Fricker, 1996; Lu et
al., 1997). Although some researchers have concluded that guard cell
chloroplasts do not reduce CO2 through the Calvin
cycle at appreciable rates (Outlaw, 1989; Reckmann et al., 1990), they
could function as part of an environmental sensing and signaling system
(Outlaw, 1989; Goh et al., 1999). For example, Melis and Zeiger (1982)
found that photosynthetic electron transport in guard cells of
Chlorophytum comosum and Aspidistra eliator
responded to changes in ambient CO2 concentration (Ca), whereas Cardon and Berry (1992)
observed modulation of the chlorophyll fluorescence signal from guard
cell chloroplasts in response to changes in
Ca and ambient O2
concentration, confirming that Rubisco was acting as a significant sink
for ATP and NADPH. In these experiments, changes in fluorescence with
ambient O2 were only observed at low
Ca (< 130 µmol
mol
1), indicating that photorespiration was the
main sink for ATP and NADPH at low Ca and
that this was inhibited at higher
Ca.
Previous measurements of chlorophyll fluorescence from guard cells have
been largely restricted to epidermal peels (Melis and Zeiger, 1982;
Ogawa et al., 1982) or guard cell protoplasts (Goh et al., 1999).
Cardon and Berry (1992) made the important advance of studying intact
tissue; however, they were only able to compare changes in the
steady-state fluorescence signal (F') from guard cell
chloroplasts located within white regions of variegated leaves of
tradescantia (Tradescantia albiflora) with those from mesophyll chloroplasts within green regions of the same leaves. Although guard cells in the white regions of leaves are capable of
opening, they are obviously not subject to the same influence of
mesophyll cell activity as they are in green areas (e.g. Scarth and
Shaw, 1951).
It is well established that the "Genty factor" fluorescence
parameter (Genty et al., 1989) can provide a good estimate of the
quantum efficiency of photosystem (PS)II photochemistry, and consequently can be used to examine changes in photosynthetic electron
transport. Images of the Genty factor (defined by
Fq'/Fm' in the
terminology used in this paper) from guard cells and mesophyll cells of
green leaves have been produced previously (Oxborough and Baker, 1997a;
Baker et al., 2001). In this study, the high resolution imaging system
described previously by Oxborough and Baker (1997a) has been combined
with an infrared gas analyzer system to compare the responses of
photosynthetic electron transport in chloroplasts of guard and
mesophyll cells in attached variegated and green leaves of tradescantia
and commelina (Commelina communis) to changes in
photosynthetically active photon flux density (PPFD), Ca, and leaf-air vapor pressure difference (VPD).
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RESULTS |
Fluorescence Parameters
The calculation of useful fluorescence parameters, irrespective of
whether fluorescence measurements are made with conventional modulated
fluorimeters or imaging systems, requires that the fluorescence signal
is recorded while the photosynthetic system is in well-defined states.
With dark-adapted material, the Fo level of
fluorescence is recorded at very low PPFD (less than 1 µmol
m2 s1), which leaves
virtually all PSII centers in the "open" state (capable of
photochemistry), whereas the Fm level of
fluorescence is recorded during a short light pulse of very high PPFD
(typically less than 1 s at several thousand µmol
m2 s1), which
transiently drives a very high proportion of PSII centers into the
"closed" state (making the capacity for photochemistry close to
zero). With light-adapted material, the equivalent terms are
Fo' and
Fm'. At any point between
Fo' and
Fm' (where a variable proportion of
PSII centers are in the open state), the fluorescence signal is termed
F'. The difference between Fm
and Fo is termed Fv and the difference between
Fm' and
Fo' is termed
Fv'. The term Fq' has recently been introduced to
denote the difference between Fm' and
F' measured immediately before application of the saturating pulse used to measure Fm' (Oxborough
and Baker, 2000; Oxborough et al., 2000; Baker et al., 2001). In theory
(Baker et al., 2001), the fluorescence parameter
Fq'/Fm'
provides a good estimate of the quantum efficiency of PSII
photochemistry at the point of measurement (hereafter referred to as
PSII operating efficiency). Fq'/Fm' is
actually the product of two other useful fluorescence parameters,
Fv'/Fm' and
Fq'/Fv':
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(1)
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Fv'/Fm'
provides an estimate of the maximum quantum efficiency of PSII
photochemistry (PSII maximum efficiency), i.e. the PSII operating
efficiency when all PSII centers are in the open state at the point of
measurement. Its value is largely determined by down-regulation, which
appears to involve the operation of one or more processes that increase
the rate constant for non-radiative decay within the PSII pigment
matrix, and which is responsible for non-photochemical quenching of
chlorophyll fluorescence. The parameter
Fq'/Fv' is a
factor (the PSII efficiency factor) that relates the PSII maximum
efficiency to the PSII operating efficiency. Its value is non-linearly
related to the proportion of PSII centers in the open state, which
determines the level of photochemical quenching of chlorophyll
fluorescence (Baker et al., 2001). The value of
Fo' is required for the calculation
of both
Fv'/Fm' and Fq'/Fv' (since
Fv' = Fm' 
Fo'). Images of
Fo' cannot be taken with the imaging
system used here (or with any other imaging system that we are aware
of). However, Oxborough and Baker (1997b) demonstrated that
Fo' can be calculated from the
following equation:
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(2)
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Recently it has been suggested that this method of estimating
Fo' cannot be applied in situations
where plants are stressed and significant amounts of photoinhibition
may occur (Maxwell and Johnson, 2000). This is not the case. The only
requirements for the calculation of
Fo' from the above equation are that
(a) all the PSII centers are open at the point at which
Fo is measured, (b) there is no reversal of
down-regulation between the measurement of
Fo and Fm, and
(c) there is no reversal of photoinhibition between the measurement of
Fm' and
Fm. In reality, given the inherent difficulty of accurately measuring
Fo', it can be reasonably argued that
the calculation of Fo' from the
above equation gives a more accurate estimate of the parameter
than does direct measurement.
