Regulation of Phytoalexin Synthesis in Jackbean Callus Cultures: Stimulation of Phenylalanine Ammonia-Lyase and o-Methyltransferase

Jackbean, Canavalia ensiformis (L.), callus tissues synthesized the phytoalexin, medicarpin (3-hydroxy-9-methoxypterocarpan), when treated with spore suspensions of Pithomyces chartarum (Berk. and Curt.) M. B. Ellis, a nonpathogen of jackbean. Medicarpin was isolated from treated callus tissue and identified by its ultraviolet and mass spectra. The minimum spore concentration found to elicit medicarpin synthesis after 26 hours was 1 × 10⁵ spores/ml; levels of medicarpin in callus tissue increased linearly up to 1 × 10⁷ spores/ml, indicating that the recognition sites for presumed elicitors were not saturated. Medicarpin was first detected in callus treated with 1 × 10⁷ spores/ml, 6 to 12 hours after application, and the concentration reached a maximum at 48 hours, slowly declining thereafter to 72 hours. In callus treated with 3.15 mm HgCl₂, medicarpin concentrations were also maximum by 48 hours. Phenylalanine ammonia-lyase (EC 4.3.1.5) activity increased 2-fold in spore-treated callus after 36 hours. Isoliquiritigenin, daidzein, and genistein o-methyltransferase (EC 2.1.1.6) activities were increased 3- to 4-fold in treated callus. Caffeic acid and naringenin were more efficient substrates for o-methyltransferase activity than the other flavonoids or apigenin, but there was no increase in these o-methyltransferase activities in spore-treated callus. The phytoalexin response in this callus tissue culture system compares well with natural plant systems and should be an excellent system for investigating regulation of phytoalexin synthesis.