Characterisation of the photosynthetic response of tobacco leaves to ozone: CO2 assimilation and chlorophyll fluorescence

J. Plant Physiol. 159. 845 – 853 (2002)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

Characterisation of the photosynthetic response of tobacco leaves to ozone: CO2 assimilation and chlorophyll fluorescence Elena Degl’Innocenti, Lucia Guidi*, Gian Franco Soldatini Dipartimento di Chimica e Biotecnologie Agrarie, Via del Borghetto 80, I – 56124 Pisa, Italy Received January 9, 2002 · Accepted March 1, 2002

Summary The present paper reports on the characterisation of the photosynthetic responses to a single pulse of ozone (150 ppb for 5 hours) in the leaves of two tobacco cultivars displaying different degrees of sensitivity to O3 (BelB, O3-resistant and BelW3, O3-sensitive). In the BelW3 cultivars, O3 induced a decrease in the photosynthetic rate, but not in actual PSII efficiency at steady-state photosynthesis. The reduction state of QA did not change in these plants while a strong decrease in intrinsic PSII efficiency was observed. The quantum yield for photosynthetic CO2 assimilation decreased more than the actual PSII efficiency, suggesting the presence of a significant fraction of electron transport to molecular oxygen or the existence of some form of cyclic electron flow. O3-treated leaves reduced the excess of absorbed light (i.e. light that couldn’t be used in photosynthesis) by dissipating a large part of the light absorbed by the PSII antenna as heath. In BelB, the CO2 assimilation rate did not change in the presence of the pollutant O3 and the only gas exchange parameter that changed was the stomatal conductance, which significantly increased. Some of the Chl fluorescence parameters changed after O3 fumigation, but returned to values similar to the controls when measured 24 hours after removing the stress. The only Chl fluorescence parameter that significantly increased during the recovery phase was the 1-qP. Key words: BelB – BelW3 – chlorophyll fluorescence – gas exchange – quenching analysis – stomatal conductance Abbreviations: Amax = CO2 assimilation rate at light saturation. – Chl = chlorophyll. – Ci = intercellular CO2 concentration. – E = transpiration rate. – ETR = electron transport rate. – F0 = minimal Chl fluorescence in dark adapted state. – Fv = variable Chl fluorescence in dark adapted state. – Fm = maximal Chl fluorescence in dark adapted state. – Fm′ = maximal Chl fluorescence with all PSII reaction centres closed in light adapted state. – Fv/Fm = photochemical PSII photochemistry in dark adapted state. – NPQ = non-photochemical quenching. – PAR = photosynthetic active radiation. – PFD = photon flux density. – qNP = non-photochemical quenching. – qP = photochemical quenching. – ROS = reactive oxygen species. – Φexc. = excitation capture efficiency of PSII (intrinsic PSII efficiency). – ΦCO2 = quantum yield for CO2 fixation. – ΦPSII = actual PSII efficiency. – %D = fraction of light absorbed in PSII that is dissipated in the PSII antenna. – %P = fraction of light absorbed that is used in photochemistry. – %X = fraction of light absorbed that is not used or dissipated in the PSII antenna. – 1-qP = PSII excitation pressure

* E-mail corresponding author: [email protected] 0176-1617/02/159/08-845 $ 15.00/0

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Elena Degl’Innocenti, Lucia Guidi, Gian Franco Soldatini

