Author for correspondence: L. Guidi Tel: + 39 50 571557 Fax: + 39 50 598614 Email:firstname.lastname@example.org
• The combined effects of the two pollutants, cadmium and ozone, on sunflower (Helianthus annuus) metabolism are analysed here.
• Photosysnthetic processes and ascorbate metabolism were studied in sunflower plants grown for 15 d in the presence of cadmium and exposed to acute O3 treatments.
• CO2 assimilation rate was reduced in plants subjected to Cd(II) and/or O3 treatments, but no alterations in stomatal conductance and Fv : Fm ratio were observed. Rubisco activity was significantly reduced only in plants grown in the presence of cadmium indicating that the photosynthetic process is mainly altered by this factor. Photochemical quenching and the quantum efficiency of PSII in steady-state conditions were significantly depressed and nonphotochemical quenching increased in stressed plants. Cd(II) and O3 also strongly affected ascorbate metabolism.
• The changes in ascorbate redox state and the increase in ascorbate-redox enzymes strongly supported an ascorbate over-utilization in Cd(II) and/or O3–treated plants. However, the increase in ascorbate-based detoxification mechanisms did not provide complete protection against the oxidative stress imposed by the two pollutants, since an increase in lipid peroxidation and protein oxidation accompanied a decrease in photosynthesis under pollutant exposure.
Ozone is a phytotoxic air pollutant able to induce visible symptoms of damage. The reduction in photosynthetic rate observed in numerous plant species induced by ozone can be due to damage of the photosynthetic apparatus or to a signal produced outside the chloroplast (Kangasjaervi et al., 1994; Pell et al., 1997). It is also known that O3 accelerates senescence (Heath & Taylor, 1997; Pell et al., 1997). Heavy metals can also increase their concentrations in the environment as a consequence of agricultural, manufacturing, mining and waste disposal practices or other anthropic factors, and reach toxic levels for plants. Heavy metals affect plant structures and functions at different levels: they have been reported to induce reduction in growth, changes in membrane structure, in metabolic processes and in water and ion uptake (Lata, 1991; Krupa & Baszynski, 1995; Masarovicova et al., 1999). An extensive review on the toxicity of cadmium in higher plants has recently been published (Sanità di Toppi & Gabbrielli, 1999). In a preliminary report we have presented data confirming a very strong dependence of the growth of sunflower plants on Cd(II) concentration in the nutrient medium (Di Cagno et al., 1999b). This was reflected as a decrease in both leaf area and leaf f. wt.
Many environmental stresses, including O3 and heavy metals, generate oxidative stress in plant tissues by inducing an over-production of reactive oxygen species (ROS), such as 1O2, ·OH, H2O2 and O2·- (Foyer et al., 1994; Gallego et al., 1996; Pell et al., 1997; Schraudner et al., 1997). The capability of plants to cope with the oxidative damage depends on whether antioxidative defences can be conformed according to the ROS overproduction. The role of glutathione in scavenging processes against heavy metals and other stress conditions has been extensively investigated (Gallego et al., 1996). In these defence strategies against abiotic stress, the ascorbate (ASC) system is also considered a crucial component, particularly in photosynthetic tissues (Noctor & Foyer, 1998). In cell metabolism, ASC contributes directly to ROS scavenging and by means of ascorbate peroxidase (APX). Since both O3 and heavy metals may induce oxidative stress, it is possible that these two kinds of stresses interact in exercising their influence on plant metabolism. Among heavy metals cadmium is probably the most harmful. However, little is known of the role of cadmium in the plant system or of its possible role in influencing O3 phytotoxicity. With the exception of a pioneer report (Czuba & Ormrod, 1974) and an our previous report (Di Cagno et al., 1999a), no studies were carried out on the effects of ozone on plants grown in the presence of cadmium. With the aim of analysing the combined effects of these two pollutants on plant metabolism, we have studied photosynthetic processes and ascorbate metabolism in sunflower plants grown in the presence of cadmium and exposed to acute O3 treatments.
