JXB Advance Access originally published online on May 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 388, pp. 1743-1752,
July 1, 2003
© 2003 Oxford University Press
The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar
Received 20 December 2002; Accepted 7 April 2003
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
* To whom correspondence should be addressed. Fax: +44 (0)1206 873416. E-mail: morisj{at}essex.ac.uk
Abbreviations: Ca, ambient CO2 concentration; F', chlorophyll fluorescence in the light-adapted state; Fm', chlorophyll fluorescence when PSII centres are maximally closed in the light-adapted state; Fo', chlorophyll fluorescence when PSII centres are maximally open in the light-adapted state; Fq', difference between F' and Fm'; Fq'/Fm', fluorescence parameter that provides an estimate of the operating efficiency of PSII photochemistry; Fq'/Fv', factor relating the operating and maximum efficiencies of PSII photochemistry; Fv', variable chlorophyll fluorescence (Fm'–Fo'); Fv'/Fm', fluorescence parameter that provides an estimate of the maximum efficiency of PSII photochemistry (when all PSII centres are open); PPFD, photosynthetic photon flux density; Rubisco, ribulose 1,5-bisphosphate carboxylase oxygenase; VPD, vapour pressure deficit.
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Abstract |
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High resolution chlorophyll a fluorescence imaging was used to compare the photosynthetic efficiency of PSII electron transport (estimated by Fq'/Fm') in guard cell chloroplasts and the underlying mesophyll in intact leaves of six different species: Commelina communis, Vicia faba, Amaranthus caudatus, Polypodium vulgare, Nicotiana tabacum, and Tradescantia albifora. While photosynthetic efficiency varied between the species, the efficiencies of guard cells and mesophyll cells were always closely matched. As measurement light intensity was increased, guard cells from the lower leaf surfaces of C. communis and V. faba showed larger reductions in photosynthetic efficiency than those from the upper surfaces. In these two species, guard cell photosynthetic efficiency responded similarly to that of the mesophyll when either light intensity or CO2 concentration during either measurement or growth was changed. In all six species, reducing the O2 concentration from 21% to 2% reduced guard cell photosynthetic efficiency, even for the C4 species A. caudatus, although the mesophyll of the C4 species did not show any O2 modulation of photosynthetic efficiency. This suggests that Rubisco activity is significant in the guard cells of these six species. When C. communis plants were water-stressed, the guard cell photosynthetic efficiency declined in parallel with that of the mesophyll. It was concluded that the photosynthetic efficiency in guard cells is determined by the same factors that determine it in the mesophyll.
Key words: Chlorophyll fluorescence, guard cell, mesophyll, photosynthesis, stomata, water stress.
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Introduction |
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The guard cells that form the stomatal pore control the flux of CO2, H2O and other gases between the plant and the atmosphere and are regulated by both internal and external factors. Stomatal movements are due to the loss or accumulation of ions, which require energy (Willmer and Fricker, 1996). In the majority of species examined guard cells contain well-developed chloroplasts, unlike the other epidermal cells from which they are formed. The role of these chloroplasts is still not clear, although they are not always essential to stomatal function since achlorophyllous guard cells do open and close (in Paphiopedilum sp., Nelson and Mayo, 1975). Guard cell chloroplasts have been proposed as significant energy sources for H+ extrusion and ion transport (Wu and Assmann, 1993; Tominaga et al., 2001), and are involved in several different light transduction pathways (Zeiger et al., 2002). Although many studies have shown that guard cells contain several of the main Calvin cycle enzymes (Shimazaki and Zeiger, 1985; Zemel and Gepstein, 1985; Gotow et al., 1988; Shimazaki et al., 1989), few have presented conclusive evidence for significant Calvin cycle activity within these cells (Outlaw, 1989; Reckmann et al., 1990), and there is continuing debate about the role and nature of guard cell photosynthesis (Zeiger et al., 2002). Chlorophyll fluorescence is a powerful, non-invasive technique to investigate photosynthetic metabolism in guard cells. The majority of chlorophyll fluorescence measurements from guard cells have been restricted to epidermal peels (Melis and Zeiger, 1982; Ogawa et al., 1982) or guard cell protoplasts (Goh et al., 1997, 1999, 2001) or have used variegated leaves; they have mainly been restricted to fluorescence induction curves or responses of the steady-state fluorescence signal (F'). However, using high spatial resolution chlorophyll a fluorescence imaging in intact green leaves of Commelina communis, it has recently been shown that guard cell quantum efficiency for PSII photochemistry (Fq'/Fm'=(Fm'–F')/Fm', ‘photosynthetic efficiency’) was approximately 70–80% of that of the mesophyll cells (Baker et al., 2001; Lawson et al., 2002) across a wide range of light intensities. It has also been shown that photosynthetic efficiency in both guard and mesophyll cells of C. communis was similarly altered by O2 concentration at low, but not at high CO2 concentration, indicating that photorespiration is a major sink for ATP and NADPH produced through electron transport in guard cells, and that Rubisco activity is significant (Lawson et al., 2002).