Introduction Amongst the air pollutants of a regional distribution, ozone (O3) is considered to have the greatest impact on vegetation (Heck et al. 1988). Chronic and acute exposures to this pollutant have been shown to stimulate leaf senescence and to decrease photosynthesis and growth in numerous plant species (Heath 1994, Farage 1996, Renaud et al. 1998). 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 site of attack of ozone within leaves has not yet been definitively identified. Susceptibility to O3 varies from species to species, and varies even among cultivars of a single species. Several mechanisms have been proposed to explain this variability. In some cases, the mechanisms linked to O3 tolerance have been attributed to a lower stomatal conductance of the tolerant species, which determines a reduction in O3 uptake. Also, an increase in respiration rate or in antioxidants may help prevent or repair damage caused by O3 (Heath 1994, Sandermann 1996). The tobacco cultivars BelW3 (sensitive) and BelB (tolerant) are widely known for their differential response to O3, the difference being attributed to stomatal conductance, intercellular leaf volume, and soluble sugar or ascorbate content (Dugger and Ting 1970, Heggestadt 1991, Sandermann 1996). However, Langebartels et al. (1991) reported that stomatal conductance is similar in the two cultivars during short-term fumigation with O3. Only a few studies have attempted to relate the inhibition of photosynthesis caused by O3 to its direct action on the mechanisms of stomatal closure or to changes in photosynthetic capacities at the mesophyll level (Nie et al. 1993, Pell et al. 1994). Few works report on the understanding of the mechanisms involved in the response at photosynthetic level of tobacco cultivars showing different susceptibility to O3. The advent of the light-doubling technique (Bradbury and Baker 1981) and the development of the instrumentation for the measurements of modulated Chl fluorescence (Schreiber et al. 1986) have allowed the identification, separation and quantification of mechanisms that quench variable Chl fluorescence emitted from PSII. Light energy absorbed by Chl molecules in a leaf can undergo one of three fates: it can be used to drive photosynthesis (photochemistry), excess energy can be dissipated as heat or it can be re-emitted as light-Chl fluorescence. These three processes occur in competition, such that any increase in the efficiency of one will result in a decrease in the yield of the other two. The aim of this work was to characterise the response of the photosynthetic process to O3 in the two tobacco cultivars, BelW3 (O3-sensitive) and BelB (O3-resistant), grown in controlled environments. The authors studied the changes in gas exchange parameters at steady-state photosynthesis, and carried out chlorophyll fluorescence analysis. Measurements

were made both at the PFD used for plant growth and at higher PFDs.

Materials and Methods Plant material and growth conditions The Nicotiana tabacum L. plants cv. Bel W3 and Bel B were grown in a growth chamber with pollutant-free air under a 12 h photoperiod at 20 ˚C with 50–70 % relative humidity and about 400 µmol photons m – 2 s –1 PAR (measured at maximum plant height). Experimentation began when the plants were about 2 months old and had five leaves. At the beginning of each experiment, leaf 2, counted from the bottom, was tagged for use in physiological analyses. These leaves had reached full expansion in both the cultivars.

Ozone Exposure Plants were transferred to a growth chamber modified for ozone fumigation and were acclimatised for 24 h before ozone treatment. Growth chamber conditions averaged 25 ˚C, 75 % relative humidity, 350 ppm CO2, with a 12 h photoperiod averaging about 400 µmol photons m – 2 s –1 at the top of the canopy. The plants were then treated with a single pulse of ozone (150 ± 20 nL L –1 for 5 h) or maintained in ambient charcoal-filtered air. Ozone was generated with a Fisher (Mod. 500, Meckenheim, Germany) ozone generator and each treatment was replicated three times. The ozone concentration inside the chamber was monitored continuously with a Monitor Labs Analyser connected to a PC (Mod. 8810, Englewood, USA).

Gas exchange Measurements of gas exchange were made on the fully expanded leaf at the end of treatment using an open system (Walz, Effeltrich, Germany). For details of the experimental procedures see Nali et al. (1998). Responses of leaf photosynthetic CO2 assimilation to irradiance (0–1000 µmol m – 2 s –1 PFD) were calculated using the method of von Caemmerer and Farquhar (1981). During gas exchange measurements in an assimilation chamber, temperature was 25 ± 3.4 ˚C, RH 65 ± 10 %, CO2 concentration was 350 ppm and O2 21 %. Responses of leaf photosynthetic CO2 uptake to irradiance were calculated using the Smith equation (Tenhuen et al. 1976). Stomatal conductance (to water vapour), transpiration rate and intercellular CO2 concentration were measured at 350 ppm CO2 at saturating light. The quantum yield for CO2 uptake (ΦCO2, µmol CO2/µmol photons) was calculated from the initial slope of the light-response curve using least-squares linear regression analysis.

