On the mechanism of apoplastic H2O2 production during lignin formation and elicitation in cultured spruce cells-peroxidases after elicitation

A cell culture of Picea abies (L.) Karst. was used for studies of H2O2 generation during constitutive extracellular lignin formation and after elicitation by cell wall fragments of a pathogenic fungus, Heterobasidium parviporum. Stable, micromolar levels of H2O2 were present in the culture medium during lignin formation. Elicitation induced a burst of H2O2, peaking at ca. 90 min after elicitation. Of exogenous reducing substrates that may be responsible for the synthesis of H2O2 from O-2, NADH stimulated H2O2 production irrespective of elicitation. Cysteine (Cys) and glutathione (GSH) partially scavenged the constitutive H2O2, but usually increased or prolonged elicitor-induced H2O2 formation. Culture medium peroxidases were not able to generate H2O2 in vitro with Cys or GSH as reductants. These thiols, however, generated H2O2 non-enzymically at pH 4.5. [S-35]Sulphate feeding to spruce cells showed that endogenous sulphur-containing compounds (including GSH, GSSG and cysteic acid) existed in the culture medium. The apoplastic levels of these were, however, undetectable by the monobromobimane method suggesting that their contribution to apoplastic H2O2 formation is probably minor. Azide, an inhibitor of haem-containing enzymes, slightly inhibited constitutive H2O2 generation but strongly delayed the elicitor-induced H2O2 accumulation. Diphenylene iodonium, an inhibitor of flavin-containing enzymes, efficiently inhibited H2O2 production irrespective of elicitation. Elicitation led to downregulation of the expression of several peroxidase genes, and peroxidase activity in the culture medium was slightly reduced. Expression of three other peroxidase genes and a respiratory burst oxidase homologue (rboh) gene were upregulated. These data suggest that both peroxidases and rboh may contribute to H2O2 generation.