Some of the previous disagreements over the role of chloroplast photosynthetic activity in guard cells could be due to differences in plant material, as a range of species are widely used in stomatal physiological studies from very different taxa with different stomatal anatomy and often grown in different conditions. As Zeiger et al. (2002) have recently emphasized, guard cell chloroplasts show remarkable functional plasticity. The aims of this study were (1) to compare guard and mesophyll cell photosynthetic efficiencies in six species previously used in stomatal physiology, and (2) to investigate the response to different growth and measurement conditions, in particular light, CO2 and O2 concentrations, and water stress. The choice of species was largely dictated by whether the stomata were large enough to be imaged clearly, but included a fern (Polypodium vulgare) and a C4 species (Amaranthus caudatus) to compare with the C3 species usually used in stomatal physiology.
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Materials and methods |
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Plant material
Seeds of Commelina communis L., Nicotiana tabacum L. and Vicia faba L. 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 transplanted into 100 mm diameter pots and used 6–7 weeks after sowing in the case of C. communis and N. tabacum and 4–5 weeks in V. faba. Cuttings of the variegated plant Tradescantia albiflora Kunth. 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 (Neutralweiss, Germany) from 06.00–21.00 h, at a constant PPFD of 530 µmol m–2 s–1 at plant height. Relative humidity was maintained at 70% through the day and 65% at night. Amaranthus caudatus L. and Polypodium vulgare L. were grown from seed in the same compost and maintained in the glasshouse where supplementary lighting was provided by high-pressure sodium lamps and the temperature was maintained between 18 °C and 30 °C. All plants were kept well watered using capillary matting, except those in the drought stress experiment.
Growth treatments
Drought stress: Plants of C. communis were grown in the controlled environment chamber until about 6-weeks-old. Water was then withheld from half the plants for 12 d, while control plants were kept well watered. Leaf water potential was measured using a pressure chamber (SKPM1400, Skye Instruments, Powys, UK). After 12 d, stressed plants were rewatered, resulting in full recovery of the water potential after 2 d.
Elevated CO2: C. communis and V. faba were grown in two controlled environment chambers as above, but CO2 concentration was maintained at either 360 or 700 µmol mol–1 using an injection system operated by an infrared gas analyser (WMA-2, PP Systems).
Growth PPFD: Plants of C. communis and V. faba were grown in the above controlled environment chambers, but half of the chamber was shaded, using neutral density tissue paper to give PPFDs at the height of the plants of approximately 640 and 260 µmol m–2 s–1.
The microscope imaging system
The optical part of the imaging microscope used in these experiments is essentially the same as that described previously (Oxborough and Baker, 1997) with the modification of the lower light source and a purpose-designed microscope cuvette attached to an infrared gas analyser to control CO2, O2 and VPD as described by Lawson et al. (2002). CO2 of known concentration was supplied through the gas analyser system (CIRAS1; PP Systems, Hitchin, UK), and known O2 concentration was supplied from external gas bottles (BOC, Surrey, UK) attached to the air inlet on the gas analyser. Unless otherwise stated, conditions in the microscope cuvette were 23–25°C with 21% O2, a Ca of 360 µmol mol–1 and a VPD of approximately 0.6 kPa. Chlorophyll fluorescence was defined by a 680 nm bandpass filter (Coherent, Watford, UK). Fo' and Fm' define the minimal and maximal fluorescence levels from leaves in the light, respectively. F' is the fluorescence level at any point between Fo' and Fm'. For the construction of parameterized images, the specific term Fq' was recently introduced (Oxborough and Baker, 1997; Oxborough et al., 2000) which denotes the difference between Fm' and F' measured immediately before application of a saturating pulse to measure Fm'. Under these conditions, Fq'/ Fm' equates to the operating quantum efficiency of PSII photochemistry. Images of Fv'/Fm' and Fq'/Fv' were generated from images of Fo, Fm and Fm' as described previously (Lawson et al., 2002). There was no attempt to estimate rates of electron transport from Fq'/ Fm' because there are uncertainties concerning the exact light absorption and contribution of PSI fluorescence for the guard and mesophyll cell chloroplasts. All images were taken from the abaxial surface of leaves (except where stated) using a 40x objective, which provided images of 310x205 µm with a pixel size of 534 nm2. Replicates are individual stomatal complexes on leaves of different plants. The mesophyll areas used for comparison were those immediately adjacent to the guard cells. Chloroplasts within guard cell pairs were isolated from images using the ends-in search and other editing tools described in Oxborough and Baker (1997) and Oxborough et al. (2000).