Images of F' from stomatal guard cell chloroplasts and the
underlying mesophyll in a tradescantia leaf are shown in Figure 1, b and c. Chlorophyll fluorescence from
individual chloroplasts in the guard cells can be clearly identified
(Fig. 1c) and isolated using the FluorImager software (see "Materials
and Methods"; Oxborough and Baker, 1997a). Consequently, images can
be generated for
Fq'/Fm' of
isolated chloroplasts in the guard cells and adjacent mesophyll tissue
(Fig. 1e; Oxborough and Baker, 1997a). Stomatal aperture (maximum pore
width) can be measured from the reflected light image (Fig.
1a).
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Figure 1.
Images of reflected light (a) and fluorescence
parameters (b-e) from stomatal guard cells and surrounding mesophyll
in a tradescantia leaf. Images of F' taken using a 695-nm
longpass filter (b) or a 680-nm bandpass filter (c); images of
Fq'/Fm' taken
using a 695-nm longpass filter (d) or a 680-nm bandpass filter (e).
Values of
Fq'/Fm' for
guard cell chloroplasts can be determined from images d and e by
isolating the chloroplasts using the FluorImager software (see
"Materials and Methods"; Oxborough and Baker, 1997).
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The main aim of this study was to compare changes in
Fq'/Fm' from
guard cell chloroplasts and mesophyll cells in response to changes in
PPFD, Ca, and VPD. While conducting
these experiments, it was clear that lower values of
Fq'/Fm' for
both guard and mesophyll cells were obtained with closed compared with
open stomata, which we attribute to the restricted
CO2 diffusion into the leaf at any given external
CO2 concentration. In addition, the differences between values of
Fq'/Fm' for
guard and mesophyll cells were usually larger for tradescantia than for
commelina, which is probably because tradescantia has fewer stomata per
area and only on the lower side, and therefore has more restricted
CO2 exchange compared with commelina, which is amphistomatous.
Imaging of Fluorescence Using 680-nm Bandpass and 695-nm Longpass
Filters
Although the editing tools built into the imaging software
(FluorImager) allow for the isolation of guard cell chloroplasts from
within an image, they cannot remove fluorescence that might be
transmitted through guard cell chloroplasts from underlying mesophyll
cells. Chlorophyll fluorescence of shorter wavelengths (between
approximately 675 and 685 nm) is strongly reabsorbed, because these
wavelengths are close to the long wavelength maximum of the chlorophyll
a absorption spectrum. Consequently, use of a 680-nm
bandpass filter (Fig. 1c), rather than a 695-nm longpass filter (Fig.
1b), gives much greater weight to fluorescence from chloroplasts that
are close to the imaged surface (including the chloroplasts within
guard cells). With the imaging system used here, the incident PPFD is
predominantly at wavelengths that are absorbed well by chlorophylls
a and b. Consequently, there is an expectation
that chloroplasts in cells at the surface will exhibit lower values of
Fq'/Fm' than
those in cells deep within the leaf, simply because they are
absorbing more photons. Using a 680-nm bandpass filter, rather than a
695-nm longpass filter, when imaging also decreases the contribution of
PSI fluorescence to the overall fluorescence signal. The yield of
fluorescence from PSI is reasonably constant and independent of PPFD
(Dau, 1994; Pfündel, 1998). A consequence of this is that PSI
fluorescence makes a proportionally larger contribution to the
fluorescence signal at F' than at
Fm'. Removing most of the PSI signal
with the 680-nm bandpass filter has the effect of increasing the
measured value of
Fq'/Fm'
(compare mesophyll regions in Fig. 1d with Fig. 1e).
Changes in
Fq'/Fm' with
increasing PPFD for guard cell chloroplasts of a variegated
tradescantia leaf are shown in Figure 2.
