Two distinct mechanisms for localized hydrogen peroxide generation in response to agar oligosaccharides in two related red algae, Gracilaria chilensis and G. conferta

WEINBERGER FLORIAN; LEONARDI PATRICIA; KLOAREG BERNARD; POTIN PHILIPPE

Bergen, Noruega

Congreso; XVIII International Seaweed Symposium; 2004

Norwegian Institute for Water Research, Institute of Marine Research y Norwegian University of Science and technology

The two agarophytes Gracilaria chilensis and Gracilaria conferta both responded with H2O2 release to the presence of oligoagars in their medium, but the release was due to distinct mechanisms. In G. conferta, a transient release of H2O2 was observed, followed by a refractory state of 6 h. This response was sensitive to diphenylene iodonium and other chemical inhibitors of NADPH oxidase, protein kinase, protein phosphatase and calcium translocation in the cell, while it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H2O2-dependent formation of ceriumperoxide from cerium chloride indicated that large amounts of oxygen were activated at the plasma membrane of G. conferta within few minutes after challenge with oligoagar. These results indicate that a system consisting of a receptor specific to oligoagar and a plasma membrane-located NADPH oxidase is responsable for the release of H2O2 in G. conferta.  In G. chilensis H2O2 release after exposure to oligoagar was located in the apoplast. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, but not to diphenylene-iodonium, and a refractory state was not observed. The release of H2O2 was correlated with accumulation of an aldehyde in the algal medium. The molar ratio of aldehyde formed and oligoagar applied was 0.85, suggesting that an oxidase is present in the apoplast of G. chilensis that oxidizes oligoagars at one specific C6 atom. The enzyme acted exclusively on oligoagars larger than the disaccharide and it was inhibited by reduced oligoagar. A method to detect oligoagar oxidase on native PAGE gels was developed. Three different isoforms of the enzyme were generally present in G. chilensis while one specific strain expressed two additional isoforms. Incubation of G. chilensis for six or more weeks at 16°C resulted in a strong expression of oligoagar oxidase, while two weeks at 12°C nearly completely supressed it. Elicitation of G. conferta with oligoagar resulted within 24 h in a strong expression of an oligoagar oxidizing enzyme on native PAGE gels and exposure of G. conferta to oligoagar resulted in the formation of 1 mol aldehyde per mol of oligoagar