Journal of Experimental Botany, Vol. 52, No. 361, pp. 1689-1696,
August 1, 2001
© 2001 Oxford University Press
Original Papers |
Primary sites of ozone-induced perturbations of photosynthesis in leaves: identification and characterization in Phaseolus vulgaris using high resolution chlorophyll fluorescence imaging
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK
Received 24 November 2000; Accepted 9 April 2001
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Abstract |
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High resolution imaging of chlorophyll a fluorescence was used to identify the sites at which ozone initially induces perturbations of photosynthesis in leaves of Phaseolus vulgaris. Leaves were exposed to 250 and 500 nmol mol-1 ozone at a photosynthetically active photon flux density of 300 µmol m-2 s-1 for 3 h. Images of fluorescence parameters indicated that large decreases in both the maximum and operating quantum efficiencies of photosystem II had occurred in cells adjacent to stomata in the upper, but not lower, leaf surfaces. However, this treatment did not produce any significant changes in the maximum or operating quantum efficiencies of photosystem II in the leaves when estimated from fluorescence parameters measured with a conventional, integrating fluorometer. The localized decreases in photosystem II photochemical efficiencies were accompanied by an increase in the minimal fluorescence level, which is indicative of photoinactivation of photosystem II complexes and a decrease in stomatal conductance. Perturbations of photochemical efficiencies were not observed in cells associated with all of the stomata on the upper leaf surface or within cells distant from the upper leaf surface. It is concluded that ozone penetrates the leaf through stomata and initially damages only cells close to stomatal pores.
Key words: Electron transport, fluorescence imaging, ozone, photosynthesis, stomata.
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Introduction |
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Pollution of the troposphere by ozone is a major problem in many regions of the world and has been associated with declines in crop productivity and changes in natural ecosystems (Heath, 1996
). Prolonged exposure of leaves to ozone produces patches of visible injury across the leaf laminae, which eventually develop into large areas of chlorotic tissue (Hill et al., 1961). While it is generally assumed that ozone produces damage to leaf tissues by moving through stomata and reacting with cell components in the sub-stomatal cavities (Heath, 1996), the primary sites of attack of ozone within leaves have not been definitively identified.
Ozone is highly reactive and is thought to react rapidly with a range of compounds associated with cell walls and membranes. Consequently, it is unlikely to move into the cytoplasm of cells and directly react with internal cellular components (Heath, 1987, 1996). Photosynthesis is particularly sensitive to ozone, with changes in stomatal conductance and decreases in the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) occurring rapidly on exposure of leaves to ozone (Heath, 1994, 1996). Changes in stomatal conductance may be associated with ozone-induced modifications of the plasmalemmae of guard cells that affect the ionic relations of the cells (Castillo and Heath, 1990; Torsethaugen et al., 1999). Decreases in Rubisco activity cannot be attributed to direct effects of ozone on the enzyme, since it is highly unlikely that ozone will reach the chloroplasts; the intercellular concentration of ozone having been estimated at very close to zero in leaves exposed to the high level of 1500 nmol mol-1 (Laisk et al., 1989). Such effects on Rubisco are likely to result from ozone-induced changes in cell signalling which affect metabolism and gene expression (Heath, 1996). Irrespective of the mechanism(s) by which ozone decreases photosynthetic activities, such rapid changes in photosynthesis offer the possibility of probing the kinetics of ozone penetration and damage in leaves.
It has long been appreciated that photosynthetic activities of leaves can be monitored non-invasively by chlorophyll fluorescence (Krause and Weiss, 1991). The recent development of high resolution imaging of chlorophyll fluorescence from intact leaves has enabled images of the relative quantum efficiency of photosynthetic electron transport in tissues, individual cells and even chloroplasts in situ to be produced (Oxborough and Baker, 1997a, b; Oxborough et al., 2000; Baker et al., 2001). In this study high resolution fluorescence imaging is used to identify the primary sites of damage and to characterize some features of the damage when amphistomatous bean leaves are exposed to ozone. Damage was detected and characterized in leaves which exhibited no significant changes in chlorophyll fluorescence parameters monitored by conventional modulated fluorometers.