These data are derived from images of guard cell chloroplasts that were
isolated from images of white and green areas of a tradescantia leaf.
These images were taken using either the 680-nm bandpass filter or the
695-nm longpass filter, and the values for
Fq'/Fm' for
white region guard cell chloroplasts were significantly lower than
those for green region guard cell chloroplasts (P < 0.001). As expected, use of the 680-nm bandpass filter resulted in higher values for
Fq'/Fm' from
the white areas (P = 0.10), due to removal of most of
the PSI signal. Over the green areas, the two filters produced very
similar values of
Fq'/Fm'. These
data suggest that the increase in
Fq'/Fm' that
results from removing most of the PSI fluorescence with the 680-nm
bandpass filter was offset by a decrease in
Fq'/Fm'
because of the decrease in the contribution of fluorescence from cells
located at depth within the leaf.
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Figure 2.
Responses of
Fq'/Fm' to
increasing PPFD of chloroplasts of guard cells of a tradescantia leaf.
Data are derived from images of F' and
Fm' taken from white areas ( ,  )
or green areas ( ,  ). Chlorophyll fluorescence was measured using
a 695-nm longpass filter (, ) or 680-nm bandpass filter (,
). Measurements were made at 25°C and a
Ca of 356 µmol
mol1. Data are the means of six replicates ± SE.
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Although use of the 680-nm bandpass filter clearly reduces
contamination of the fluorescence signal from guard cell chloroplasts by fluorescence from the underlying mesophyll cells, it is inevitable that some contamination will result. The extent of this contamination is difficult to quantify precisely, although a number of observations suggest that it is not large. First, if there was a significant level
of contamination of the guard cell chloroplasts fluorescence with
fluorescence from underlying mesophyll cells,
Fq'/Fm' values from guard cell chloroplasts in intact leaves would be higher than
those from the guard cell chloroplasts in epidermal peels. Figure
3 shows that
Fq'/Fm' values
from guard cell chloroplasts of intact leaves were indistinguishable
those from peels but significantly different from those of mesophyll
cells within the same leaves (P = 0.02; Fig. 3).
Further evidence for a low level of contamination came from comparison
of the fluorescence signal (F') from guard cell chloroplasts
of green and white areas of tradescantia leaves. The values of
F' from the guard cell chloroplasts in the green, compared
with the white, areas were indistinguishable (96.6 ± 33.7 compared with 91.2 ± 4.28 at PPFD of 93 µmol
m2 s1 and 154.2 ± 50.9 compared with 145.4 ± 57.4 at 256 µmol
m2 s1), which would not
be the case if there had been significant contamination from the
mesophyll cells.
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Figure 3.
Responses of
Fq'/Fm' to
increasing PPFD for mesophyll () and guard cells () of a
commelina leaf, and for guard cells in a peel from a commelina leaf
(). Measurements were made at 25°C and a
Ca of 360 µmol
mol1. Data are the means of four
replicates ± SE.
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Responses to Changing PPFD
Images from green and white areas of an intact tradescantia leaf
were taken over a range of PPFDs between 0 and 680 µmol
m2 s1 with the leaf at
steady-state photosynthesis. The changes in Fq'/Fm',
Fv'/Fm', and
Fq'/Fv' with
increasing PPFD, calculated from the images, were qualitatively similar
for both guard and mesophyll cells (Fig.
4). There were significantly lower values of Fq'/Fm'
from guard cell chloroplasts within both green and white areas of
leaves, compared with mesophyll cells, at PPFDs above 20 µmol
m2 s1 (Fig. 4A;
P = 0.031 and P = 0.005, for green and
white areas respectively). Values of
Fq'/Fm' from
guard cell chloroplasts in the white area were noticeably lower than
from those in the green area at PPFDs between 46 µmol
m2 s1 and 446 µmol
m2 s1
(P = 0.13). The lower values of
Fq'/Fm' from
guard cell chloroplasts in the white area resulted from lower values of
Fv'/Fm'
(P = 0.14), suggesting a higher level of
down-regulation in these cells. The differences in
Fq'/Fm'
between the mesophyll and guard cell chloroplasts in green
areas at PPFDs above 20 µmol m2
s1 were due to higher values of
Fq'/Fv' within
the mesophyll cells (P = 0.013; Fig. 4C), as there were
no statistically significant differences in
Fv'/Fm'. There
was a close linear relationship between
Fq'/Fm' for
guard and mesophyll cells over the PPFD range (see inset in Fig. 4A),
suggesting that the response of the photosynthetic apparatus to
changing PPFD was similar in both cell types.
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Figure 4.
