Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing cucumber ascorbate oxidase (EC.1.10.3.3) were used to examine the role of extracellular ascorbic acid in mediating tolerance to the ubiquitous air pollutant, ozone (O 3 ). Three homozygous transgenic lines, chosen on the basis of a preliminary screen of AO activity in the leaves of 29 lines, revealed up to a 380-fold increase in AO activity, with expression predominantly associated with leaf cell walls. Overexpression of AO resulted in no change in the total ascorbate content recovered in apoplast washing fluid, but the redox state of ascorbate was reduced from 30% in wild-type leaves to below the threshold for detection in transgenic plants. Levels of ascorbic acid and glutathione in the symplast were not affected by AO overexpression, but the redox state of ascorbate was reduced, while that of glutathione was increased. AO overexpressing plants exposed to 100 nmol mol -1 ozone for 7 h day -1 exhibited a substantial increase in foliar injury, and a greater pollutant-induced reduction in both the light-saturated rate of CO 2 assimilation and the maximum in vivo rate of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation, compared with wild-type plants. Transgenic plants also exhibited a greater decline in CO 2 assimilation rate when exposed to a brief ozone episode (300 nmol mol -1 for 8 h). Stomatal conductance, hence O 3 uptake, was unaffected by AO over-expression. Our findings illustrate the important role played by ascorbate redox state and sub-cellular compartmentation in mediating the tolerance of plants to ozone-induced oxidative stress.Abbreviations AO: ascorbate oxidase AE A sat : CO 2 assimilation rate under light-saturated conditions AE AA: L-ascorbic acid AE AWF: apoplast washing fluid AE c a : atmospheric CO 2 concentration AE c i : intercellular CO 2 concentration AE CFA: charcoal/Purafil-filtered air AE DAT: days after transfer to the respective treatments AE DHA: dehydroascorbate AE GSH: reduced glutathione AE GSSG: oxidised glutathione AE g H2O : stomatal conductance to water vapour AE PPFD: photosynthetic photon flux density AE ROS: reactive oxygen species AE V cmax : maximum in vivo rate of Rubisco carboxylation Planta (2003) 216: 918-928

This paper presents a mathematical model which enables the semi-quantification of ozone (O3) detoxification, based upon the direct reaction of the pollutant with ascorbate (ASC) located in the aqueous matrix associated with the cell wall (i.e. the apoplast). The model describes the uptake of ozone into the leaf and its direct reaction with ASC, taking into consideration the regeneration of dehydroascorbic acid in the cytosol, the rate of replenishment of cell wall ASC and the distribution of ASC between sub-cellular compartments based upon the permeability of biomembranes to the neutral species, ascorbic acid and the pH of various sub-cellular compartments. The importance of various physico-chemical characteristics (e.g. stomatal conductance, mesophyll cell wall thickness and tortuosity, chloroplast volume, apoplast pH, ASC:O3 reaction stoichiometry) in mediating the flux of ozone to the plasmalemma is analysed. Model simulations, supported by experimental observations, suggest that the ASC concentration in the leaf apoplast is high enough to scavenge a significant proportion of the O3 taken up into the leaf interior, under environmentally relevant conditions. However, there is considerable variation between taxa in the potential degree of protection afforded by apoplastic ASC, emphasizing the need for an improved understanding of the reaction chemistry of O3 in the cell wall.