Materials and Methods
Seeds of Helianthus annuus L. (hybrid Select) were sown in watered sand. After 10 d, the seedlings were individually transplanted into pots containing expanded clay and a nutrient solution as defined by Di Cagno et al. (1999b). The plants were homogeneously divided into two groups, one of which had 20 µM Cd(II) added in the form of Cd(II)Cl2 to the nutrient solution. The plants were grown for a further 15 d in a chamber with temperatures of 25/20°C day/night, 50–80% rh and a 12-h photoperiod at c. 400 µmol m−2 s−1 (PAR). Plants were grown with or without cadmium in the chamber for 15 d in ambient air where the O3 concentration was lower than 2 nl l−1. After this preliminary period, plants were exposed to O3 fumigations and photosynthetic processes and antioxidant status were analysed.
All the analyses were performed on fully expanded leaves of the same age and at the same stage of development. Moreover, in order to avoid differences due to the peculiarity of a single leaf, each analysis was performed on leaf tissue taken from single (not pooled) leaves belonging to different plants. The leaf tissue was immediately used for noninvasive analyses (gas exchange and chlorophyll fluorescence) or stored at −80°C for the other biochemical analyses following the immersion into liquid N2.
The ozone treatment was performed in a controlled-environment chamber (0.483 m3) (Cavallo, Milan, Italy). Ambient air supplied to the chamber was passed through a charcoal filter. Details of the experimental design have been described by Guidi et al. (1999). Briefly, temperature was maintained at 25 ± 1.5°C, RH at 85 ± 2%. Ozone was generated by electric discharge with an air-cooled generator (Fisher Model 500, Meckenheim, Germany) supplied with pure oxygen and was mixed with the air entering the chambers. The O3 concentration was continuously monitored with a photometric ML8810 analyser (Monitor Labs, San Diego, CA, USA). Plants grown with or without cadmium in the nutrient solution were exposed to a single pulse of O3 with a target concentration of 160 nl l−1 for 2 h. The control plants were exposed to charcoal-filtered air only. The ozone-treated plants and controls were previously conditioned 24 h in the growth chambers at the same temperature and rh conditions utilized during the fumigation.
Measurements of gas exchange were made on fully expanded leaves at the end of the experiment, using an open system (Heinz Walz, Germany). For details of the experimental procedures see Soldatini & Guidi (1992). The gas exchange parameters determined at light saturation level (800–900 µmol m−2 s−1) were photosynthetic rate (Amax, µmol CO2 m−2 s−1), stomatal conductance to water vapour (Gw, mmol H2O m−2 s−1), transpiration rate (E, mmol H2O m−2 s−1) and intercellular CO2 concentration (Ci, ppm).
Chlorophyll fluorescence and content
Chlorophyll a fluorescence analysis was also carried out on leaves of similar age to those utilized for gas exchange. The ratio of variable to maximal chlorophyll fluorescence (Fv : Fm) of the leaves was measured using an amplitude-modulated fluorometer (PAM-2000, Heinz Walz, Germany) after 40 min dark adaptation (Di Cagno et al., 1999b). The system was used for the analysis of quenching by the saturation pulse method (Schreiber et al., 1986). F′0 was measured in the presence of far-red light (7 µmol m−2 s−1) in order to fully oxidize the PSII acceptor. The actual PSII efficiency (ΦPSII) and intrinsic PSII efficiency (Φexc.) were calculated as (Fm′ − Fs) : Fm′ and Fv′ : Fm′, respectively (Genty et al., 1989). Photochemical quenching (qP) was calculated as (Fm′ − Fs) : Fv′ according to Van Kooten & Snel (1990). The term (1 − qP) was used as an estimate of the reduction state of the primary quinone electron acceptor (QA). Nonphotochemical quenching was estimated as (Fm − Fm′) : Fm.
For determination of chlorophyll contents, leaf samples (0.3 g FW) were ground in 80% acetone (1 : 30 w/v). The homogenate was centrifuged at 2700 g for 10 min. The supernatant absorbances at 663, 646 and 470 nm were measured. Contents of Chl a and b as well as total carotenoids (xanthophyll and β-carotene) were calculated by the formulae of Lichtenthaler (1987).