Responses of the fluorescence parameters
Fq'/Fm' (A),
Fv'/Fm' (B),
and Fq'/Fv'
(C) of a variegated tradescantia leaf to increasing PPFD. Data were
obtained from guard cells in white () areas of the leaf and from
mesophyll () and guard cells () in green areas. Measurements were
made at 25°C and a Ca of 360 µmol
mol1. Data are the means of six replicates ± SE. The inset in A shows the relationship
between
Fq'/Fm' for
mesophyll and guard cells in green areas of the leaf over the range of
PPFDs (the linear relationship is defined by y = 0.76x; r2 = 0.92).
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Responses to Changes in Ambient CO2 and
O2 Concentration
To further examine whether there may be differences in
photosynthetic metabolism between the two cell types, the responses of
the cells to changes in ambient CO2 and
O2 concentration were studied. The changes in
Fq'/Fm' within
guard cell chloroplasts and mesophyll cells of attached leaves of
tradescantia and commelina, in response to increasing
Ca, are shown in Figure
5.
Fq'/Fm'
increased with Ca up to approximately 350 µmol mol1, after which the response curves
flattened out. Values of
Fq'/Fm' of
guard cell chloroplasts in the green regions of tradescantia and in
commelina leaves were lower than those from the adjacent mesophyll
cells over the Ca range. In tradescantia
(Fig. 5A), stomata started fully open in the white area, with apertures
of approximately 30 µm, and partially open in the green area (with apertures of approximately 18 µm compared with 30 µm for fully open
stomata). Values of
Fq'/Fm' of
guard cell chloroplasts within the white area of tradescantia were very
much lower than for mesophyll cells (by 50%-60%) or guard cell
chloroplasts in green areas (by 45%-50%). The stomata in the
commelina leaves (Fig. 5b) started fully open with apertures of
approximately 35 µm, therefore the difference between
Fq'/Fm' values
of guard cell chloroplasts and mesophyll was not so large; however, the
response to CO2 in the two cell types was the
same and similar to that of tradescantia.
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Figure 5.
Response of
Fq'/Fm' of
tradescantia (A) and commelina leaves (B) to increasing
Ca. A, Data were obtained from guard cells
in white () areas of the tradescantia leaf and from mesophyll ()
and guard cells () in green areas. B, Data are from mesophyll ()
and guard cells () of the commelina leaf. Measurements were made at
an ambient temperature of 25°C and at PPFDs of 215 and 265 µmol
m2 s1 for tradescantia
and commelina leaves, respectively.
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The responses of
Fq'/Fm' of
guard and mesophyll cells of a green area of a tradescantia leaf to
changes in Ca at 2% and 21% O2 are shown in Figure
6.
Fq'/Fm' of
both cell types was lower at 2% O2, compared
with 21% O2, over a
Ca range of 0 to 600 µmol mol1 (Fig. 6). As
Ca increased, the difference in
Fq'/Fm' at the
two O2 concentrations decreased, and became close
to zero at 974 µmol mol1. As would be
expected, the values of
Fq'/Fm' in
21% O2 when the stomata were closed (Fig. 6)
were much lower at any given Ca than was
the case when the stomata were open (Fig. 5A), because of restricted
CO2 diffusion.
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Figure 6.
Response of
Fq'/Fm' of
mesophyll (, ) and guard cells (, ) to increasing
Ca in the green areas of a tradescantia
leaf in an atmosphere containing 2% (, ) or 21%
O2 (, ). Measurements were made at a PPFD
of 215 µmol m2 s1 and
an ambient temperature of 25°C.
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The responses of photosynthetic electron transport, as monitored by
Fq'/Fm', in
guard and mesophyll cells in response to changes in ambient
CO2 and O2 were
qualitatively similar (Figs. 5 and 6) suggesting that photosynthetic
metabolism in the two cell types is similar.
Responses to Changes in VPD
The effects of changes in VPD on stomatal aperture and
Fq'/Fm' of
guard cells in white areas of a tradescantia leaf at a
Ca of 80 or 220 µmol
mol1 are shown in Figure
7. Stomatal closure was stimulated by
rapidly decreasing the humidity in the leaf cuvette so that VPD
increased from approximately 1.0 to 1.5 to 3.0 to 3.5 kPa. At low
Ca (80 µmol
mol1), the initial effect of the increase in
VPD was a rapid hydropassive opening of the stomata over approximately
5 min (Fig. 7A), because of changes in the balance between epidermal
and guard cell turgor pressures (Ståfelt, 1955; Kappen et al., 1987;
Willmer and Fricker, 1996). The initial increase in stomatal aperture
was followed by a much slower decrease (over 2-3 h), which was
accompanied by a decrease in
Fq'/Fm'. When
VPD was decreased to the original level, both stomatal aperture and
Fq'/Fm'
increased to within a few percent of their original values (over a
period of approximately 1 h). At a higher
Ca of 200 µmol
mol1, the same increase in the VPD induced a
much more rapid closure of the stoma with little effect on
Fq'/Fm' (Fig.
7B). Returning the VPD to original levels at this higher
Ca resulted in a slow increase in stomatal
aperture over approximately 2 h (Fig. 7B).