Chlorophyll fluorescence measurements Modulated chlorophyll a fluorescence measurements were made with a PAM-2000 fluorometer (Walz, Effeltrich, Germany) on leaves identical in age to those utilised for the gas exchange. Measurements were carried out on the apical portion of the leaf, paying careful attention to exclude the principal vein. Dark-adapted leaves (40 min) were initially exposed to a weak, modulated measuring beam, followed by exposure to saturated white light at a PFD of about 15,000 µmol m – 2 s –1. In

Ozone, photosynthesis and energy dissipation this way, an estimate of the minimal (F0) and maximal fluorescence levels (Fm) (the latter corresponding to a transient maximal reduction in the pool of primary PSII electron acceptor, QA) were determined. The saturation pulse method was used for the analysis of quenching components (Schreiber et al. 1986). Intermittent, abrupt illumination by sufficiently strong light causes the transient, but complete removal of photochemical quenching, giving rise to a corresponding increase in variable fluorescence, Fv to Fv′; any residual quenching is assumed to be non-photochemical. The intensity of the actinic light was maintained at about 400 µmol m – 2 s –1 and saturating flashes of white light 15,000 µmol m – 2 s –1 and 800 m s duration were given every 20 s. After the saturating pulse, the maximal fluorescence reached the Fm′ value and the actinic light allowed steady-state photosynthesis and modulated fluorescence yield to be reached at this steady-state (Fs). Determination of quenching components qP and qNP were calculated as defined by Schreiber et al. (1986). Measurements of F0′ were carried out in the presence of far-red light (7 µmol m – 2 s –1) in order to fully oxidise the PSII acceptor side. The actual PSII efficiency (ΦPSII) and the intrinsic PSII efficiency (Φexc.) were calculated as (Fm′ – Fs)/Fm′ and Fv′/Fm′ respectively (Genty et al. 1989, Harbinson et al. 1989). Approx-

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imations of the electron transport rate through PSII were calculated as ΦPSII × PAR × 0.5 × 0.8, by multiplying the quantum efficiency of PSII according to Genty et al. (1989) by the incident photon flux density and an average factor of 0.8 for leaf absorptance, and dividing by a factor 2 to account for the sharing of absorbed photons between the two photosystems. Non-photochemical fluorescence quenching (NPQ) was calculated from the Stern-Volmer parameter, that represents relative measurement of thermal dissipation at the PSII level, calculated according to the equation NPQ = Fm/Fm′ – 1 of Bilger and Björkman (1990). The fractions of light absorbed that are dissipated in the PSII antennae (%D) and utilised in PSII photochemistry (%P), were estimated from 1-(Fv′/Fm′) and (Fv′/Fm′) · qP (Demmig-Adams and Adams 1996). It should be noted that although the parameter %P is equivalent to ΦPSII, both are still commonly used in the literature. The fraction of light absorbed by PSII that is not utilised in photochemistry or dissipated in the PSII antennae (%X), was estimated from (Fv′/Fm′) · (1-qP) according to Demmig-Adams and Adams (1996). To measure the fluorescence-PFD response, tobacco leaves were held horizontally in a leaf-clip holder (2030-B, Walz, Effeltrich, Germany). The PFD on the leaves, provided by a halogen lamp (2050-H, Walz, Effeltrich, Germany), was adjusted from darkness to 1000 µmol m – 2 s –1 in steps of 50 – 200 µmol m – 2 s –1. The halogen lamp was equipped with a heat-reflecting filter to reduce heat generation by the lamp. PFD on the leaf was monitored with a microquantum sensor installed on the leaf-clip holder next to the spot where fluorescence is measured. After the leaf was exposed to the desired PFD for 10 – 20 min, the chlorophyll a fluorescence of PSII was measured using the PAM-2000.

Statistics Experiments were repeated 3 times and averaged. Data were tested for the analyses of variance (ANOVA) and Student’s t-test.