The level of lipid peroxidation in the leaf tissue was measured in terms of malondialdehyde (MDA) content determined by the thiobarbituric acid (TBA) reaction (Heath & Packer, 1968; Guidi et al., 1999). The amount of MDA-TBA complex was calculated from the extinction coefficient 155 mmol l−1 cm−1.
Protein carbonyl content was determined by reaction with 2,4-dinitrophenylhydrazine (DNPH) as described by Reznick & Packer (1994) with little modification. After treatment with DNPH and precipitation, the final protein pellets were dissolved in 6 M guanidine hydrochloride, pH 2.3. The protein was quantified by reading the absorption at 280 nm. The amount of proteins was calculated from a bovine serum albumine standard curve (0.25–2 mg ml−1) dissolved in guanidine hydrochloride and read at the same wavelength. Each sample was read against the sample treated with HCl (2.5 M) and the carbonyl content was calculated from the peak absorbance (360 nm) using an absorption coefficient ɛ of 22 000 M−1 cm−1.
Determination of cadmium
At the end of the 15-d Cd(II)-stress period the plants were divided into different portions (leaves, stems and roots), dried at 80°C for 48 h and the d. wt measured. The cadmium determinations were made on nitric-perchloric acid (3 : 1, v/v) digests of three replicate plant tissue samples. Cadmium concentration was determined by atomic absorption spectrophotometer (Perkin-Elmer 373, Norwalk, CT, USA).
Extraction and assay of Rubisco
Rubisco activity (EC 18.104.22.168) was determined spectrophotometrically in a coupled reaction by monitoring NADH oxidation at 340 nm and 25°C (Usuda, 1985). Frozen leaf tissues (0.3 g) were rapidly homogenized in a chilled mortar with 3 ml of ice-cold extraction buffer solution containing 0.25 M tris-HCl (pH 7.8), 0.05 M MgCl2, 0.0025 M EDTA and 37.5 mg dithiotreitol. The homogenate was centrifuged for 10 min at 10 000 g at 4°C. The resulting supernatant was collected and used for the enzymic assays outlined below.
Following a 30-min light incubation at room temperature, Rubisco activity was assayed in a medium containing 50 mM Tris-HCl (pH 7.8), 10 mM MgCl2, 30 mM NaHCO3 and 5 mM β-meSH, 5 units phosphoglycerate kinase, 5 units of glyceraldehyde 3-phospho dehydrogenase, 6 units of triose phosphoisomerase, 1 unit of glicerol phosphate dehydrogenase, 0.15 mM NADH, 5 mM ATP. The activity was measured, after the addition of 50 µl of extract and 0.5 mM ribulose 5 phosphate, at 340 nm and measurements were completed within 15 min.
Enzyme activity was expressed as µmol CO2 fixed min−1 mg−1 total proteins.
Ascorbate/dehydroascorbate redox status
ASC and DHA were measured according to Kampfenkel et al. (1995) on a deproteinized supernatant obtained by homogenization of 2 g leaf samples with ice cold 5% metaphosphoric acid (w/v) and centrifugation at 20 000 g for 15 min. A recovery test for ASC was made by adding a known amount of ASC in the metaphosphoric acid used for the tissue homogenization, in order to verify the suitability of the extraction medium. Because the recovery test had shown that 20–25% of ASC was oxidized to DHA, such a value was taken into account for the calculation of the ASC and DHA content.
Ascorbate redox enzymes and peroxidase activity
Spectrophotometric determination of ASC redox enzymes was assayed as reported in Arrigoni et al. (1992) with minor modifications, by using a Beckman DU 7000 spectrophotometer.
Leaf samples (3–4 g) were homogenized in an ice-cold porcelain mortar with a grinding medium consisting of 50 mM Tris-HCl (pH 7.8), 0.3 M mannitol, 1 mM EDTA, 10 mM MgCl2, 1 mM ASC, 0.1% BSA and 0.05% (w/v) cysteine; 1 : 4 ratio (w/v). The homogenates were centrifuged for 15 min at 30 000 g, and the supernatant was used for the enzymatic determinations.