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Materials and methods |
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Growth and treatment conditions
Seeds of Phaseolus vulgaris cv. Vilbel (Nickerson-Zwaan, Barendrecht, The Netherlands) were sown in 500 ml pots containing modular compost (F2, Levington, Ipswich, UK). Plants were grown in a controlled environment cabinet (SGC066, Fitotron, Sanyo Gallenkamp, Leicester, UK) under a 16 h photoperiod at a PPFD of 350 µmol m-2 s-1, 70/80% (day/night) relative humidity, and 23/20 °C (day/night). The first leaves were investigated as soon as they were fully expanded.
Leaves were fumigated individually inside a leaf chamber (ADC, Hoddeson, Herts., UK) connected to a heater/circulator (R20, Haake, Berlin, Germany) and refrigeration unit (Tecam, Cambridge, UK), which maintained air temperature in the chamber at 23 °C. Ozone was generated from a UV source by a combined generator and analyser unit (1008-PC, Dasibi Environmental Corp., Glendale, CA). Prior to ozonation air was then passed through a water trap at 15 °C to maintain a relative humidity of 60%. The leaves were irradiated with a 75 W halogen lamp (Halogen Xenophot, Osram, Germany) placed above the leaf chamber. In all experiments leaves were subjected to a PPFD of 300 µmol m-2 s-1 for 1 h prior to exposure to ozonated air containing either 250 or 500 nmol mol-1 ozone.
Chlorophyll a fluorescence measurements and imaging
During ozone fumigation, chlorophyll fluorescence was monitored using a PAM-2000 fluorometer (Walz, Effeltrich, Germany). With dark-adapted material, the minimal (Fo) level of fluorescence was recorded at a very low PPFD of less than 1 µmol m-2 s-1, while the maximal (Fm) level of fluorescence was recorded during a 0.8 s pulse at a PPFD of c. 6000 µmol m-2 s-1. Fv/Fm gives the maximum quantum efficiency of PSII photochemistry in the dark-adapted state. This was measured before and after ozone fumigation following a 20 min dark adaptation. The term 
has recently been introduced to denote the difference between 
and F' measured immediately before application of the saturating pulse used to measure (Oxborough et al., 2000; Baker et al., 2001). / is theoretically proportional to the operating quantum efficiency of PSII photochemistry (Genty et al., 1989), often referred to as the PSII operating efficiency. / has previously been termed 
F/, (-Ft)/ and (-Fs)/ (where F=, and Ft and Fs equate to F'), and has been widely used to estimate the relative quantum efficiency of linear electron transport through PSII (
PSII). A more detailed description of this new, improved nomenclature has been given previously (Baker et al., 2001).
After ozone fumigation, treated plants were transferred to the high resolution chlorophyll fluorescence imaging system. Images were taken from two areas of the upper surface and one area of the lower surface of each leaf. Leaves were allowed to adapt to a change in PPFD for at least 15 min before images were taken. The optical part of the instrument used in these experiments is essentially the same as that described previously (Oxborough and Baker, 1997a). Fluorescence was detected using either a 680 nm bandpass filter (Coherent, Watford, England) or a 695 nm longpass filter (Schott, Mainz, Germany). Fluorescence emitted at 680 nm by cells within the leaf is strongly re-absorbed by chloroplasts within the overlying cells. Consequently, use of the 680 nm bandpass filter gives greater weighting to fluorescence emitted from cells at the surface of the leaf. Reflected light images were taken using a 630 nm short pass filter. The fluorescence imaging system was controlled by a computer program called FluorImager, which was developed in-house using Microsoft Visual C++. This program was run on a dual Pentium PC under the Microsoft Windows NT 4.0 operating system. Images were taken from both surfaces of leaves using a 40x objective, which provides images of 310x205 µm with a pixel resolution of (534 nm)2. FluorImager was used to generate images of Fv/Fm and / from images of Fo, Fm, F ' and .
Stomatal conductance
Leaf conductance measurements were taken from both leaf surfaces using a diffusion porometer (Delta-T AP4, Delta-T Devices, Cambridge, UK) fitted with a standard cuvette.