Statistical analysis
Mean values of chloroplast photosynthetic efficiencies (Fq'/Fm') were calculated from the images, and differences between species, cell type (mesophyll or guard cell, abaxial or adaxial) or treatments (light, CO2 or O2 concentration) were compared using ANOVAs with mixed ‘between subjects’ (e.g. species) and ‘within subjects’ (e.g. cell type, CO2 or O2 concentration) designs as appropriate. The data in the regression in Fig. 6b were examined for the effect of any treatments using analysis of covariance. All statistical analyses were carried out with SPSS v. 10, or Systat v. 5.
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Results |
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Fluorescence images and guard cell chloroplast appearance
Images of steady-state fluorescence (F') from guard cells in the six species examined are shown in Fig. 1, and demonstrate the variation in stomatal pore and guard cell size in the different species and the differences in chloroplast number and orientation within individual guard cells. For example, in N. tabacum (Fig. 1d) the chloroplasts are located on the outer wall of the guard cells, whereas in T. albiflora (Fig. 1e) chloroplasts appear to be distributed evenly throughout the cells around the vacuole. Guard cells of the fern P. vulgare (Fig. 1c) contain many chloroplasts, as do the other epidermal cells, as previously noted (Willmer and Fricker, 1996). Despite these differences, all the chlorophyll fluorescence parameters were readily measured and hence photosynthetic electron transport efficiency (estimated by Fq'/Fm') could be calculated for both guard cells and underlying mesophyll cells in all of the species.
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Photosynthetic efficiencies in guard and mesophyll cells in different species
There were significant differences between species in Fq'/Fm' of guard and underlying mesophyll cells during steady-state photosynthesis under identical measurement conditions (species difference P <0.001; Fig. 2a). The fern P. vulgare exhibited the lowest efficiencies, with guard cell and mesophyll Fq'/Fm' values of 0.24 and 0.31, respectively, while the maximum values of 0.62 and 0.65, respectively, were found in V. faba. The low values of Fq'/Fm' observed in P. vulgare are notable, particularly as the large number of chloroplasts present might have been expected to result in a high degree of self-shading, which would reduce the average incident-light intensity and, consequently, result in a higher photosynthetic efficiency. One explanation might be the higher light level during growth reducing photosynthetic efficiency as these plants were glasshouse-grown, but A. caudatus was grown in a similar environment and yet showed higher efficiencies. For P. vulgare, N. tabacum and T. albiflora guard cell Fq'/Fm' values were 79%, 92% and 87% of the values of the adjacent mesophyll cells, respectively, although no significant differences were observed for A. caudatus, V. faba and C. communis (overall cell type difference P <0.001; speciesxcell type interaction P=0.049), and there was a close linear correlation between mesophyll and guard cell Fq'/Fm' (Fig. 2b).
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Effect of growth and measurement PPFD
Photosynthetic efficiency is dependent both on measurement PPFD and on growth conditions which affect the development of the photosynthetic apparatus, as exemplified by the large differences observed for sun and shade leaves (Pearcy, 1998). In species that are amphistomatous, stomatal guard cells provide an interesting system to examine the effect of growth PPFD on photosynthetic behaviour, as guard cells in the upper surface are exposed to much higher PPFD than those on the lower surface. In C. communis and V. faba, plants grown at moderate light intensity (530 µmol m–2 s–1) guard cell Fq'/Fm' in both upper (adaxial) and lower (abaxial) leaf surfaces decreased with increasing incident PPFD (Fig. 3; P <0.001). The two species differed in the response of the photosynthetic efficiency of the upper and lower guard cells to PPFD (P=0.003). In C. communis the decrease of Fq'/Fm' was substantially larger in guard cells in the lower surface (Fig. 3a). In V. faba at the lowest measurement PPFD of 93 µmol m–2 s–1 guard cells in the lower surface operated at a higher efficiency than those in the upper surface (Fig. 3b). However, as PPFD increased there were large decreases in efficiency in guard cells in both surfaces and no significant differences were observed between the guard cells on the lower and upper surfaces (Fig. 3b). Fq'/Fm' was lower in guard cells of V. faba than those from C. communis at the higher PPFD levels, particularly in the upper surface.