Results Visible injury Two-to four hours after the end of the O3 treatment, leaves of the BelW3 cultivar showed typical symptoms of injury represented by water-logging. These initial chlorotic spots in BelW3 developed into necrotic lesions 24 hours after the end of fumigation. BelB showed only sporadic lesions on old leaves which became evident only 20 – 24 hours after the end of the exposure to O3.

CO2 assimilation at increasing PFDs Figure 1. Rates of photosynthesis (µmol CO2 m – 2 s –1) versus PFD in BelB (A) and BelW3 (B) tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours) (open squares). Controls were represented by plants maintained in filtered air (open circles). All measurements were made in filtered air at T = 25 ˚C, RH 65 %, 345 ppm CO2 and O2 21%.

CO2 assimilation showed typical response curves at increasing PFDs in control leaves of both cultivars (Fig. 1), but a strong decrease of the CO2 assimilation rate in BelW3 leaves was observed following the O3 treatment (Fig. 1 B). The CO2 fixation rate per unit leaf area of control BelW3 leaves was approximately 2.5 µmol CO2 m – 2 s –1 at light saturation level. In

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Elena Degl’Innocenti, Lucia Guidi, Gian Franco Soldatini changed following O3 exposure was stomatal conductance, which significantly increased. A different picture was seen in the BelW3 cultivar, where all parameters changed significantly. The CO2 fixation rate decreased by about 57% in comparison with the controls and a reduction of approximately 30 % was found for transpiration rate and stomatal conductance in O3-treated leaves. A significant increment in Ci was also recorded in ozonated leaves. To evaluate the recovery ability of the tobacco plants, a set of measurements was carried out 24 hours after the end of O3 exposure. The Bel B cultivar did not show alterations when compared to the controls. The CO2 assimilation rate remained low at values of approximately 1 µmol m – 2 s –1 in BelW3 leaves. A further decrease was observed for stomatal conductance, which reached a value of 55 mmol H2O m – 2 s –1. A further increase was shown by intercellular CO2 concentration (350 ppm). The quantum yield for CO2 uptake (ΦCO2) was the same in controls of both BelB and BelW3 cultivars. Following the treatment, ΦCO2 appeared to be reduced more in BelW3 (0.0028 µmol CO2/µmol photons) than in BelB (0.0081 µmol CO2/µmol photons) (Table 1).

Table 1. Gas exchange parameters determined in BelB and BelW3 tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours). Controls were represented by plants maintained in filtered air. Amax: CO2 assimilation rate (µmol CO2 m – 2s –1); E: transpiration rate (mmol H2O m – 2s –1); Gw: stomatal conductance (mmol H2O m – 2s –1); Ci: intercellular CO2 concentration (ppm). ΦCO2: quantum yield for CO2 uptake (µmol CO2/µmol photons). All measurements were made in filtered air at light saturation, RH 65 % and T = 25 ˚C. Each value is the mean of three replicates followed by the standard deviation. In the last row, the significance of the difference between control and ozonated leaves is reported (one-way ANOVA test). NS: not significant; *: P ≤ 0.05; **: P ≤ 0.01. Amax

E

Gw

ΦCO2

Ci

BelB cultivar Control Ozonated P

1.53 (0.29) 1.48 (0.62) NS

1.70 (0.30) 2.07 (0.96) NS

83 (6.55) 122 (9.5) *

304 (7.09) 313 (10.00) NS

0.0137

2.26 (0.31) 0.97 (0.23) **

2.37 (0.058) 1.71 (0.29) *

125 (8.33) 86 (11.5) **

307 (4.36) 316 (3.00) *

0.0137

0.0081

BelW3 cultivar Control Ozonated P

0.0028

Chl fluorescence quenching parameters at plant growth PFDs

leaves of ozonated BelW3 plants, the photosynthetic rate per unit leaf area decreased at about 1.2 µmol CO2 m – 2 s –1. In Table 1, all parameters related to gas exchanges are reported. In the BelB cultivar the only parameter which

The analysis of quenching was carried out at a light intensity similar to those of plant growing conditions (approximately 400 µmol photons m – 2 s –1).