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Results |
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Ozone-induced effects on fluorescence parameters
The effects of exposure to 250 and 500 nmol mol-1 ozone for 3 h at a PPFD of 300 µmol m-2 s-1 on the photosynthetic performance of bean leaves were assessed by monitoring Fv/Fm and
/ using a modulated, integrating fluorometer (Table 1). After 3 h exposure to both 250 and 500 nmol mol-1 ozone, there were no significant changes in these fluorescence parameters, indicating that ozone was not inducing any significant perturbations of leaf photosynthesis. Significant decreases were observed when the incident PPFD during the ozone exposure was increased to 480 µmol m-2 s-1 (Table 1), thus demonstrating the onset of the expected light-dependent inhibition of photosynthesis by ozone in the leaves.
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/
) of leaves of Phaseolus vulgaris at actinic PPFDs of 300 and 480 µmol m-2 s-1 after 3 h exposure to 250 and 500 nmol mol-1 ozone
Identification of sites of ozone damage
A primary objective of this study was to identify the primary sites of ozone damage in leaves. In order to do this, it was important to examine leaves that were not showing significant levels of ozone damage. From the integrated measurements of fluorescence parameters using a modulated fluorometer reported above, it was evident that leaves exposed to 250 and 500 nmol mol-1 ozone for 3 h at a PPFD of 300 µmol m-2 s-1 exhibited no measurable perturbation of photosynthesis. Consequently, these treatments were used in the fluorescence imaging studies with the aim of identifying primary sites of ozone attack in the leaves prior to the onset of severe damage throughout the leaves.
The fluorescence parameters, / and Fv/Fm, were routinely used to estimate the operating and maximum quantum efficiencies of PSII photochemistry, respectively, of leaves. Images of / and Fv/Fm from control leaves are shown in Fig. 1. Images of / from the upper surfaces of leaves were produced using the 680 nm bandpass (Fig. 1D) and 695 nm longpass (Fig. 1J) filters. The fluorescence signal at 680 nm arises primarily from chloroplasts in cells in the upper cell layers of the leaf. This is because fluorescence at this wavelength is strongly absorbed by chlorophyll within the overlying cells. Consequently, this fluorescence from cells distant from the leaf surface has a high probability of being absorbed by chlorophyll in cells between the site of emission and the leaf surfaces. Chlorophyll does not absorb strongly above c. 685 nm. Consequently, the images produced using the 695 nm longpass filter will contain a much higher contribution from cells below the surface. Images of / produced using the 680 nm bandpass filter (Fig. 1D) show lower efficiencies of PSII electron transport and a greater heterogeneity than is the case for images constructed from fluorescence measured using the 695 nm longpass filter (Fig. 1J). The heterogeneity does not appear to be associated with the stomata, whose positions can be identified from reflected light images of the leaf surface.
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After exposure of a leaf to 250 nmol mol-1 ozone for 3 h at a PPFD of 300 µmol m-2 s-1, images of
/ using the 680 nm bandpass filter from leaves exposed to a PPFD of 270 µmol m-2 s-1 contained regions of low photosynthetic efficiencies (Fig. 1E
). A similar situation was observed for images of / constructed using the 695 nm longpass filter (Fig. 1K), however, the areas of reduced efficiencies were not as great as for the 680 nm bandpass fliter images (Fig. 1E), suggesting that the primary sites of ozone damage are close to the leaf surface. Although the majority of the large ozone-induced depressions in the PSII operating efficiency were occurring in cells adjacent to stomata, occasional patches of damage remote from the stomata were identified. Examples of this are shown in Fig. 1E and K. It was also evident that ozone damage does not occur around every stomate. The nature of the heterogeneity of photosynthesis induced by the ozone treatment is clearly quite different from heterogeneity observed in control leaves, since the distribution of / values around the mean was normal in control leaves (Fig. 1G), but skewed to lower values, producing an asymmetric distribution, in the ozone-treated leaves (Fig. 1H). The heterogeneity in photosynthetic efficiency was also found to be greater when monitored using the 680 nm bandpass filter (Fig. 1E) rather than the 695 nm longpass filter (Fig. 1K), suggesting that the effects of the ozone are mediated closer to the leaf surface. Decreases in the efficiency of PSII electron transport are associated with decreases in Fv/Fm (Fig. 1N), suggesting that photoinhibition of PSII is associated with the ozone-induced loss in photosynthetic efficiency.