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The influence of growth PPFD on the lower surface stomata and any possible acclimation to light was studied by growing C. communis and V. faba at PPFD of 260 and 640 µmol m–2 s–1 and measuring photosynthetic efficiency at PPFDs of 93 and 428 µmol m–2 s–1 (Fig. 4). While Fq'/Fm' declined in higher measurement PPFD in both cell types in both species (P <0.001), overall the photosynthetic efficiencies measured in V. faba were about 0.10 lower than those of C. communis (overall species difference P=0.026), and there was a larger relative response of Fq'/Fm' to measurement PPFD in V. faba (P=0.012). There was also a small (but significant, P=0.018) difference between species in the response to growth PPFD, with C. communis plants grown at 640 µmol m–2 s–1 having a higher Fq'/Fm' for both guard and mesophyll cells, regardless of the measurement PPFD (Fig. 4a). In contrast, V. faba showed no differences in Fq'/Fm' between plants grown under the two different light intensities (Fig. 4b). While there were no significant differences between Fq'/Fm' of guard and mesophyll cells in each combination of growth and measurement PPFD treatments for C. communis, guard cells in V. faba had significantly lower Fq'/Fm' values (approximately 8%) than the mesophyll cells, for all growth and measurement PPFDs (overall cell typexspecies interaction P=0.018). Both the short-term responses of Fq'/Fm' to measurement PPFD and the longer term acclimation to growth PPFD were very similar for both guard and mesophyll cells (Fig. 4c).
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Effect of CO2 and O2 concentration
The effects of CO2 and O2 concentration on the photosynthetic efficiencies of the guard and mesophyll cells were determined for the six species (Table 1). As in the previous experiments, Fq'/Fm' of the guard cells were lower than those of the adjacent mesophyll in some, but not all, species (significant cell typexspecies interaction P=0.003). Overall, there was a more pronounced increase of Fq'/Fm' with an increase of [CO2] from 150 to 360 µmol mol–1 for the C3 species than for the C4 species Amaranthus caudatus (overall CO2xspecies interaction P=0.036; if data from A. caudatus are excluded there is no significant interaction, P=0.274). In mesophyll cells in the five C3 species, decreases of [O2] from 21% to 2% decreased Fq'/Fm' substantially at the lower [CO2] (7–26% reduction), but not in higher [CO2] (there were no significant species differences in response to CO2, P=0.232, but there was a significant overall CO2xO2 interaction, P=0.036). The reduction in Fq'/Fm' in low [O2] or low [CO2] was almost entirely due to a decrease in Fq'/Fv', with little change being observed in Fv'/Fm' (data not shown), indicating that the reduced availability of terminal electron acceptors for electron transport from PSII was primarily responsible for the decrease in photosynthetic efficiency. In the C4 species no effects of [O2] on Fq'/Fm' of the mesophyll at either [CO2] were observed. Clearly, in low [CO2] in the mesophyll of C3, but not C4 species, O2 is acting as a sink for the products of photosynthetic electron transport due to Rubisco oxygenase activity and photorespiratory metabolism. However, low [O2] decreased Fq'/Fm' of the guard cells in all six species (range from 7–24%) at both CO2 concentrations (O2 effect P <0.001, no CO2xO2 interaction, P=0.301), indicating that Rubisco is active in the guard cells of the C4 species as well as of the C3 species.
