Early production and scavenging of hydrogen peroxide in the apoplast of sunflower plants exposed to ozone.

The present work set out to define the processes involved in the early O3-induced H2O2 accumulation in sunflower plants exposed to a single pulse of 150 ppb of O3 for 4 h. Hydrogen peroxide accumulation only occurred in the apoplast and this temporally coincided with the fumigation period. The inhibitor experiments suggested that both the plasma membrane-bound NAD(P)H oxidase complex and cell-wall NAD(P)H PODs contributed to H2O2 generation. To investigate the mechanisms responsible for O3-induced H2O2 accumulation further, both production and scavenging of H2O2 were investigated in the extracellular matrix after subcellular fractionation. The results indicated that H2O2 accumulation is a complex and highly regulated event requiring the time-dependent stimulation and down-regulation of differently located enzymes, some of which are involved in H2O2 generation and degradation, not only during the fumigation period but also in the subsequent recovery period in non-polluted air. Owing to the possible interplay between H2O2 and ethylene, the time-course of ethylene emission was analysed too. Ethylene was rapidly emitted following O3 exposure, but it declined to control values as early as after 4 h of exposure. The early contemporaneous detection of increased ethylene and H2O2 levels after 30 min of exposure does not allow a clear temporal relationship between these two signalling molecules to be established.

Assimilation of CO2 , enzyme activation and photosynthetic electron transport in bean leaves, as affected by high light and ozone.

• Bean seedlings (Phaseolus vulgaris cv. Pinto) were grown in the greenhouse at a light intensity of 400 µmol m-2  s-1 . When the primary leaf was fully expanded, plants were divided into four groups and subjected to one of the following treatments: light intensity of 400 µmol m-2  s-1 and filtered air (control); light intensity of 400 µmol m-2  s-1 and ozone (O3 ) (150 nl l-1 for 5 h) (ozonated); light intensity of 1000 µmol m-2  s-1 for 5 h and filtered air (HL); and light intensity of 1000 µmol m-2  s-1 and O3 (150 nl l-1 ) for 5 h (HL + O3 ). • At the end of the treatments (HL and/or O3 ) a strong decrease in CO2 assimilation rate as well a decrease in stomatal conductance were observed, while no changes in intercellular CO2 concentration were recorded. In addition the Fv  : Fm ratio (maximal quantum yield for PSII photochemistry) decreased in the stressed leaves (HL and/or O3 ), indicating photoinhibition, and they showed a corresponding increase in minimal fluorescence (F0 ), indicating a higher number of deactivating photosystem II (PSII) centres. • The maximum catalytic activity of the Benson-Calvin cycle enzymes, fructose-1,6-bisphosphate phosphatase (FBPase) and Rubisco, decreased following HL + O3 stress but activation was enhanced. A linear relation was found between activation state of NADP-malate dehydrogenase (MDH) and the flux of electrons through PSII and in HL + O3 -treated plants NADP-MDH activity decreased at high irradiance levels, indicating a limitation in linear electron flux.

Effect of chronic ozone fumigation on the photosynthetic process of poplar clones showing different sensitivity.

Rooted cuttings from two poplar clones (Populus x euramericana, I-214, and Populus deltoides x maximowiczii, Eridano) were exposed for 15 days to diurnal square-wave treatment with ozone (60 nL L-1 for 5 h day-1). Completely fully expanded leaves exposed to ozone showed a reduction in net CO2 assimilation rate as compared to the control leaves during whole exposure period in both the clones. The reduction was related to a strong stomatal closure in clone I-214, but also to an altered mesophyll activity ascribed to limitation of the dark reactions of photosynthetic process. The results obtained in leaves of I-214 subjected to long-term fumigation seem to support the view that the decrease in quantum yield of electron transport may be a mechanism to down-regulate photosynthetic electron transport so that production of ATP and NADPH would be in equilibrium with the decreased demand in the Calvin cycle. In Eridano the CO2 assimilation was reduced because of the exposure and any alteration in stomatal conductance was observed. Thus, chlorophyll fluorescence parameters showed that an inhibition of photosystem II had occurred (reduction in Fv/Fm ratio), while no alterations in quenching parameters were observed upon illumination. The results seem to indicate that an alternative sink for reducing equivalent, other than carbon metabolism is present.