When the ambient concentration of ozone was increased from 250 to 500 nmol mol-1, the effects on the leaf photosynthetic performance were, surprisingly, slightly reduced after 3 h of exposure (Fig. 1, compare E and F). This could indicate that stomata close more rapidly at the higher ozone concentration. The greatest inhibitions of photosynthetic efficiency were observed in cells immediately adjacent to stomata and located close to the leaf surface (Fig. 1F). As with the 250 nmol mol-1 ozone treatment, not all stomata were associated with affected cells, suggesting that some stomata were closed during the treatment and did not allow movement of ozone into the leaf. An estimate of the number of stomata associated with adjacent cells which had large decreases in photosynthetic efficiencies indicated that for the 250 nmol mol-1 ozone treatment 63% of stomata were affected compared to only 38% in the 500 nmol mol-1 ozone treatment (data not shown).
By focusing on cells in the palisade parenchyma, it was observed that parenchyma cells with low PSII operating efficiencies exhibited a considerably higher fluorescence (F ') signal (Fig. 2). These cells also appeared to have their fluorescence emissions concentrated into a smaller area of the cell than was the case for cells with higher photosynthetic efficiencies, suggesting that they may have become plasmolysed. These cells also exhibited Fv/Fm values of below 0.6 and much greater Fo yields (Fig. 3). A decrease in Fv/Fm and an increase in Fo suggest that photoinactivation of PSII complexes is occurring (Bradbury and Baker, 1986).
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When images of
/ of the lower surfaces of leaves exposed to ozone were examined using the same actinic PPFD of 300 µmol m-2 s-1, it was observed that the PSII operating efficiencies were considerably lower than those found in upper surface cells and that there was considerably less heterogeneity (Fig. 4). There was no indication that cells close to stomata were being affected by ozone, as was the case for the upper leaf surface.
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Stomatal conductance
To investigate the effects of the 250 and 500 nmol mol-1 ozone treatments at a PPFD of 300 µmol m-2 s-1 on stomatal opening, stomatal conductance (gs) was measured for upper and lower leaf surfaces over a 3 h period (Fig. 5). Both ozone treatments produced large decreases in gs of both upper and lower leaf surfaces over 3 h with the effects being greater at the higher ozone concentration. Ozone at 500 nmol mol-1 induces rapid stomatal closure on both leaf surfaces within the first hour of exposure.
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) and 500 nmol mol-1 ozone (
) at PPFD of 300 µmol m-2 s-1. Values are given as a percentage of the gs value at time 0 h and are the means±SE of three independent replicates. At time 0 h values of gs for upper and lower leaf surfaces were 120±20 and 464±56 mmol m-2 s-1, respectively. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The application of high resolution fluorescence imaging to the study of ozone perturbation of leaf photosynthesis has demonstrated that ozone has a rapid effect in highly localized regions of the leaf which cannot be detected by conventional integrating fluorometers. The majority of these primary perturbations were associated with palisade parenchyma cells around stomata on the upper leaf surface and close to the leaf surface (Fig. 1
). This would support the suggestion that ozone enters the leaf by passing through stomata and then rapidly reacts with palisade cell surfaces close to stomatal pores. Such ozone perturbations were not found to be associated with all stomata on the upper leaf surface. This would be consistent with the well-established spatial heterogeneity in stomatal aperture in many leaves (Weyers and Lawson, 1997), although rapid closure of some stomata on exposure to ozone would also explain reduced ozone penetration into sub-stomatal cavities.
Occasionally ozone-induced perturbations were identified at sites within leaves remote from stomata (Fig. 1E). It is not clear from these studies why such perturbations should occur. They may be the consequence of ozone diffusion through cracks in the cuticle, which could not be resolved with light microscopy (Grace and van Gardingen, 1996). Alternatively, they may result from rapid diffusion of ozone from open stomata through the internal air spaces to particularly susceptible cells. This second possibility would seem unlikely since such perturbations are very infrequent.
