Dark-induced ascorbate deficiency in leaf cell walls increases plasmalemma injury under ozone

To assess protection of the mesophyll cell plasmalemma against O3 by apoplasmic reduced ascorbate (AA), its concentration in the leaf cell wall of common bean (Phaseolus vulgaris L.) was lowered from 0.6 mM to 0.1 mM by pre-exposing plants to continuous darkness for up to 48 h. Subsequent ozonization of ascorbate-deficient leaves with 350-450 nmol O3 mol-1 resulted in a rapid rise of apoplasmic AA within the second hour of the treatment, the concomitant appearance of cytoplasmic marker enzymes in cell wall solute extracts and the development of water-logged spots on leaves. Prior to these events, stomatal conductances had just reached values close to those observed in AA- nondeficient leaves, whereas AA concentration in the cell wall was still 24 times lower than in leaves pre-exposed to the normal 10-h dark period. In AA-nondeficient leaves the initial apoplasmic AA level of 0.6 mM was maintained under O3 for 2.5 h; thereafter, it increased moderately. There appeared to be no signs of injury even 2 d after the whole 4.5-h treatment. During the period of equal stomatal conductances, the O3 decay rate in direct reaction with AA in AA-deficient cell walls was estimated to be 50-70% of that occurring in AA-nondeficient leaves. It is suggested that under AA deficiency some threshold for the stability of the plasmalemma was surpassed owing to the more "O3-permeable" cell wall. The mesophyll conductance was found to be stable throughout O3 exposure, indicating that the cytoplasmic O3 defense barrier was not exceeded. Possible changes in oxyradical reactions and in cell wall phenolics are discussed. It is suggested that after prolonged darkness the flow rate of reactive oxygen intermediates to the plasmalemma may also be higher because they are less trapped in direct and peroxidase-catalyzed reactions.