Table 2. Chl fluorescence parameters determined in BelB and BelW3 tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours). Ozonated leaves were measured about 1 hour after the end of fumigation. A set of measurements was carried out 24 hours after the end of exposure to O3. Controls were represented by plants maintained in filtered air. Each value is the mean of three replicates followed by the standard deviation. In the last row the significance of the difference between control and ozonated leaves is reported (one-way ANOVA test). NS: not significant; P ≥ 0.05; *: P ≤ 0.05; **: P ≤ .01. For each column, means flanked by the same letter are not significantly different. F0

Fm

Fv/Fm

1-qP

qNP

ΦPSII

Φexc.

ETR

BelB Control Ozonated Ozonated (after 24 hrs) P

121 a (13) 119 a (10) 100 a (11) NS

541 a (57) 362 b (74) 439 a (45) *

0.771 a (0.039) 0.706 a (0.059) 0.772 a (0.028) NS

0.363 b (0.004) 0.478 b (0.103) 0.622 a (0.002) *

0.499 b (0.037) 0.649 a (0.041) 0.674 a (0.035) **

0.409 a (0.022) 0.269 b (0.018) 0.386 a (0.012) **

0.640 a (0.030) 0.515 a (0.080) 0.560 a (0.028) NS

69 a (3.5) 40 b (5.2) 66 a (4.5) **

90 b (0.07) 120 a (0.001) 109 b (6) **

412 (54) 472 (36) 510 (190) NS

0.778 (0.047) 0.744 (0.019) 0.778 (0.141) NS

0.579 a (0.034) 0.423 b (0.046) 0.433 b (0.087) **

0.588 b (0.047) 0.756 a (0.003) 0.687 a (0.065) **

0.255 (0.013) 0.289 (0.020) 0.315 (0.061) NS

0.604 a (0.037) 0.499 c (0.005) 0.548 b (0.129) **

39 (6.5) 48 (4.0) 53 (11.6) NS

BelW3 Control Ozonated Ozonated (after 24 hrs) P

Ozone, photosynthesis and energy dissipation The ratio Fv/Fm in BelB and BelW3 leaves of control plants ranged from 0.77 to 0.78 (Table 2). This is below the mean value of 0.843 obtained from Björkman and Demmig (1987) for dicotyledonous species measured at 77K. Neither the ratio Fv/Fm nor F0 changed in O3-treated leaves of BelB plants in comparison to the control. Control leaves showed actual PSII efficiency (ΦPSII) values of about 0.400 (Table 2). O3 treatment led to significant changes in some fluorescence parameters: as compared to the controls, treated-BelB leaves showed reduction in ΦPSII, and ETR and increase in qNP values and in the 1-qP parameter. The recovery in fluorescence in BelB leaf parameters at 24 hours after the end of fumigation are also reported in Table 2 and were as follows: non-photochemical quenching remained high, while a recovery of the ΦPSII, was observed. The 1-qP values strongly increased during the recovery while ETR returned to a value similar to those of the control leaves. In BelW3 the picture appears quite different; F0 changed significantly while no differences were found in Fm and Fv/Fm ratio values in ozonated leaves as compared to the controls (Table 2). Because of the presence of O3, Chl fluorescence changed as follows: 1-qP values decreased together with the intrinsic photochemical PSII efficiency (Φexc.), while no change in ΦPSII was observed in these leaves following O3 fumigation. In BelW3 leaves, O3 exposure determined an increase in qNP values. The measurements carried out 24 hours after the end of the treatment on BelW3 leaves are showed in Table 2. The F0 values partially recovered; the 1-qP parameter diminished and so the Φexc. value significantly decreased as compared to the controls. The non-photochemical quenching coefficient was increased as compared to the controls, while no changes were observed in ETR values. The large variability observed in Table 2 for BelW3 leaves may be attributed to variability in the proportion of chlorotic to green area on the leaf even though the methods were standardised.

