Plant defense metabolism is increased by the free radical-generating compound AAPH

Effects of the free radical-generating substance 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) on defense systems in plant tissue cultures were investigated. Exposure of Catharanthus roseus, C. tricophyllus, and Pisum sativum cultures to AAPH caused altered levels of reduced and oxidized glutathione. An increased total glutathione content in C. roseus was prevented by the glutathione biosynthesis inhibitor buthionine-sulfoximine. The specific phenylalanine ammonia-lyase activity in a C. roseus culture was increased from 4 to 34 mukat(kg protein)-1 by 1 mM AAPH. 5 mM AAPH increased the excretion of phenolic substances into the culture medium of a Pisum sativum culture, from 18 to 67 micrograms ml-1. The level of thiobarbituric acid reactants in a C. tricophyllus culture was increased from 46 to 93 nmol(g fresh weight)-1 by 0.4 mM AAPH. The present results, which constitute the first report on effects of the radical-generator AAPH on plant tissue, were achieved with cultures of various plant species and various types of tissue differentiation and demonstrate that AAPH is a suitable agent for the stimulation of the defensive and secondary metabolism in plant tissue cultures. It is proposed that the effects caused by AAPH are mediated by the generation of free radicals and oxidative stress, and that this agent may be used as a model substance for ozone and UV-B exposure.