When the ozone concentration was increased from 250 to 500 nmol mol-1 a decrease in the frequency of perturbations of the photosynthetic parameters was observed. Measurements of stomatal conductance demonstrated that stomatal closure occurred much more rapidly at the higher ozone concentration (Fig. 5), which might account for a reduced rate of ozone penetration into the leaf and the reduced frequencies of perturbation of photosynthesis.
No ozone-induced perturbations of photosynthesis were found on the lower leaf surfaces (Fig. 4). This cannot be attributed to lack of ozone penetration through stomata, since stomatal conductance of the lower leaf surface of the control bean was c. 3.7-fold greater than that for the upper surface, and the closure of stomata in response to ozone exposure was similar on both leaf surfaces (Fig. 5). Consequently, the lack of ozone perturbations on the lower leaf surface is attributable to the lower PPFD incident on these cells.
The initial perturbations of photosynthesis in the upper mesophyll cell layers in the bean leaves, on exposure to ozone, are associated with decreases in / and Fv/Fm (Fig. 1) and an increase in Fo (Fig. 3). The decrease in / is indicative of a decrease in the PSII quantum efficiency, which reflects a decrease in the quantum efficiency of electron flux through PSII at steady state. The decline in Fv/Fm demonstrates that a decrease in the maximum quantum efficiency of PSII photochemistry is occurring. This could be due to an increase in non-photochemical quenching of excitation energy in the light-harvesting antennae of PSII and/or photoinactivation of PSII reaction centres (Baker and Horton, 1987; Krause and Weiss, 1991). The increase in Fo that accompanies the decline in Fv/Fm is consistent with photoinactivation of PSII reaction centres (Bradbury and Baker, 1986). Studies with Chlorella cells have shown that the initial effects of exposure to ozone were changes in the permeability of the plasmalemma to ions and solutes (Heath, 1987). Decreases in photosynthetic oxygen evolution then occurred and were accompanied initially by an increase and then a decrease in Fo; the decrease in Fo occurred at about the same time as the general disruption of the cells (Heath et al., 1982; Heath, 1987). Consequently, the observation in this study that fluorescence emissions in bean leaves exposed to ozone were concentrated into smaller regions of the cells than those in control leaves when increases in Fo were occurring (Fig. 4), would be consistent with plasmalemma dysfunction and cell plasmolysis.
It is evident from this study that for an ozone concentration of 250 nmol mol-1 there are effects on stomata and photosynthetic electron transport occurring during a 3 h exposure. From the measurements made it is not possible to determine whether the decline in electron transport is due to stomatal closure limiting CO2 supply or a loss of Rubisco activity. However, photoinactivation of PSII is likely to contribute to the reduction in electron transport. Currently, a fumigation chamber is being constructed that will allow high resolution imaging of a leaf during the fumigation. This should enable the kinetics of stomatal closure, decline in electron transport and PSII photoinactivation to be determined during the exposure to ozone and thus allow assessment of the effects of stomatal closure and PSII photoactivation on electron transport.
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Acknowledgments |
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This research was supported by grants to NRB from the Biotechnology and Biological Sciences Research Council of the UK and to JL from the Deutsche Forschungsgemeinschaft (Grant no. LE1181/1-1). We also thank Tracy Lawson and James Morison for helpful discussions.
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Notes |
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1 Present address: Institute of Plant Sciences, Swiss Federal Institute of Technology, Universitätstrasse 2, 8092 Zürich, Switzerland.
2 To whom correspondence should be addressed. Fax: +44 1206 873416. E-mail: baken{at}essex.ac.uk
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Abbreviations |
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F', fluorescence level in the light; Fm, maximal fluorescence level from dark-adapted leaves;
, maximal fluorescence level from leaves in light; Fo, minimal fluorescence level from dark-adapted leaves; 
, difference in fluorescence between and F (
=-F'); Fv, variable fluorescence level from dark-adapted leaves (Fv=Fm-Fo); gs, stomatal conductance. |
References |
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