Relationship between photosystem II electron transport and CO2 assimilation in ozonated leaves at increasing PFDs The relationship between the values of ΦPSII and ΦCO2 in leaves of both the tobacco cultivars at different PFDs could be described as linear (Fig. 2). In BelB ozonated leaves, a strong increase in ΦPSII was observed with changing ΦCO2 (Fig. 2 A). In BelW3 leaves fumigated with O3 at almost all PFDs measured, relatively high ΦPSII values occurred when ΦCO2 values were very low (Fig. 2 B).

Effect of ozone on the distribution of absorbed energy The response of control leaves of BelB to increasing light intensity from 400 to 1000 µmol m – 2 s –1 enhanced the amount of

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Figure 2. Relationship between actual PSII efficiency (ΦPSII) and quantum yield for CO2 uptake (ΦCO2) in BelB (A) and BelW3 (B) tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours) (open squares). Controls were represented by plants maintained in filtered air (open circles). All measurements were made in filtered air at T = 25 ˚C, RH 65 %, 345 ppm CO2 and O2 21%. The linear equations were: Control BelB, Y = 0.379 + 37.62X (r = 0.933**); Ozonated BelB, Y = 0.185 + 57.21X (r = 0.722*); Control BelW3, Y = 0.051 + 41.28X (r = 0.984***); Ozonated BelW3 Y = – 0.018 + 196.70X (r = 0.874**).

thermally dissipated light absorbed by PSII (D) from approximately 44 % to 70 % (Fig. 3 A). A parallel decrease of light absorbed by PSII and used in photochemistry (P, equivalent to ΦPSII) was observed with a reduction of 54 % to 25 %. Leaves of O3-treated BelB plants showed a greater dissipation from 66 to 77% and used 31 and 18 % of the light absorbed by PSII at about 400 and 1000 µmol m – 2 s –1, respectively (Fig. 3 B). In control leaves of the BelW3 cultivar, the amount of dissipated light absorbed ranged from 65 % at 400 µmol m – 2 s –1 and 79 % at 1000 µmol m – 2 s –1 (Fig. 3 C). Under the same conditions, the amount of light absorbed and used for photochemistry decreased from 31 % at 400 µmol m – 2 s –1 and to 15 % at 1000 µmol m – 2 s –1. With O3 treatment, leaves of BelW3 dissipated approximately 45 % of the absorbed light and used 54 % at the light intensity during plant growth (400 µmol m – 2 s –1), whereas at 1000 µmol m – 2 s –1 they dissipated and used approximately 92 % and 6 % of the absorbed light, respectively (Fig. 3 D). Thermal energy dissipation can also be estimated through non-photochemical quenching (NPQ). The data obtained are

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Elena Degl’Innocenti, Lucia Guidi, Gian Franco Soldatini

Figure 3. Fractions of light absorbed by the PSII antenna used in photochemistry (%P) and thermally dissipated (%D) versus PFD in BelB (B) and BelW3 (D) tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours). Controls were represented by plants maintained in filtered air (A, BelB and C, BelW3). In each graph the open circles indicate controls and open squares O3-treated plants.

shown in Figure 4. NPQ increased in response to increasing light intensity in control BelB leaves. At high PFD (1000 µmol m – 2 s –1), values of 1.75 and 1.46 were found for control and O3-treated leaves, respectively. At low light intensity (400 µmol m – 2 s –1), an increase in NPQ value was observed in O3-treated leaves as compared to the controls. In BelW3 cultivars, a strong increase of NPQ was found as an effect of the O3 treatment.

Discussion These experiments permit an insight into the characterisation of O3-induced changes in the gas exchanges and chlorophyll fluorescence quenching parameters in two tobacco cultivars well known for their differential response to this pollutant. This report represents the first investigation into the effect of O3 on the photosynthetic activity of the two tobacco cultivars BelW3 and BelB, which are widely studied for many other biochemical and physiological parameters (Rhoads and Brennan 1978, Heggestad 1991, Örvar et al. 1997, Torsethaugen et al. 1997).

