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).