Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.)

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Abstract
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 (O3) for 7 h d−1 (O3‐grown plants) for 28 d, and then exposed to 150 nmol mol−1 O3 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 CO2 assimilation (Asat) was recorded as an indicator of O3‐induced intracellular damage. Measurements of Asat revealed enhanced tolerance to 150 nmol mol−1 O3 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 (ASCapo) declined upon O3‐treatment in both CFA‐ and O3‐grown plants, consistent with the oxidation of ASCapo under O3‐stress. Furthermore, the decline in ASCapo was reversible in O3‐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 (ASCsymp) was observed in plants exposed to 150 nmol mol−1 O3, and there was no difference in the constitutive level of ASCsymp between CFA‐ and O3‐grown plants. Model calculations indicated that the reaction of O3 with ASCapo in the leaves of Vicia faba is potentially sufficient to intercept a substantial proportion (30–40%) of the O3entering the plant under environmentally‐relevant conditions. The potential role of apoplastic ASC in mediating the tolerance of leaves to O3 is discussed.