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Effect of growth CO2 concentration
The effects of growth [CO2] (360 or 700 µmol mol–1) on Fq'/Fm' was examined for C. communis and V. faba to determine if photosynthetic acclimation resulted in different behaviour of guard cells compared with mesophyll cells (Fig. 5). Fq'/Fm' of guard cells was slightly (2–9%) lower than that of mesophyll cells, except for V. faba grown in high [CO2] where there was no difference (overall cell type effect, P=0.002). Fq'/Fm' in both guard and mesophyll cells increased by approximately 10% with an increase in measurement [CO2] from 150 to 360 µmol mol–1, but not in higher [CO2]. The effect of growth [CO2] on Fq'/Fm' of both guard and mesophyll cells differed between the two species (P=0.014). In C. communis, growth in high [CO2] substantially reduced Fq'/Fm' of both guard and mesophyll cells in all CO2 measurement concentrations (Fig. 5a; P <0.001), whereas V. faba showed no significant effect of growth CO2 concentration (Fig. 5b). The decrease in Fq'/Fm' in C. communis was attributable to a decrease in Fq'/Fv' since Fv'/Fm' did not change significantly (data not shown); again this indicates that the decrease in photosynthetic efficiency is due to a decrease in the ability to use the products of electron transport.
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Effect of water stress on the relationship between guard and mesophyll cells
Close correlations were found between the values of Fq'/Fm' of guard and adjacent mesophyll cells for different species and in differing measurement and growth conditions (Figs 2c, 4c, 5c). To examine if such correlations between the guard and mesophyll cell photosynthetic efficiencies are conserved during water stress, C. communis plants were subjected to mild water stress by withholding water for 12 d. Leaf water potentials decreased to approximately –0.75 MPa in the drought-stressed plants, while well-watered controls remained at c. –0.05 MPa throughout. Stomatal aperture showed a steady decline as water was withheld, but recovered within 48 h after rewatering (data not shown). Both guard and mesophyll cell Fq'/Fm' declined during the stress, but recovered along with stomatal aperture 48 h after rewatering (Fig. 6a). This decrease in Fq'/Fm' was again largely due to a decrease in Fq'/Fv' not in Fv'/Fm' (data not shown), again indicating the limitation on photosynthetic efficiency being the ability to use the products of electron transport. However, there was a close linear relationship between guard and mesophyll cell Fq'/Fm' as the values changed through the drying cycle which was not distinguishable from that of the control plants (Fig. 6b, pooled regression for all treatments r2=0.902). Increasing measurement Ca from 360 to 700 µmol mol–1 resulted in a substantial increase in Fq'/Fm' of both guard and mesophyll cells in the water-stressed plants (mean increase at the end of the cycle was from 0.23 to 0.37 for mesophyll cells), but only a small increase was observed in control plants (Fig. 6b). However, even with increased CO2 concentration Fq'/Fm' of drought-stressed plants did not increase to that of the control plants. This suggests that as water stress increased, stomata adjusted to reduce water loss, and restricted CO2 diffusion into the leaf, resulting in a decrease in Fq'/Fm' through a reduction in the capacity for the consumption of the products of electron transport. The relationship between Fq'/Fm' of the guard and mesophyll cells was not affected by CO2 or water stress (although there is a statistically significant difference of the intercept for the water stress treatment (P <0.001) at 0.028 it is negligible).
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Discussion |
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Much of the previous work investigating guard cell photosynthesis has been confined to species such as C. communis or V. faba due to the ease with which the epidermis can be removed from the leaf and the ability to obtain uncontaminated guard cell protoplasts (Willmer and Fricker, 1996). In this study, photosynthetic efficiency in guard cells during steady-state photosynthesis has been measured in intact leaves from a number of species. Taxonomically, these species are very diverse, including one fern and five angiosperms, and the angiosperms include two monocots (C. communis and T. albiflora, both in the Commelinaceae) and three dicots, each from families in different subclasses (V. faba: Fabaceae, subclass Rosidae; A. caudatus: Amaranthaceae, Caryo phillidae; and N. tabacum: Solanaceae, Asteridae). Consequently, it is believed that these conclusions on the responses of the photosynthetic efficiency of guard cells to environmental factors have considerable general application. In leaves of all species the values of photosynthetic efficiency for guard cells were either indistinguishable from or only slightly lower (minimum of 79%) than those of the underlying, spongy mesophyll cells. In all species examined the responses of guard and mesophyll photosynthetic efficiency to changes in light, [CO2] and [O2] were similar, although there were demonstrable differences between species in the actual values of guard cells and mesophyll, and their responses to growing conditions (Figs 2–5; Table 1). Some of these differences may be due to the plants being grown under different environmental conditions, as it has been demonstrated that photosynthetic efficiency can vary with the growth environment (Figs 4– 6). However, the lower values of Fq'/Fm' for T. albiflora than for four other species from the chambers is consistent with previous work (Lawson et al., 2002). The lower efficiencies observed in T. albiflora and P. vulgare are also reflected in lower photosynthetic rates compared with the other species, indicating lower intrinsic light use efficiencies for photosynthesis. These two species also tend to have a greater number of chloroplasts in the guard cells than other species, which may result in increased light absorption and a reduced efficiency at any given incident PPFD.
