Antigenic cross-reactivity among monoterpene cyclases from grand fir and induction of these enzymes upon stem wounding

Abstract

A major wound response in grand fir (Abies grandis) sapling stems is the rapid increase in monoterpene production at the site of injury. Monoterpene cyclases (synthases) catalyze the formation of monoterpenes from geranyl pyrophosphate, and total cyclase activity increases markedly on wounding. At least six distinct cyclases, producing different monoterpene products, have been isolated from wounded grand fir saplings and characterized. The predominant wound-inducible cyclase produces both alpha- and beta-pinene. This pinene cyclase was purified, and polyclonal antibodies were generated in rabbits against the sodium dodecyl sulfate-denatured protein. The antibody preparation was found to cross-react by Western blotting with other grand fir monoterpene cyclases that produce different olefinic products, but not with monoterpene cyclases from related conifer species (Pinus contorta and P. ponderosa) or from angiosperms (Mentha piperita and M. spicata). The increase in monoterpene cyclase activity after wounding was closely correlated with the appearance of new cyclase protein as determined by immunoblotting. These results indicate that the wound-dependent increase in monoterpene cyclase activity is a consequence of de novo synthesis of cyclase protein.