THE INFLUENCE OF APOPLASTIC ASCORBATE ON THE ACTIVITIES OF CELL WALL-ASSOCIATED PEROXIDASE AND NADH OXIDASE IN NEEDLES OF NORWAY SPRUCE (PICEA-ABIES L)

Cell wall-associated peroxidases (EC 1.11.1.7) were extracted from the current year's needles of Norway spruce trees (Picea abies L.) in two fractions, namely soluble apoplastic peroxidases and covalently wall-bound peroxidases. Peroxidase activities were determined with two substrates: coniferyl alcohol, which is important for lignification, and NADH, which is necessary for the production of H2O2. Coniferyl alcohol peroxidase activity was detected in both the soluble apoplastic fraction and the wall-bound fraction, whereas NADH oxidase activity was found only in the soluble apoplastic fraction. Net oxidation of coniferyl alcohol and NADH was inhibited by ascorbate, which reduced the oxidized intermediates of the peroxidase- and oxidase-catalyzed reactions. Since ascorbate itself was oxidized in these reactions, the inhibition was not persistent and it was released once the ascorbate present in the assay mixture had been oxidized. Ascorbate delayed the oxidation of NADH 10-fold more efficiently than the oxidation of coniferyl alcohol. Although the level and the redox state of apoplastic ascorbate were lower in lignifying needles than in mature needles, the concentration, which was 1.17 mM in apoplastic washing fluids, was sufficiently high to inhibit peroxidase activity in vitro. These results suggest that peroxidases can catalyze lignification only if local differences exist in the concentration of reduced ascorbate between lignifying and non-lignifying tissues.