Effect of light
Some differences were observed in the values and responses of photosynthetic efficiency between the guard cells on the adaxial and abaxial surfaces, and the way it was affected by PPFD (Fig. 3). In V. faba a difference was observed only at low PPFD, agreeing with the report of similar photosynthetic rates per unit chlorophyll for adaxial and abaxial guard cell protoplasts from the same species by Goh et al. (1997). In C. communis the different light responses of the guard cells in adaxial and abaxial surfaces were similar to those seen in leaf photosynthetic responses for sun and shade leaves, respectively (Pearcy, 1998). In addition, photosynthetic efficiency of C. communis guard and mesophyll cells was affected by the PPFD during growth, while this was not the case in V. faba. The contrast between the two species may be due to the growth habit. Leaves of C. communis are more horizontal, consequently the adaxial surface is better illuminated than in V. faba, and the abaxial surface only receives the light that is transmitted though the leaf. On the other hand, leaves of V. faba are less rigid in their angle to the stems, and are frequently twisted, exposing both surfaces to similar, but reduced, PPFD. Such differences in photosynthetic efficiency may lie behind the higher sensitivity of abaxial stomata to light (Travis and Mansfield, 1981), and may be associated with differences in guard cell pigment content (Lu et al., 1993; Goh et al., 1997).
Effect of changes in CO2 and O2
In the C3 species, the photosynthetic efficiencies of the guard and mesophyll cells were reduced at low CO2 concentration (Fig. 4; Table 1). Similar reductions were observed in Fq'/Fm' of both the guard and the mesophyll cells in five C3 species when the ambient O2 was decreased from 21% to 2% at low CO2 concentrations (Table 1). We argue, as others have done (Cardon and Berry, 1992), that such effects of CO2 and O2 on photosynthesis indicate that Rubisco is a major sink for the products of photosynthetic electron transport in guard cells, and confirm and extend earlier evidence for this from T. albiflora (Lawson et al., 2002). The argument is strengthened by the lack of response of Fq'/Fm' to [O2] in the mesophyll in Amaranthus caudatus, a C4 species, which is as expected as there is no Rubisco activity in mesophyll cells of C4 leaves. However, there was an O2 response in the guard cells (Table 1) which agrees with recent immunogold labelling studies on Amaranthus viridis which showed substantial amounts of Rubisco in guard cells, but no labelling in mesophyll cells (Ueno, 2001). Consequently, it appears that although the mesophyll cells of the C4 leaves lack Rubisco and do not operate a carbon reduction cycle as expected, the guard cells do exhibit Rubisco and Calvin cycle activity.
Furthermore, the guard cells in C. communis showed the same reduction in Fq'/Fm' that occurred in the mesophyll cells in response to growth in high [CO2] (Fig. 5). The current explanations for photosynthetic acclimation during growth in high CO2 are either that increased carbohydrate supply is not matched by sink demand, thus inhibiting Calvin cycle activity either by limiting RuBP regeneration or by causing the down-regulation of Rubisco or that the amount of Rubisco is reduced due to a change in N allocation (Drake et al., 1997). Therefore, the matching reduction of photosynthetic efficiency in the guard and mesophyll cells in high CO2 also argues for comparable Calvin cycle activity in guard and mesophyll cells. Such Rubisco regulation in both guard and mesophyll cells may be the mechanism behind the parallel acclimation of stomatal conductance and mesophyll photosynthesis to high [CO2] sometimes observed (Morison, 1998; Assmann, 1999; Lodge et al., 2001).