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Figure 7.
The effects of changes in VPD on stomatal aperture
( ) and
Fq'/Fm' ()
from guard cells in white areas of a tradescantia leaf at a
Ca of 80 µmol
mol1 (A) or 220 µmol
mol1 (B). Stomatal aperture was determined from
reflected light images. Measurements were made at a PPFD of 215 µmol
m2 s1 and an ambient
temperature of 25°C.  indicate the time at when VPD was increased
from 1.0 to 1.5 kPa to 3.0 to 3.5 kPa;  indicate the time when VPD
was decreased back to 1.0 to 1.5 kPa.
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Similar experiments were conducted on a commelina leaf, but using
higher Ca values (180 and 330 µmol
mol1). At the Ca of
180 µmol mol1, the effect of increasing the
VPD on stomatal aperture was qualitatively the same (Fig.
8A) as was observed for the white areas
of the tradescantia leaf (Fig. 7A). However, the response was much more rapid and more extreme; a large, transient hydropassive movement being
followed by almost complete closure within 10 min. Decreases in
Fq'/Fm' within
both guard cell chloroplasts and mesophyll cells occurred almost as
rapidly within 15 min (Fig. 8A). Before the increase in VPD,
Fq'/Fm' of
guard cells was approximately 10% lower than from the mesophyll cells.
After the increase in VPD, this difference increased to approximately
15%. When the VPD was decreased to the original level, stomatal
aperture and
Fq'/Fm' of
both cell types also increased to the pretreatment levels. The recovery
of Fq'/Fm' in
both guard and mesophyll cells of commelina from the VPD-induced
decrease occurred considerably before the stomata were fully open. This
could reflect patchy stomatal opening on the lower (imaged) surface or
a more rapid opening of stomata on the upper surface of the leaf.
Commelina leaves are amphistomatous, unlike tradescantia, with the
ratio of stomatal density between upper and lower surface in our plant
material being approximately 1:3. At the higher
Ca of 330 µmol
mol1, a similar stomatal closing response to
VPD was observed, with Fq'/Fm' in
both the guard and mesophyll cells showing a small initial decrease,
which returned to its initial value within about 30 min, well before
any increase in stomatal aperture (Fig. 8B).
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Figure 8.
The effects of changes in VPD on stomatal aperture
() and
Fq'/Fm' from
mesophyll () and guard cells () in a commelina leaf at a
Ca of 180 µmol
mol1 (A) or 330 µmol
mol1 (B). Stomatal aperture was determined from
reflected light images. Measurements were made at a PPFD of 265 µmol
m2 s1 and an ambient
temperature of 25°C. indicate the time at when VPD was increased
from 1.0 to 1.5 kPa to 3.0 to 3.5 kPa; indicate the time when VPD
was decreased back to 1.0 to 1.5 kPa.
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DISCUSSION |
This is the first study, to our knowledge, to show the effect of
changing environmental conditions on PSII photochemical efficiency (measured by
Fq'/Fm')
within guard and mesophyll cells of intact, green leaves. Although
there have been a number of chlorophyll fluorescence studies on stomata
in white areas of variegated leaves (Zeiger et al., 1981; Cardon and
Berry, 1992), the PPFD response curves in Figure 4A show
Fq'/Fm' of
guard cells to be substantially lower in the white than in the green
areas of variegated tradescantia leaves. We agree with Scarth and Shaw
(1951) that the responses of the guard cells in white areas of leaves
cannot be considered to be indicative of the responses in green
tissues. Figure 7 also confirms that guard cells from albino portions
of leaves are slower to move than those in the green areas (Scarth
1932).
We found (Figs. 3 and 4A) that there was no difference in the dark
adapted Fv/Fm of
guard and mesophyll cells, whereas in the light
Fq'/Fm' of
guard cell chloroplasts was lower than that of mesophyll cells, which
confirms for intact, photosynthesizing leaves the results of Goh et al.
(1999) with Vicia faba protoplasts. A marked decrease
in Fv'/Fm'
upon illumination was observed for both mesophyll and guard cell
chloroplasts (Fig. 4B), which is consistent with the substantial
light-induced, non-photochemical quenching observed previously in
V. faba guard cell protoplasts (Goh et al., 1999). The above
results agree with other studies on guard cells of both V. faba and Arabidopsis, which indicated that some zeaxanthin was
maintained in the dark and that additional zeaxanthin was produced on
exposure to low light levels (Frechilla et al., 1999). The accumulation
of zeaxanthin would explain the observed rapid drop in
Fv'/Fm' at low
PPFD levels, because this could result in an increase in non-radiative
decay of excitation energy. Our data for both intact leaves (Figs. 2
and 4-8) and epidermal peels (Fig. 3) showing high PSII operating
efficiencies support the suggestion from a number of earlier studies
that guard cell chloroplasts are capable of photophosphorylation (Melis
and Zeiger, 1982; Shimazaki and Zeiger, 1985; Mawson and Zeiger, 1991;
Goh et al., 1999). The fact that
Fq'/Fm' is
only 20% to 30% lower in the guard cells compared with the mesophyll
cells (Figs. 3 and 4) would imply that the photosynthetic electron
transport rate in the guard cell chloroplasts is likely to be 70% to
80% of that in chloroplasts of mesophyll cells, if light absorption was similar, which seems a reasonable assumption. However, we have not
attempted to estimate electron transport rates from
Fq'/Fm', because there are uncertainties in the exact light absorption and
contribution of PSI fluorescence for guard and mesophyll chloroplasts.