Iron deficiency differently affects peroxidase isoforms in sunflower.

The response of both specific (ascorbate peroxidase, APX) and unspecific (POD) peroxidases and H(2)O(2) content of sunflower plants (Helianthus annuus L. cv. Hor) grown hydroponically with (C) or without (-Fe) iron in the nutrient solution were analysed to verify whether iron deficiency led to cell oxidative status. In -Fe leaves a significant increase of H(2)O(2) content was detected, a result confirmed by electron microscopy analysis. As regards extracellular peroxidases, while APX activity significantly decreased, no change was observed in either soluble guaiacol or syringaldazine-dependent POD activity following iron starvation. Moreover, guaiacol-dependent POD activity was found to decrease in both ionically and covalently-cell-wall bound fractions, while syringaldazine-POD activity decreased only in the covalently-bound fraction. At the intracellular level both guaiacol-POD and APX activities underwent a significant decrease. The overall reduction of peroxidase activity was confirmed by the electrophoretic separation of POD isoforms and, at the extracellular level, by cytochemical localization of peroxidases by diaminobenzidine staining. The electrophoretic separation, besides quantitative differences, also revealed quantitative changes, particularly evident for ionically and covalently-bound fractions. Therefore, in sunflower plants, iron deficiency seems to affect the different peroxidase isoenzymes to different extents and to induce a secondary oxidative stress, as indicated by the increased levels of H(2)O(2). However, owing to the almost completely lack of catalytic iron capable of triggering the Fenton reaction, iron-deficient sunflower plants are probably still sufficiently protected against oxidative stress.

Combined cadmium and ozone treatments affect photosynthesis and ascorbate-dependent defences in sunflower.

• 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.

Screening of bean cultivars for their response to ozone as evaluated by visible symptoms and leaf chlorophyll fluorescence.

Fourteen Italian cultivars of Phaseolus vulgaris were exposed to a single pulse of ozone (O(3), 150 nl l(-1)) or to filtered air (<3 nl l(-1)) for 3.5 h. O(3) sensitivity was assessed by recording the extent of visible symptoms, effects on chlorophyll (Chl) content and changes in Chl a fluorescence parameters. This paper reports the results of an initial screening of 14 bean cultivars that was used to select a small number of cultivars for further work. Seven cultivars showed visible symptoms of injury in the range of 2-60 h after the end of the O(3) fumigation. O(3) significantly depressed total Chl content in most cultivars and a significant correlation was found between Chl content and visible symptoms. Most cultivars showed a significant change in the F(v)/F(m) ratio, even when there were no visual symptoms. There was no relationship between the extent of visual symptoms and quenching coefficients, indicating that these parameters were of no use in the determination of sensitivity to O(3) stress.

Two-dimensional electrophoresis of thylakoid protein patterns in two wheat cultivars with different sensitivity to sulfur dioxide.

Plants of two wheat cultivars (Triticum aestivum L., cultivars Mec and Chiarano), differing in sensitivity towards sulfur dioxide, were fumigated with 96 nL L-1 of SO2 for four months. Thylakoid membranes were subjected to two-dimensional electrophoresis in order to detect possible changes in the protein patterns. We found the greatest alterations following treatment in the polypeptide profiles of the most sensitive cultivar, Mec, even if some significant changes were also detected in the plants of the cultivar Chiarano.

Sulfite: Preferential sulfur source and modifier of CO2 fixation in Chlorella vulgaris.

Sulfite was added at the time of inoculation to a standard and to a sulfate deficient medium of Chlorella vulgaris. It was not only used as a sulfur source, but besides this, at concentrations <1.0 mmol l(-1), the growth yield was enhanced up to 30% compared to sulfate saturated conditions. Higher sulfite concentrations increasingly inhibited cell growth. Growth rate determinations indicated that the enhancement, and the inhibition respectively, were confined to the very beginning of culture growth; the time period during which the sulfite was not yet oxidized (5-10 h). In contrast, an increased CO2 fixation rate/unit of protein, occurring up to 5.0 mmol l(-1) sulfite and a shift towards the ß-carboxylation pathway, are persisting at least during the growth period of 4 days. The preferential uptake of sulfite, also indicated by a marked increase in methionine content of algal protein, presumably causes an increase in thylakoidal sulfolipids, and is such modifying the CO2 fixation.