BelB cultivar Our data indicate that fumigation of BelB plants with a single pulse of O3 did not determine any changes in the CO2 assimilation rate (Fig. 1 A). The only significant differences were an increase in stomatal conductance and a reduction in quantum yield for CO2 uptake. The data obtained indicate that the greater O3 tolerance of BelB cultivar is not attributable to lower absorption of the pollutant. These results contrast with the findings of some authors (Fuhrer et al. 1993, Heagle et al. 1993), but are in accord with those of Pell and Pearson (1983), Furukawa et al. (1984) and Langebartels et al. (1991) who found that O3 influenced photosynthetic processes directly with little or no stomatal closure. The reduction of the CO2 assimilation rate observed at low light intensity, i.e. the quantum yield for CO2 uptake, can be linked to a reduction in the efficiency with which the antenna of PSII absorb light energy and then transfer that energy to the reaction centres. However, no significant effects of O3 treatment on F0 were observed and the similarity in Fv/Fm ratio in treated BelB leaves and the controls, also points to the lack of effects on PSII itself.

Ozone, photosynthesis and energy dissipation

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The remaining unaltered centres appear to be able to carry out normal photochemistry. Generally speaking, very little is known about the molecular basis of the competitive de-excitation process and its regulation. Decreased lumen pH values and zeaxanthin formation both have been implicated in this process (Horton et al. 1996). Experimentally, this regulation of PSII is measured as the decrease in the efficiency of excitation capture by open PSII reaction centres (Φexc.) and by an increase in qNP. In BelB leaves exposed to O3, only qNP increased significantly. The fact that Φexc. did not change in ozonated BelB leaves indicates that the open reaction centres of PSII are able to utilise excitation energy captured by the antennae. However, the 1-qP parameter increased because of the presence of O3. These results suggest that only a fraction of PSII reaction centres are able to carry out an electron flow through PSII. Moreover ΦPSII, which decreased immediately after the end of fumigation, partially recovered within 24 hours. This may reflect the ability of BelB to maintain high electron transport rates. The change of NPQ in the leaves at higher PFD might be explained by an increase of energy-dependent quenching connected with the energisation of the thylakoid membrane (Fig. 4 A) (Krause and Weis 1991). In ozone-treated leaves of BelB plants, an increase in NPQ at higher PFD is evident and may indicate an increase in dissipation processes.

Figure 4. Non-photochemical quenching (NPQ) versus PFD in BelB (A) and BelW3 (B) tobacco cultivars exposed to a single pulse of O3 (150 nL L –1 for 5 hours) (open squares). Controls were represented by plants maintained in filtered air (open circles).

The fact that the fraction of light absorbed was thermally dissipated at low light intensity can be linked to the decrease in ΦCO2. Our data indicate that during light-limited CO2 assimilation and at high oxidation state of QA, the quantum yield of CO2 uptake and the ΦPSII significantly changed (Fig. 2 A). At the same moment, dissipation increased and these results indicate that ΦCO2 at low light was mainly controlled by nonphotochemical and non-radiative dissipation of excitation energy rather than by accumulation of reduced acceptor QA. The reduction state of QA (i.e. (QA)red/[(QA)red + (QA)ox] can be estimated by steady state Chl a fluorescence as 1-qP at the irradiance value during growth, reflecting PSII excitation pressure (Dietz et al. 1985, Demmig-Adams et al. 1990). The rate of QA reduction significantly changed in BelB leaves following O3 exposure, indicating modifications in electron flow around PSII. It is interesting to note the values of 1-qP recorded during the recovery. In fact, 1-qP increased significantly, indicating a further increase in reduction state of primary acceptor QA. Thus, it seems that an increasing proportion of centres are converted to fluorescence quenchers that are acting as traps for excitons and convert the excitation energy to heat.