DHA reductase (EC 22.214.171.124) activity was performed following the increase in absorbance at 265 nm due to the GSH-dependent production of ASC. The reaction mixture contained 0.1 M phosphate buffer, pH 6.2, 2 mM GSH and 50–100 µg proteins. The reaction was started upon addition of 1 mM DHA. The rate of nonenzymatic DHA reduction was subtracted (extinction coefficient 14 mM−1 cm−1).
AFR reductase (EC 126.96.36.199) was tested by measuring the oxidation rate of NADH at 340 nm in a reaction mixture composed of 0.2 mM NADH, 1 mM ASC, 50–100 µg proteins and 50 mM Tris-HCl, pH 8.0. The reaction was started by adding 0.2 units of ASC oxidase to generate a saturating concentration of AFR (extinction coefficient 6 mM−1 cm−1).
APX (EC 188.8.131.52) activity was determined following the H2O2-dependent oxidation of ASC at 265 nm in a reaction mixture composed of 50 µM ASC; 90 µM H2O2, 50–100 µg proteins and 0.1 M phosphate buffer, pH 6.4. The activity of APX was corrected by subtracting the nonenzymatic H2O2-dependent ASC oxidation and the H2O2-not dependent ASC oxidation. An extinction coefficient of 14 mM−1 cm−1 was used.
Total peroxidase activity was measured according to Ferrer et al. (1990) using 4-methoxy-α-naphtol as the substrate.
Protein determinations for the assays of the activity of Rubisco, ASC-redox enzymes and peroxidases were performed according to the method of Lowry et al. (1951) using bovine serum albumine as standard.
Experiments were carried out three times with 10 plants for each treatment. Data were subjected to a two-way ANOVA in which cadmium and ozone represented the two factors. Statistical differences between mean values were determined with LSD. For comparisons between two means the Student’s t-test was applied.
Sunflower plants grown for 15 d in the presence of cadmium showed visible symptoms of damage represented by diffuse chlorosis on the youngest leaves, whereas ozone treatments alone did not induce visible symptoms of damage. The presence of cadmium in the nutrient solution induced a remarkable increase in Cd(II) levels in the whole plant. As expected Cd(II) concentration was much higher in the roots (40-fold over control plants) than in the leaves (about 10-fold), with intermediate values (28-fold) in the stems (Table 1).
Table 1. Cadmium concentrations (µg g−1 d. wt) in different organs of Helianthus annuus grown for 15 d in nutrient solution enriched with 20 µM Cd(II)
The control plants were maintained in the complete nutrient solution without cadmium. The values are the mean of three replicates ± SE. The last row indicates the significance of the difference between the means following Student’s t-test. ***, P < 0.001; **, P < 0.01.
Maximum CO2 assimilation rate (Amax) decreased because of the presence of cadmium and/or ozone (Table 2). In plants grown with cadmium in the nutrient medium the decrease was of 16% compared with the control and a similar decrease (12%) was induced by O3 fumigation. However, the strongest reduction was observed in leaves subjected to cadmium plus ozone treatment (−30%). Neither stomatal conductance (Gw), nor the transpiration rate (E, data not shown), were affected by the stresses, and furthermore the intercellular CO2 concentration did not change significantly in the presence of cadmium and/or ozone (data not shown).
Table 2. Gas exchange in leaves of Helianthus annuus grown for 15 d in the presence or absence of cadmium and then exposed or not to a single pulse of O3 (160 nl l−1 for 2 h)
The results reported are referred to the values reached at saturation light intensity (c. 800 µmol m−2 s−1). Amax, CO2 assimilation rate (µmol CO2 m−2 s−1); Gw, stomatal conductance to water vapour (mmol H2O m−2 s−1); each value is the average of three replicates ± SE. Means flanked by the same letters are not significantly different for P = 0.05 following the two-way ANOVA test.
6.22 ± 0.03a
5.59 ± 0.06b
5.23 ± 0.05b
4.33 ± 0.02c
173 ± 1.6a
173 ± 12.7a
160 ± 16.3a
153 ± 3.5a
In Fig. 1 the activity of Rubisco in leaves of plants exposed to cadmium and/or O3 is reported. The Rubisco activity was significantly reduced only in plants grown in the presence of cadmium. No further effects were recorded in plants grown in the presence of cadmium and then treated with ozone.
