Diterpene biosynthesis in maize seedlings in response to fungal infection

A cell-free system which catalyzes the biosynthesis of terpene hydrocarbons when supplemented with mevalonate, Mn²⁺, and ATP was prepared from the scutellum-embryonic axis region of maize seedlings. The capacity of this system for the production of terpene hydrocarbons was enhanced 50- to 100-fold when the seedlings were exposed for 48 hours to the fungus Rhizopus stolonifer prior to tissue homogenization. The fungi Aspergillus niger, Fusarium moniliforme, and Verticillium albo-atrum also elicited this biosynthetic enhancement. The terpene hydrocarbon products were separable into six fractions by argentation thin layer chromatography. Radioactivity was contributed to five of these fractions when either geranylgeranyl pyrophosphate or copalyl pyrophosphate was supplied as substrate, suggesting that polycyclic diterpenoid hydrocarbons were the main products. Large scale biosynthetic reactions led to the acquisition of about 1 milligram of terpene hydrocarbon products plus some more polar terpenoid products. Analysis of the hydrocarbon products by gas chromatography and mass spectrometry led to the separation of six distinct diterpene hydrocarbons plus a fraction with a molecular weight of about 550. Three of the diterpene hydrocarbons were identified as kaur-16-ene, kaur-15-ene (isokaurene), and pimara-8(14),15-diene. None of the terpene hydrocarbon fractions tested displayed antifungal activity in the Cladosporium cucumerinum thin layer plate assay.