BelW3 cultivar The decrease in CO2 fixation recorded in the O3-treated leaves of this cultivar was associated with a strong reduction in stomatal conductance and an increase in intercellular CO2 concentration. The results obtained from gas exchange seem to indicate that in these plants not only did O3 induce stomatal closure, but also an alteration in the mesophyll activity as indicated by the increase in Ci. The decrease of the CO2 assimilation rate was observed both at low and high light intensity (Fig. 1 B). Indeed, the quantum yield for CO2 fixation, ΦCO2, decreased significantly following the exposure to O3. The changes in CO2 assimilation curves versus light intensity are accompanied by changes in Chl fluorescence parameters. The effects of O3 on Chl fluorescence were biphasic: O3 induced an increase in F0 and a partial recovery 24 hours after the end of fumigation. The rise in F0 under unfavorable environmental conditions is usually due to the inability of the reduced plastoquinone acceptor, QA, to be oxidised completely because of retardation of the electron flow through PSII (Krause and Weis 1991) or to the separation of light-harvesting Chl a/b protein complexes of PSII from the PSII core complexes (Schreiber and Armrod 1978). The significant increase in F0 values could also indicate damage to PSII centres not readily reversible (Krause 1988). The unchanged Fv/ Fm ratio observed in ozonated BelW3 leaves indicates that no PSII photoinhibition occurs.

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Elena Degl’Innocenti, Lucia Guidi, Gian Franco Soldatini

O3-treated leaves, however, apparently are capable of maintaining the PSII acceptor side oxidised, as indicated by the reduction of the 1-qP values. This suggests the existence of an alternative electron acceptor in PSII that would consume electrons from QA and/or the PQ pool, maintaining the PSII acceptor side oxidised. Furthermore, in these leaves there was significant electron transport through PSII which did not allow assimilation of CO2, thus suggesting the presence of a significant fraction of electron transport to molecular oxygen or the existence of some form of cyclic electron flow. These results also account for the unchanged ΦPSII. However, the Φexc., which is a measure of PSII photochemical efficiency under steady state light conditions, was considerably depressed by O3. This result implies that a lightinduced non-photochemical quenching may become established in BelW3 tobacco leaves as a result of O3 treatment. Indeed, the qNP values significantly increased after O3 exposure and also remained high during the recovery. Ozone-treated leaves of BelW3 showed a higher capacity for nonphotochemical quenching (NPQ) at growth and in saturating light and this probably prevented photoinhibitory damage (Fig. 4 B). This fact and the F0 parameter increase indicate that photoprotective capacity is unable to prevent damage. The picture of BelW3 cultivar response to O3 stress appears quite complex. One of the main causes of O3 stress is the production of reactive oxygen species (ROS), which are highly stimulated in light inducing photooxidation (Hippeli and Elstner 1996, Schraudner et al. 1997). ROS production may also be enhanced during O3 stress because of the slowing down in enzyme activities of the Calvin-Benson cycle. This implies a restriction in electrons to be accepted by NADP + , leading to excess absorption by oxygen (Hippeli and Elstner 1996). Three mechanisms are involved in diminishing photooxidation during stress (including O3 stress) (Elstner and Osswald 1994): i) avoiding production of ROS by diminishing the electron transport chain, ii) dissipating excess energy as heat via the xanthophyll cycle, and iii) scavenging ROS formed by antioxidant compounds and enzymes. Our data indicate that electron transport did not diminish following O3 exposure. Furthermore, the non-photochemical quenching coefficient, qNP, changed significantly in these leaves when measured at growing-light intensity. This indicates that in BelW3 leaves the presence of O3 determines the mechanisms involved in diminishing the O3-induced photooxidation. On the other hand, other authors (Pasqualini et al. 2001) have reported that antioxidant compounds are effective in O3 tolerance only in the BelB cultivar, while no activation of scavenging mechanisms are triggered in BelW3 leaves.

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Acknowledgements. The authors would like to thank Prof. G. Lorenzini for seeds of the two tobacco cultivars. This research was financially supported by MURST (National Projects, 2000), Rome (Italy).

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