Chlorophyll content and fluorescence
Effects of cadmium and/or ozone on chlorophyll and carotenoid contents are reported in Table 3. The chlorophyll a and b contents decreased significantly in leaves of plants grown with cadmium (−61 and −79% than controls, respectively). Leaves subjected to the O3 fumigation had a lower content of Chl a when compared with the untreated plants, but a stronger reduction in Chl a level was observed in plants subjected to both the stresses. O3 also induced a decrease in Chl b content even if no further decrease was observed in the plants subjected to both Cd(II) and O3 treatments. Since both Cd(II) and O3 affected Chl b more than Chl a, the Chl a : b ratios were higher under Cd(II) or O3 treatments than in the controls. The interaction between the two stresses induced a significant decrease in Chl a : b ratio alongside the strong decreases of Chl a and b in these plants. Carotenoid content was reduced only in plants grown in the presence of cadmium in nutrient solution (Table 3).
Table 3. Chlorophyll and carotenoid contents (µg g−1 f. wt) in leaves of Helianthus annuus grown with or without Cd(II) in nutrient solution and then exposed or not to a single pulse of O3 (160 nl l−1 for 2 h)
Each value is the average of three replicates ± SE. Means flanked by the same letters are not significantly different for P = 0.05 following the two-way ANOVA test.
840 ± 51.6a
646 ± 15.3b
325 ± 11.3c
202 ± 14.4d
398 ± 0.1a
224 ± 15.9b
83 ± 5.0d
112 ± 13.2c
2.11 ± 0.13c
2.89 ± 0.14b
3.92 ± 0.05a
1.81 ± 0.08d
129 ± 9.0a
136 ± 5.9a
68 ± 5.6b
117 ± 17.3a
The values of F0 and Fm did not change in the cadmium-treated plants and no effect was recorded in the ratio Fv : Fm. In the same way, O3 did not affect either F0 and Fm and Fv : Fm ratio (data not shown).
The Φexc. which is a measure of PSII photochemical efficiency under steady-state light conditions, was depressed considerably in the presence of cadmium or ozone (Table 4). These results imply that a light-induced nonphotochemical quenching may become established in sunflower leaves as a result of cadmium or ozone treatment. Moreover, this quenching mechanism was not related to inhibition of PSII photochemistry since the Fv : Fm ratio remained stable. To investigate this possibility, the values of 1 − qP and qNP under steady-state light conditions were calculated. The coefficient 1 − qP increased three times in Cd(II)-treated plants and an even more remarkable rise (19 times) was induced by O3 exposure. The combined treatments with Cd(II) and O3 did not determine any additive effect. qNP similarly increased under Cd(II) or O3 treatments, reaching values higher than controls (45% and 36%, respectively). Moreover, the acute O3 exposure of Cd(II)-treated plants induced a 95% increase in qNP, thus determining a clear additive effect on this quenching parameter. The decrease in Φexc. is responsible for the considerable decrease in actual quantum yield for PSII electron transport (ΦPSII) which is visible in plants grown in the presence of cadmium and/or exposed to O3. Also for this parameter the combined effects of the two stresses are additive.
Table 4. Quenching coefficients in leaves of Helianthus annuus grown with or without Cd(II) in nutrient solution and then exposed or not to a single pulse of O3 (160 nl l−1 for 2 h)
The coefficients were obtained at steady state conditions of photosynthesis at a light intensity of c. 300 µmol m−2 s−1. Each value is the average of six replicates ± SE. Means flanked by the same letters are not significantly different the two-way ANOVA test (P = 0.05).