Effect of water stress
During slowly imposed water stress, there were parallel declines in the photosynthetic efficiencies of the guard and mesophyll cells over a time-course of days (Fig. 6). Goh et al. (2001) have described declines in photosynthetic efficiency in the guard and mesophyll cell protoplasts under hypertonic osmotic stress, but this was accompanied by declines in photochemical and non-photochemical quenching. In their experiment there were differences between the guard and mesophyll cells, but they imposed rather severe osmotic stress on cells without walls. Some of the decline in photosynthetic efficiency in this study was due to reductions in CO2 supply since doubling Ca increased Fq'/Fm' and reductions of Ca below ambient reduced Fq'/Fm' markedly, as shown in Fig. 5 (see also Lawson et al., 2002). However, it is also likely that some of the decline in Fq'/Fm' was due to other water stress effects causing ‘impaired photosynthetic metabolism’ (see review by Lawlor, 2002) because increased Ca did not completely offset the decline in Fq'/Fm'. Previous work indicates that Ca, and by inference intercellular [CO2], would have to be very low to cause the declines observed here (Lawson et al., 2002; Fig. 5b). While these results cannot distinguish which of the possible mechanisms are involved in this photosynthetic inhibition, an important result is that both the guard and mesophyll cells are similarly affected. This suggests that if guard cell photosynthetic electron transport or Calvin cycle activity is important to aperture maintenance, then there must be an additional positive feedback of reduced photosynthesis in guard cells during water stress, contributing to reductions in stomatal aperture.
Photosynthesis in guard cells
There is a long-standing controversy over the role and activity of the Calvin cycle in guard cells (see recent review by Zeiger et al., 2002). A possible explanation for the discrepancy between results indicating substantial Calvin cycle activity and those concluding that there is little activity is that photosynthetic regulation in guard cells reflects the pretreatment of leaves and the measurement conditions (Zeiger et al., 2002). For example, it has been proposed that the accumulation of sucrose near the guard cells in the apoplastic phloem loader V. faba may suppress Calvin cycle enzymes (Lu et al., 1997) so that studies of guard cell photosynthetic activities using intact leaves may give different results to those with peels. Secondly, Talbott and Zeiger (1996) observed changes in the osmotic regulation of guard cells of V. faba during the day, with K+ being the main osmoticum early in the day, but replaced by sucrose later. Thirdly, the light regulation of stomatal function is complex: both blue and red light stimulate photosynthesis and sucrose accumulation in guard cells (Tallman and Zeiger, 1988; Talbott and Zeiger, 1993), but the relative proportion of red and blue light appears to change the balance between the starch–sugar and the K+–malate osmotic mechanisms (Talbott and Zeiger, 1993). The present work provides strong evidence for Calvin cycle activity in guard cells of six species, including V. faba, during steady-state photosynthesis in intact leaves, using moderate to high fluence rates of blue light when sucrose supply in the apoplast should have been substantial. Such moderate and high fluence rates will have driven normal photosynthetic activity in the guard cells (Wu and Assmann, 1993) and this probably contributed to stomatal opening in addition to any low intensity, chlorophyll-independent blue light response (Tallman and Zeiger, 1988; Taylor and Assmann, 2001). Furthermore, these measurements of guard cell photosynthetic efficiency agree well with those of Goh et al. (1999, 2001) using chlorophyll fluorescence in the guard cell protoplasts.
An important result emerging from these studies is that photosynthetic efficiency of guard cells in intact leaves responded quantitatively to light, CO2, O2 and water stress in a similar way to adjacent mesophyll cells. As highlighted by Wong et al. (1979), there is often a close positive correlation at the leaf scale between stomatal conductance and mesophyll CO2 assimilation rate across a range of environmental conditions. This close relationship has been attributed to the influence of internal CO2 concentration (Raschke, 1976), but there have also been suggestions that there is another signal transmitted from the mesophyll cells to the guard cells such that mesophyll photosynthesis controls the degree of stomatal opening (Heath and Russell, 1954; Wong et al., 1979; Lee and Bowling, 1995). However, the nature of any such messengers is not clear. Sucrose movement within the transpiration stream has been suggested recently by Outlaw and colleagues, as this has been shown to be a major source of organic carbon for the guard cells, and can also exert an osmotic effect by accumulation in the cell apoplast (Lu et al., 1997; Outlaw and De Vlieghere-He, 2001). Alternatively, photosynthetic metabolism in the guard cells may be behind the co-ordination of the stomata and the mesophyll (Farquhar and Wong, 1984; Jarvis and Davies, 1998). Figures 2c, 4c, 5c, and 6b show that a close, linear correlation between the guard cell and mesophyll photosynthetic activity exists at the cell level. This suggests that the guard cell photosynthetic activity may provide the sensing mechanism linking stomatal movement to mesophyll photosynthetic rate.
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Acknowledgement |
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This work was funded by the BBSRC (grant 84/P10409).
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References |
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