CELL-SUSPENSION CULTURES OF SPRUCE (PICEA-ABIES) - INACTIVATION OF EXTRACELLULAR ENZYMES BY FUNGAL ELICITOR-INDUCED TRANSIENT RELEASE OF HYDROGEN-PEROXIDE (OXIDATIVE BURST)

Elicitation of suspension culture cells of spruce [Picea abies (L.) Karst] with a fungal cell wall preparation of the spruce pathogenic fungus Rhizosphaera kalkhoffii Bubak induced inactivation of extracellular enzymes. Extracellular peroxidase, beta-glucosidase and acid phosphatase, secreted by the cells during growth, and also alpha-amylase and pectinase from Aspergillus strains, added to an elicited cell culture, were inactivated. Inactivation is caused by an elicitor-mediated transient release of H2O2 from the cells (oxidative burst). H2O2 released into the medium was determined with ABTS (2,2'-Azino-bis-(3-ethylbenthiazoline-6- (formation of blue colour) and with phenol red (destruction of pH indicator). The release started only minutes after beginning of elicitation and its inactivating effect existed for more than 1 day. Release of H2O2 is a biphasic process with a first smaller maximum at 1 h, followed by a second larger increase, peaking at 5-6 h and returning to approximately the control levels thereafter. Also H2O2 is transiently released in small quantities from cell incubations in the absence of elicitor as a stress response of the cells to manipulations of the cultures. Extracellular enzymes secreted into the medium could also be inactivated by direct addition of exogenous H2O2 . Catalase prevents inactivation of the secreted extracellular enzymes, however, to a limited extent only because, as a result of contact of cells and medium, catalase becomes inactivated. The ionophores A 23187 and cycloheximide induced release of H2O2 and, when present together with elicitor, induction was synergistically increased.