0.638 ± 0.0005a
0.516 ± 0.0002b
0.524 ± 0.0001b
0.315 ± 0.0005c
0.004 ± 0.0001c
0.076 ± 0.0003a
0.015 ± 0.0001b
0.078 ± 0.0004a
0.452 ± 0.0002c
0.613 ± 0.0003b
0.657 ± 0.0010b
0.883 ± 0.0006a
0.642 ± 0.0006a
0.496 ± 0.0005b
0.535 ± 0.0002ab
0.335 ± 0.0006c
Ascorbate redox state and ascorbate redox enzymes
Cd(II) and O3 had different effects on the ASC content (Fig. 2a). In Cd(II)-treated plants a remarkable decrease in ASC occurred, whereas in O3–fumigated plants in the absence of Cd(II) treatment, a significant increase of ASC occurred. Combined treatments with Cd(II) and O3 induced a reduction in ASC content compared with the control, but significantly smaller than that induced by Cd(II) alone (Fig. 2a).
Plants grown with cadmium showed a DHA content always higher than that of untreated plants. No effects were found on DHA content in ozonated plants. In Cd (II)-treated plants, DHA content was remarkably higher than that of the controls (Fig. 2b).
APX, a key enzyme in the ROS scavenging processes, was strongly affected by both Cd(II) and O3 treatments, its activity being about two and three times higher in O3- and Cd(II)-treated plants, respectively, than in untreated ones (Fig. 3a). When plants were subjected to the two pollutants, the APX activity reached values 4.5 times higher than that of the control plants.
As far as the ASC-regenerating enzymes are concerned, AFRR was strongly affected by Cd(II), O3 and Cd(II) plus O3 treatments: Cd(II) induced a 57% increase in AFRR activity and O3 an increase of 100%. Further rises in this ASC-recycling enzyme (more than three times) were induced when Cd(II)-treated plants were also exposed to O3 fumigation (Fig. 3b). A remarkable increase (five times) was shown in DHAR activity in plants subjected to both stresses, while no significant change was observed for cadmium or ozone single treatment (Fig. 3c). Because, other than APX, many different peroxidases (PODs) are active in plant cells and several kinds of stresses affect their activities (Casano et al., 1999; Gabrielli et al., 1999; Kristensen et al., 1999), changes induced by Cd(II), O3 or Cd(II) plus O3, in the activities of these isoenzymes were analysed by utilizing 4-methoxy-α-naphthol as substrate, an unspecific electron donor. O3 had a much higher effect on POD activity than Cd(II) since it determined a rise of more than six times compared with the twofold rise induced by the heavy metal. O3 fumigation of the Cd(II)-treated plants induced a further remarkable increase of POD activity, accounting to almost 16 times higher than that of the untreated plants (Fig. 3d).
Lipid peroxidation and protein oxidation
Because cadmium and O3 treatments were expected to generate active oxygen species in sunflower plants, the level of cellular injury induced by oxidative stress was determined by analysing lipid peroxidation, measured as the amount of TBA-reacting substances, and protein oxidation, measured as proteic carbonyl content.
The level of TBA-reacting substances increased under both the stress conditions (Table 5) with the higher increase detected in plants grown with cadmium in nutrient solution (fourfold vs threefold induced by O3).
Table 5. Thiobarbituric acid reactive substances (TBARS) (µmol g−1 FW) and total protein carbonyl content (TPC) (nmol carbonyl mg−1 protein) in leaves of Helianthus annuus grown with or without Cd(II) in nutrient solution and then exposed or not to a single pulse of O3 (160 nl l−1 for 2 h)
Each value is the average of six replicates ± SE. Means flanked by the same letters are not significantly different for P = 0.05 following the two-way ANOVA test.
11.28 ± 0.21d
32.77 ± 1.22c
44.67 ± 2.17a
40.67 ± 3.41b
10.08 ± 0.99d
30.45 ± 2.91a
16.41 ± 0.03c
20.17 ± 1.65b
In ozonated plants the level of carbonyl content was threefold greater than the controls while in plants grown with cadmium the carbonyl content was about twofold (Table 5). No additive effects on this parameter were observed.
The increase in Cd(II) concentration in the leaves of sunflower grown in a nutrient solution enriched with Cd(II), and the gradient in the metal concentration between roots and shoots clearly indicates that cadmium was absorbed by the roots and translocated to the leaves, confirming previous reports (Di Cagno et al., 1999b). Substantial decreases in pigment level, that is chlorophylls and carotenoids, were also observed in Cd(II)-treated plants as already reported by other authors (Krupa et al., 1993; Chugh & Sawhney, 1999).