Modulation of the steady-state fluorescence signal (F') from
guard cells within white areas of Tradescantia leaves by
ambient O2 concentration was observed by Cardon
and Berry (1992), and was taken as evidence for photorespiration (and
hence Rubisco oxygenase activity) within these cells. A major
difficulty with the interpretation of these data was that F'
is affected by both photochemistry (photochemical quenching) and
down-regulation (non-photochemical quenching). Consequently,
decreases in F' induced by increases in
O2 concentration, although consistent with an
increase in photochemical quenching of chlorophyll fluorescence
(because of increased photorespiration producing an increased rate of
electron transport), could also be attributable to increases in
non-photochemical quenching (down-regulation). However, we have
demonstrated a very clear codependence of the more definitive parameter
Fq'/Fm' in
guard cell chloroplasts on CO2 and
O2 concentration (Fig. 6). Decreasing the
concentration of ambient O2 at low
CO2 markedly decreased the PSII operating efficiency. As Cardon and Berry (1992) did, we interpret this O2-CO2 interaction as
strong evidence for Rubisco-mediated carbon assimilation as a sink for
the products of PSII electron transport in guard cell chloroplasts.
Furthermore, as the fluorescence in guard cells showed very similar
O2-CO2 sensitivity to that
of the mesophyll this must indicate that the proportion of electron transport being used by Rubisco is similar in the two cell types. This
Calvin cycle activity may have an important role in producing signal
metabolites (Outlaw, 1989; Reckmann et al., 1990; Goh et al., 1999).
Alternatively, if the above logic on the actual rates of photosynthetic
electron transport is accepted, the similarity of the responses of
mesophyll and guard cell chloroplasts suggests that Rubisco activity in
guard cell chloroplasts is broadly comparable with that of the
mesophyll cells. When extrapolating to the whole cell, the
photosynthetic activity in guard cells would be much lower compared
with the underlying spongy mesophyll (our results are from the abaxial
surface) due to the 20- to 50-fold lower chlorophyll content per cell
(Table III.1 in Willmer and Fricker, 1996). However, the guard cell
volume is approximately a tenth that of the spongy mesophyll cells, so
the possible contribution of such CO2
assimilation to the guard cell carbon balance might be about one-third
to one-tenth that of mesophyll cells. We can offer no explanation for
the difference between this fluorescence-derived, in vivo estimate and
the biochemical assays of Outlaw and colleagues (Outlaw et al., 1979;
Outlaw, 1989) and Reckmann et al. (1990), which have suggested minimal
Rubisco activity and Calvin cycle contribution (in broad bean
[V. faba] and pea [Pisum sativum], respectively).
Our consistent observation was that
Fq'/Fm' for
guard cell chloroplasts was lower than for mesophyll cells. The
difference between the two cell types varied considerably depending
upon the species and to a smaller extent on stomatal aperture, which ranged from approximately 5% lower in commelina leaves with fully open
stomata (Fig. 5B) to more than 40% lower in green areas of tradescantia with closed stomata (Fig. 6). Small differences may be
simply attributable to differences in the amount of light absorbed by
the two cell types. In addition, in green areas of tradescantia and
commelina with reduced stomatal aperture, the lower
Fq'/Fm' values
from guard cells compared with mesophyll cells could reflect reduced
availability of CO2 to the guard cells. However,
guard cells are obviously closer to the external atmosphere, and have numerous mitochondria (Willmer and Fricker, 1996), so this would be
surprising. Alternatively, differences in the response of the two cell
types to environmental conditions could produce differences in
organization and/or poising of the photosynthetic systems, which may
result in differences in the PSII operating efficiency.
The time courses of
Fq'/Fm' and
stomatal aperture changes shown in Figures 7 and 8 indicate that
Fq'/Fm'
of both the mesophyll and guard cells is strongly affected by
CO2 supply through the stomatal aperture.
Similarly, when the internal CO2 concentration was changed by manipulation of Ca changes
in Fq'/Fm'
were observed (Fig. 5). The simplest conclusion is that the change in
guard cell
Fq'/Fm' as
aperture changed was not due to any change in guard cell metabolism but
solely due to the change in CO2 diffusion through
the stomatal pore, because there was no change in
Fq'/Fm' at
high Ca even though aperture changes were
as large as those observed at low Ca (Figs.
