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

• Published in: Planta 2009-08, Vol.230 (3), p.553-567
• Main Authors: Kärkönen, Anna, Warinowski, Tino, Teeri, Teemu H, Simola, Liisa Kaarina, Fry, Stephen C
• Format: Article
• Language: English
• Subjects: Agriculture; Apoptosis - drug effects; Biological and medical sciences; Biomedical and Life Sciences; Cells, Cultured; Ecology; Forestry; Fundamental and applied biological sciences. Psychology; Hydrogen peroxide; Hydrogen Peroxide - pharmacology; Life Sciences; Lignin - biosynthesis; Original Article; Picea - cytology; Picea - metabolism; Plant Sciences
• ISSN: 0032-0935; 1432-2048
• DOI: 10.1007/s00425-009-0968-5

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 O₂, 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. [³⁵S]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.