CO2 assimilation rate in the mesophyll was reduced by cadmium but stomatal conductance was unaffected, though a strong decrease of Rubisco activity was observed. The inhibition of Rubisco activity as an effect of cadmium has already been reported (Sheoran et al., 1990; Siedlecka et al., 1997; Krupa & Moniak, 1998). In plants exposed to O3 the reduction in CO2 fixation rate was linked to limitations at mesophyll level even if in this case no alteration in Rubisco activity was observed.
The Φexc., which is a measure of photochemical efficiency in steady state light conditions, was considerably depressed in plants grown in the presence of cadmium. This result implies that a light-induced nonphotochemical quenching may become established in sunflower leaves as a result of the stress. Moreover, this quenching mechanism was not due to inhibited photochemistry since the Fv : Fm ratio remained quite stable. Similar results were also reported by Krupa et al. (1993) in bean plants grown with different concentrations of cadmium.
Cadmium treatment caused an increase in 1 − qP and a concomitant increase in qNP; this indicates that energy consumption was inhibited by Cd(II), most probably through a decrease in the Calvin cycle as indicated also by the reduction in Rubisco activity. Inhibition of Rubisco activity apparently determined a lower utilization of NADPH and ATP generated by the primary light reaction and then also a reduced electron flow. The increase in qNP is indicative of an increased pH gradient under steady-state conditions (Krause & Weis, 1984). Thus, it should be ascribed to nonphotochemical quenching of fluorescence due to high-energy state (Genty et al., 1989; Krause & Weis, 1991). This may cause a down-regulation of PSII to avoid over-reduction of QA (Genty et al., 1990). The drops in Φexc. and in qP were responsible for the considerable decrease in actual quantum yield for PSII electron transport (ΦPSII).
Plants not pretreated with cadmium and exposed to O3 quenching parameters, that is ΦPSII, Φexc., 1 − qP, qNP, were altered in the same way as in cadmium treated plants, but Rubisco activity was not modified in comparison to the untreated plants. It is known that O3 affects CO2 fixation ability more than electron transport. Specific targets of O3 have been proposed to be Rubisco activity (Pell et al., 1997) or the regeneration of ribulose bisphosphate (Wallin et al., 1992). In our case, due probably to a single O3 pulse, Rubisco activity in ozonated plants was not altered and it is presumed that the regeneration of RuBP was the target. This result is in agreement with those obtained by Farage et al. (1991) and Grandjean Grimm & Fuhrer (1992) for wheat leaves after acute O3 exposures.
In order to study the combined effects of the two stresses, plants grown with cadmium in nutrient solution for 15 d were exposed to ozone. A strong decline in chlorophyll content was observed in O3-treated plants grown in presence of cadmium even if the effect of two stresses was not additive. Negative effects on chlorophyll content were also observed in plants not pretreated with cadmium and subjected to ozone as already established (Paakkonen et al., 1996; Reichenauer et al., 1997). Mechanisms involved in the response of plants to combined treatments with Cd(II) and O3 were not substantially different to those recorded in plants exposed to a single stress (cadmium or ozone).
The photochemical efficiency of PSII was, however, not affected even if the CO2 assimilation rate diminished to a major extent in plants subjected to both the stresses. Stomatal conductance and intercellular CO2 concentration did not change, indicating major effects at mesophyllic level. On the other hand Rubisco activity was depressed in these leaves and an increase in 1 − qP coefficient and a parallel increase in qNP were observed. This indicates that energy consumption was inhibited by the two stresses most likely through a decrease in CO2 fixation ability.
It should be underlined that for CO2 fixation and for the actual quantum yield the combined effects of the two stresses were synergic, and this may be important from an ecological point of view, since O3 and cadmium pollution do coexist in natural conditions.
Although O3 is unlikely to penetrate to the chloroplast, the ROS generated by this pollutant affect chloroplastic metabolism, since they induce pigment co-oxidation and Calvin-cycle enzyme inhibition (Hippeli & Elstner, 1996). Heavy metals, such as cadmium can also determine a reduction in chlorophyll content which can be due to a reduced synthesis but also to oxidative stress as reported by Gallego et al. (1999).
