Measurement of the ascorbate content of spinach leaf protoplasts and chloroplasts during illumination

Protoplasts prepared from spinach leaves in May and June contained substantial amounts of ascorbate (1.33±0.28 μmol mg(-1) chlorophyll), of which 30-40% was localised in the chloroplasts. During illumination, the ascorbate content was maintained at approximately the same concentration as in the dark in both protoplasts and chloroplasts, even in the absence of CO2 when pseudocyclic electron flow would be expected to be maximal. The addition of the Mehler reagent, methyl viologen, to isolated chloroplasts caused a rapid oxidation of stromal ascorbate in the light such that less than 95% of the ascorbate was oxidised after illumination for 1 min. Similarly the stromal ascorbate pool was rapidly oxidised upon the addition of H2O2. We conclude that when the intracellular ascorbate concentration is high, photosynthetically generated H2O2 can be reduced at rates comparable to its synthesis via the ascorbate-glutathione cycle. The addition of methyl viologen which catalyses rapid production of the superoxide anion, O 2 (-) or the addition of excess H2O2, overwhelms the reductive cycle and the ascorbate system becomes partially or totally oxidised.