Polyphenol oxidase and herbivore defense in trembling aspen (Populus tremuloides): cDNA cloning, expression, and potential substrates

The biochemical anti-herbivore defense of trembling aspen (Populus tremuloi des Michx.) was investigated in a molecular analysis of polyphenol oxidase (PPO; EC 1.10.3.2). A PPO cDNA was isolated from a trembling aspen wounded leaf cDNA library and its nucleotide sequence determined. Southern analysis indicated the presence of two PPO genes in the trembling aspen genome. Exp ression of PPO was found to be induced after herbivory, by forest tent cate rpillar, by wounding, and by methyl jasmonate treatment. Wound induction wa s systemic. and occurred in unwounded leaves on wounded plants. This patter n of expression is consistent with a role of this enzyme in insect defense. A search for potential PPO substrates in ethanolic aspen leaf extracts usi ng electron spin resonance (ESR) found no pre-existing diphenolic compounds . However. following a brief delay and several additions of oxygen, an ESR signal specific for catechol was detected. The source of this catechol was most likely the aspen phenolic glycosides tremulacin or salicortin which de composed during ESR experiments. This was subsequently confirmed in experim ents using pure salicortin.