Cell suspension cultures of spruce (Picea abies): inactivation of extracellular enzymes by fungal elicitor-induced transient release of hydrogen peroxide (oxidative burst)

Abstract

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, β-glucosidase and acid phosphatase, secreted by the cells during growth, and also α-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-sulfonate)) (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.