Broad bean (Vicia faba L.) plants were exposed, in duplicate controlled environment chambers, to charcoal/Purafil-filtered air (CFA-grown plants) or to 75 nmol mol(-1) ozone (O(3)) for 7 h d(-1) (O(3)-grown plants) for 28 d, and then exposed to 150 nmol mol(-1) O(3 )for 8 h. The concentration of ascorbate (ASC) was determined in leaf extracellular washing fluid (apoplast) and in the residual leaf tissue (symplast) after 0, 4 and 8 h acute fumigation, and after a 16 h "recovery" period in CFA. Changes in stomatal conductance were measured in vivo in order to model pollutant uptake, while the light-saturated rate of CO(2) assimilation (A:(sat)) was recorded as an indicator of O(3)-induced intracellular damage. Measurements of A:(sat) revealed enhanced tolerance to 150 nmol mol(-1) O(3) in plants pre-exposed to the pollutant compared with equivalent plants grown in CFA, consistent with the observed reduction in pollutant uptake due to lower stomatal conductance. The concentration of ASC in the leaf apoplast (ASC(apo)) declined upon O(3)-treatment in both CFA- and O(3)-grown plants, consistent with the oxidation of ASC(apo) under O(3)-stress. Furthermore, the decline in ASC(apo) was reversible in O(3)-grown plants after a 16 h "recovery" period, but not in plants grown in CFA. No significant change in the level and/or redox state of ASC in the symplast (ASC(symp)) was observed in plants exposed to 150 nmol mol(-1) O(3), and there was no difference in the constitutive level of ASC(symp) between CFA- and O(3)-grown plants. Model calculations indicated that the reaction of O(3) with ASC(apo) in the leaves of Vicia faba is potentially sufficient to intercept a substantial proportion (30-40%) of the O(3)entering the plant under environmentally-relevant conditions. The potential role of apoplastic ASC in mediating the tolerance of leaves to O(3) is discussed.

$ on growth and reproductive performance confirmed the sensitivity of the population to the pollutant. In the absence of the development of typical visible symptoms of foliar damage, the total d. wt of plants maintained in O $ over a 56-d period was 35 % lower than that of control plants. However, the impact of the pollutant was found to decrease as plants aged. Plant relative growth rate (R E ) was only affected in seedlings, suggesting that effects of O $ on seedling growth were largely responsible for the decrease in accumulated biomass ; the growth rate of older plants was not affected by O $ . The observed shift in O $ resistance with plant age was mediated by both ' acclimation ' and ontogenetic changes. ' Acclimation ' was not associated with changes in O $ uptake, and there was some evidence to support the existence of compensatory growth responses. In addition to effects on vegetative growth, plants exhibited an O $ -induced decline in reproductive performance ; O $ reducing the number of flower spikes and seed capsules produced per plant. Ozone episodes administered at different stages of development indicated that reproductive development was particularly sensitive to O $ during the early stages of flowering.The findings of this study are discussed in relation to evolutionary adaptation to O $ in natural plant communities.The importance of plant age, prior exposure to the pollutant and the timing of O $ episodes in relation to plant developmental stage are highlighted.

SUMMARYThe relative ozone resistance of 20 European and two American populations oi Plantago major was examined, and relationships with climatic factors at the source of the plant material were explored using data provided by participants in the ICP-Crops initiative (International Co-operative Programme to Investigate the Effects of Air Pollutants and Other Stresses on Agricultural and Semi-Natural Vegetation). Plants grown from seed were exposed to either charcoal/Purafil® filtered air (CF < 5 nmol mol"^ O3) or CF 4-ozone (70 nmol mol"^ O3 7 h d~^) over a 2-wk period in controlled environment chambers, and effects on mean plant relative growth rate (R) and allometric root/shoot growth (K) determined. Ozone resistance (R%) was calculated from (Ro^/R^,^) x 100.Populations exhibited contrasting sensitivities to ozone, without the development of typical visible symptoms of injury. A positive relationship was found between relative ozone resistance and descriptors of the ozone-climate at the site of seed collection for the year of, and the 2 yr before, seed collection. The best predictors of inherent ozone resistance were shown to be cumulative ozone exposure indices calculated according to current United Nations Economic Commission for Europe (UN-ECE) critical level guidelines for the pollutant (i.e. the accumulated hourly average ozone exposure over a threshold level of 40 nmol mor^ (AOT40) or 30 nmol mol"( AOT30) calculated during daylight hours for the consecutive 3-month period of the year experiencing the highest ozone concentrations). No relationships were found between ozone resistance and climatic factors (temperature, precipitation, sunshine hours, humidity) or the concentrations of other air pollutants (SOg, NOg, NO).These findings support the view that current ambient levels of ozone in many regions of Europe are high enough to promote evolution of resistance to the pollutant in native plant populations. The significance of these findings to the debate over the establishment of separate critical levels for the protection of natural and semi-natural vegetation is discussed.