7A and 8B). During preliminary studies it was observed that
illumination of a large area of the adaxial surface of the leaf was
necessary to determine reliable and reproducible responses of
Fq'/Fm'and
stomatal aperture to CO2,
O2, and humidity. When only a small area on the
abaxial leaf surface was illuminated through the microscope lens, then
the effects of high internal CO2concentrations
(presumably resulting from respiration in surrounding dark areas of the
leaf) overrode any influences of changes in Ca.
The highly sensitive response of
Fq'/Fm' to
CO2 supply mediated via the guard cell pore
emphasizes that stomatal aperture must to be taken into account in
assessments of photosynthetic activity using fluorimeters (Maxwell and
Johnson, 2000). It also reveals another key aspect of the sensitivity
of stomatal metabolism to CO2. Bulk stomatal
conductance derived from gas exchange measurements with attached leaves
has been shown to respond to changes Ci, not Ca (Mott, 1988). The data presented in
Figures 7 and 8 indicate that Ci must be
sensed by a guard cell metabolic process directly. If stomata responded
to Ca (or a combination of
Ca and Ci)
there would be either no (or a reduced) response of
Fq'/Fm' to
changes in stomatal aperture, and hence
Ci. However, the changes in guard cell
Fq'/Fm'
matched those of the adjacent mesophyll cells in all cases, indicating
that the CO2 supply route and sensitivity are the
same for both guard and mesophyll cells.
The responses of guard cell
Fq'/Fm' to
changing Ca (Figs. 5 and 6) indicate that
the largest changes occur below a Ca of 400 µmol mol1, and closely mirror the previously
observed response of stomatal conductance to changing
Ca (e.g. Morison, 1998). Many of the
physiological studies seeking to elucidate the mechanism behind the
CO2sensitivity of stomata have focused on large
changes and comparisons between Ca levels
of 350, 700, or 1,000 µmol mol1 and
CO2-free air, whereas leaf conductance
measurements show high sensitivity over changes of 50 µmol
mol1 or less (see reviews by Morison, 1998;
Assmann, 1999). Our measurements on guard cells in intact leaves
reveal, for the first time, large changes in
Fq'/Fm' in
response to changing Ca, and demonstrate that small changes in Ca produce large
changes in photosynthetic electron transport rate in guard cells.
However, whereas stomatal conductance declines with increasing
Ca, PSII operating efficiency increases.
Farquhar and Wong (1984) and Jarvis and Davies (1998) have proposed
that a carbon metabolite pool in the guard cells may be the link
between increased carbon assimilation and reduced stomatal conductance.
Our results showing CO2 and
O2 modulation of photosynthetic electron
transport and, by inference, Calvin cycle activity in the guard cells
are compatible with this hypothesis. In addition, our evidence for
Calvin cycle activities in both guard and mesophyll cells provides an
explanation for the parallel acclimation of CO2
assimilation rate and stomatal conductance sometimes observed when
plants are grown at high Ca (Morison, 1998;
Assmann, 1999; Lodge et al., 2001).
| |
CONCLUSIONS |
This study has shown that guard cell chloroplasts have a 20% to
30% lower quantum efficiency of photosynthetic electron transport, as
estimated from
Fq'/Fm', than
mesophyll cells. This measure of photosynthetic efficiency exhibited
similar responses in these two cell types to changes in light-,
CO2-, O2-, and
humidity-induced changes in stomatal aperture. We infer that
photosynthetic electron transport rates (on a per chlorophyll basis) in
guard cell chloroplasts are of similar magnitude to those in mesophyll
cells, and that the Calvin cycle and Rubisco are active in guard cell
chloroplasts of the two species examined. Photosynthetic electron
transport in guard cell chloroplasts was found to respond to
intercellular CO2 concentration
(Ci), not Ca,
and the sensitivity to changes in Ci was
similar to that of the response of stomatal conductance.
| |
MATERIALS AND METHODS |
Plant Material
Seeds of commelina (Commelina communis) were sown
in a peat- and loam-based compost (F2, Levington, Horticulture Ltd,
Ipswich, UK) in a controlled environment chamber (SGC066, Fitotron,
Sanyo Gallenkamp, Leicester, UK). After 3 weeks, seedlings were potted up into 100-mm pots and used 6 to 7 weeks after sowing. Cuttings of the
variegated plant tradescantia (Tradescantia albiflora) were grown in the same compost and environment chamber. The chamber air
temperature was maintained at 18°C at night, and 22°C through the
day. Light was provided by halogen quartz iodide lamps (Powerstar HQI-TS 250 W/NDL, Osram, Munich) from 6 AM to 10 PM, at a constant PPFD of 530 µmol m2
s1. Relative humidity was maintained at 70% through the
day and 65% at night. Plants were kept well watered using capillary matting.