Data obtained in this paper indicate that sunflower plants tried to cope with the oxidative stress induced by Cd(II) and O3 treatments by strengthening their antioxidant capabilities, since the ascorbate pool (ASC and DHA) was significantly increased under both stress conditions. Our results also support the involvement of APX in the defence mechanisms against the oxidative stress induced by Cd(II) and/or O3, since the activity of this H2O2-scavenging enzyme increased markedly both in Cd(II)- and in O3-treated plants, and a further increase occurred in plants simultaneously exposed to the two pollutants.
In Cd(II)- or O3-treated plants an increase in ASC oxidation is clearly indicated also by the increase in DHA content, the final product of ASC oxidation, and in the activity of AFRR, the enzyme responsible of the first product of ASC oxidation. Cd(II) and O3 treatments seem to have a different effect on the reduced form of the ascorbate pool, because O3 fumigation induced an increase in ASC content, whereas cadmium determined a decrease. The reduction in ASC content induced by Cd(II) could be due to a less efficient recycling of the ASC oxidized forms. The DHAR activity did not significantly change; on the other hand, AFRR had lower activity in Cd(II)- than in O3-treated plants. This confirms that AFRR, more than DHAR, plays a key role in ASC-recycling in accordance with previous observations (De Gara & Saracino, 1997; De Gara & Tommasi, 1999).
As far as the combined effect of Cd(II) and O3 is concerned, our data suggest that when sunflower plants must cope with both pollutants, their ASC utilization is higher than that occurring under the stress conditions induced by a single pollutant. This is substantiated by the behaviour of APX, AFRR and DHAR, the activities of which were more enhanced in plants subjected to both the stresses than in those subjected to a single stress.
The increase in the antioxidant capability induced by Cd(II) and/or O3 is not enough to overcome the toxic effect of the two pollutants completely. Physiological data obtained from gas exchange and chlorophyll fluorescence, reported in this paper, indicate that plants subjected to Cd(II) and/or O3 respond at photosynthetic level by showing a reduced CO2 assimilation. Moreover, the persistence of oxidative stress induced by Cd(II) and/or O3 exposure is clearly indicated by the increase in lipid peroxidation and in the protein carbonyl content. This is because ROS can be considered to be the initiators of cell damage through lipid peroxidation, providing steady generation of free radicals in vivo. Protein degradation, which follows oxidative protein modifications, seems also to be an index of oxidative stress, even more sensitive than lipid peroxidation (Gallego et al., 1999). On the other hand, Torsethaugen et al. (1997) reported that an overproduction of APX in transgenic tobacco plants did not provide complete protection against O3 stress. Another well documented metabolic event occurring under stress conditions is the increase in the activity of PODs. Many POD isoenzymes are involved in cell wall stiffening (Barcelò, 1997), a well known event occurring under a wide range of stress conditions. Moreover, recently a POD isoenzyme has also been localized in chloroplasts (Zapata et al., 1998). Interestingly, the activity of this POD increases greatly when overproduction of ROS is induced (Casano et al., 1999). In plants subjected to Cd(II) and/or O3, POD activity rose compared to the control. Similar results are reported in tobacco plants exposed to acute O3 (Schraudner et al., 1997) and in sunflower cotyledons grown in presence of Cd(II) (Gallego et al., 1996).
By contrast to ASC redox enzymes, Cd(II) plus O3 treatment had a synergic effect on POD activity that increased 16 times, remarkably more than the sum of the increases induced by the single treatments. The prolonged Cd(II) treatment that preceded the acute O3 fumigation, could constitute a sort of adaptation which develops a major capability for raising the activity of specific peroxidase/s when another different stress condition also intervenes. The rise in POD activity does not seem to be correlated to an increase in resistance against both stresses, since, in spite of POD increase, the effects at physiological and morphological levels were not overcome. This agrees with the results obtained in tobacco plants exposed to O3 fumigation, where POD activity increases both in resistant and sensitive cultivars (Schraudner et al., 1997).
This research was partially supported by the Murst (National Projects) Rome (Italy).