In Schizaeaceous ferns, antheridiogens have been reported to be GA-related compounds. Antheridic acid ( Fig. 1) is a major antheridiogen in four Anemia species, i.e. A. phyllitidis (Nakanishi et al., 1971;Corey et al., 1986), A. hirsuta (Zanno et al., 1972), A. rotundifolia, and A . flexuosa (Yamane et al., 1987). In A. mexicana, lp-hydroxy-9,15-cyclo-GA, (GA,,,; Fig. 1) was isolated as a major antheridiogen (Nester et al., 1987;Furber et al., 1989;Oyama et al., 1996). The principal antheridiogen in three Lygodium ferns (L. japonicum, L. circinnatum and L.flexuosum) was found to be GA,,-Me ( Fig. 1) (Yamane et al., 1988;. GA,-Me ( Fig. 1) was also identified in L. japonicum as an antheridiogen . In L. circinnatum, GA,-Me and monohydroxylated derivatives of GA,,-Me and GA,-Me were detected as minor antheridiogens .The occurrence of three skeletal types as the principal antheridiogens in the Schizaeaceous ferns, i. 20-norgibberell-9(11),16-diene derivative], suggests that they might be related biosynthetically. The ent-9,15-cyclo-20-norgibberell-16-ene structure could be a precursor of either 20-norantherid-16-ene and/ or ent-20-norgibberell-9(11),16-diene derivatives. Alternatively, the ent-20-norgibberell-16-ene structure could be a common precursor to each of the three classes of compounds. We reported previously that antheridic acid is biosynthesized from 9,15-cyclo-GA9 (GA,,,) via 3a-hydroxy-GA,,, (GA,,,) (Yamauchi et al., 1991;Oyama et al., 1996). However, almost no information has been obtained so far on the biosynthetic origin of GA,,-Me. The only information on the biosynthesis of antheridiogens in Lygodium ferns is that exogenously applied GA, was converted into GA,-Me and then into 13-OH-GA9-Me (GA,,-Me), 12p-OH-GA9-Me (GA,,-Me), and 12a-OH-GA,-Me (GA,,-Me) in L. japonicum prothallia Sato et al., 1985).In the present work, we investigated the biosynthetic pathway of GA,,-Me in L. circinnatum by the analyses of the endogenous GAs and GA methyl esters in prothallia and by feeding experiments using possible precursor candidates.
MATERIALS A N D M E T H O D S Analyses of Endogenous C A s and C A M e t h y l Esters in Fern ProthalliaSpores of Lygodium circinnatum were collected from sporangia-bearing pinnae harvested in the greenhouse at Ulm University, Ulm, Germany, and stored in a glass tube at 4°C until use. Spores of L. circinnatum were sterilized with 0.5% NaOCl and washed repeatedly with sterile water. They were then sown onto 0.5% agar-solidified medium (Mohr, 1956) and cultured under continuous white light (10 W m-') at 21°C. After about 5 weeks of culture, the prothallia were transferred individually to plastic Petri dishes (3.5 cm in diameter) containing 2 mL of the above medium and cultured further under the same conditions. After 10 weeks of culture, the prothallia were collected from the agar medium. The fresh weight of prothallia was 7.09 g.Abbreviations: AE, acidic ethyl acetate; DEA, diaminoethylpropyl; EtOAc, ethyl acetate; GA,-Me, GA, methyl ester; HOAc, acetic acid; MeOH, methanol; MSTF...