The Imaging System
The optical part of the instrument used in these experiments is
essentially the same as that described previously (Oxborough and Baker,
1997a). One change has been modification of the lower light source
(which is used to illuminate the opposite side of the leaf to the one
being imaged) so that a much larger area of leaf (a circle of 1.5 cm in
diameter) is illuminated. This was required because only a small area
is illuminated by the upper light source (through the lens), and this
left the Ci dependent on diffusion within
the leaf from the surrounding (non-illuminated) tissue, rather than on
changes in stomatal aperture. With most leaves, very little of the
light from the lower light source is transmitted through to the upper
surface. However, the system has been designed in such a way that the
upper and lower light sources can be controlled independently. This
allows the change in light output from the upper and lower light
sources during a saturating pulse to be matched or for the lower light
source to be shuttered out completely while imaging is taking place. Chlorophyll fluorescence was defined by either a 680-nm bandpass filter
(Coherent, Watford, UK) or an RG 695 longpass filter (Schott, Mainz,
Germany). These filters were located within a filter wheel, located
between the camera and microscope, along with the 630-nm shortpass
filter used for reflected light images (Oxborough and Baker,
1997a).
A purpose-designed microscope cuvette attached to a portable
photosynthesis system (CIRAS2, PP Systems, Hitchin, Hertsfordshire, UK)
was used to control Ca and VPD. Cuvette and
leaf temperatures were maintained at between 25°C and 28°C for all
experiments. The stirred chamber was sealed to a modified
objective lens using a condom with the end cut off, providing an
uninterrupted light path between the lens and leaf.
All images were taken from the abaxial surface of leaves using a 40×
objective, which provides images of 310 × 205 µm with a pixel
size of (534 nm)2. This area covers an average of one to
two stomatal complexes on the surface of a tradescantia leaf or two to
three stomatal complexes on the surface of a commelina leaf. A
reflected visible light image was used to measure the maximum width of
the stomatal pore as an indicator of aperture. Chloroplasts within
guard cell pairs were isolated from images using the ends-in search and
other editing tools described in Oxborough and Baker (1997a) and
Oxborough et al. (2000). Figures 6 through 8 showing the effect of
CO2 and humidity on stomatal and mesophyll responses use
one representative example out of three experiments, because of the
considerable variability between plants. This variability is most
likely the result of varying internal CO2 concentrations
because of heterogeneity in stomatal aperture (not only those guard
cells being measured but also those in the surrounding area) and
photosynthetic activity.
Images of
Fv'/Fm'
and
Fq'/Fv'
were generated from images of Fo,
Fm, and
Fm'. This was achieved
through production of a virtual image of
Fo' (in computer memory),
calculated as described previously (Oxborough and Baker, 1997b). In the
specific case of chloroplasts within guard cells, stomatal movements
between the dark and light-adapted states often make it very difficult
to match up the locations of chloroplasts within all three required
images. Although this can prevent the generation of images of
Fv'/Fm'
and
Fq'/Fv',
it is still possible to generate mean values of
Fo, Fm, and
Fm' for the guard cell
chloroplasts within each image, which can then be used in the
calculation of mean values of
Fv'/Fm'
and
Fq'/Fv'.
The fluorescence imaging system was controlled by a computer program
called FluorImager (Technologia Ltd., Colehester, UK), which was
written in house using Microsoft Visual C++. FluorImager was run on a
dual Pentium PC under the Windows NT 4.0 operating system (Microsoft,
Redmond, WA).
Epidermal Peels
Epidermal peels were removed from commelina leaves using the
method of Weyers and Johansen (1985). The lower epidermis was removed
from intact leaves of 6-week-old commelina plants and floated on an
incubation buffer (50 mM KCl in 10 mM PIPES at
pH 6.8; Weyers, 1994) in 50-mm diameter Petri dishes. The Petri dishes were placed on a water bath, to maintain a temperature of 25°C, under
fluorescent lights, which provided an incident PPFD of 200 µmol
m2 s1. A small syringe needle attached to a
pump via tubing was inserted into the lid of the Petri dish to maintain
a constant air supply to the peels. The bubbles produced by the air
pumping also ensured a constant mixing of the buffer solution.
Statistical Analysis
Data shown in Figures 2 to 4 are means (±1 SE) from
three to five replicates (stomata on different leaves). The differences between cell type (Figs. 2 to 4) or filters (Fig. 2) was analyzed using
ANOVA, with a repeated measures design for the different PPFD levels,
except for the comparison of epidermal peel with intact leaf guard
cells (Fig. 3) where different PPFD values were used, and therefore
regression comparison was carried out.
We are grateful to Drs. Jörg Leipner and Ian McKee for
helpful discussions.
Received April 3, 2001; returned for revision June 17, 2001; accepted September 13, 2001.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.010317.
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ABSTRACT
INTRODUCTION