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Young shoots of normal maize (Zea mays L.) were used to determine both the stepwise metabolism of ent-kaurene to gibberellin A(12)-aldehyde and the endogenous presence of the members in this series. Each of the five steps in the sequence was established by feeds of 17-(13)C, (3)H-labeled kauranoids to cubes from the cortex of elongating internodes, to homogenates from the cortex of elongating internodes, and/or to homogenates from dark-grown seedlings. The (13)C-metabolites were identified by Kovats retention indices (KRI) and full-scan capillary gas chromatography-mass spectrometry (GC-MS). Five substrates and the final product in this sequence were shown to be native by the isotopic dilution of 17-(13)C, (3)H-labeled substrates added as internal standards to extracts obtained from elongating internodes. Evidence for the isotopic dilution was obtained by KRI and full-scan capillary GC-MS. Thus, we document the presence in young maize shoots of the metabolic steps, ent-kaurene --> ent-kaurenol --> ent-kaurenal --> ent-kaurenoic acid --> ent-7 alpha-hydroxykaurenoic acid --> gibberellin A(12)-aldehyde.

[17-(13)C,(3)H]-Labeled gibberellin A(20) (GA(20)), GA(5), and GA(1) were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). (13)C-Labeled GA(29), GA(8), GA(5), GA(1), and 3-epi-GA(1), as well as unmetabolized [(13)C]GA(20), were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-(13)C, (3)H]GA(20) to all three genotypes. (13)C-Labeled GA(8) and 3-epi-G(1), as well as unmetabolized [(13)C]GA(1), were identified by GC-SIM from feeds of [17-(13)C, (3)H]GA(1) to all three genotypes. From feeds of [17-(13)C, (3)H]GA(5), (13)C-labeled GA(3) and the GA(3)-isolactone, as well as unmetabolized [(13)C]GA(5), were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-(13)C, (3)H]GA(5) to [(13)C]GA(1), either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA(20), as follows: (a) GA(20) --> GA(1) --> GA(8); (b) GA(20) --> GA(5) --> GA(3); and (c) GA(20) --> GA(29). The in vivo biogenesis of GA(3) from GA(5), as well as the origin of GA(5) from GA(20), are conclusively established for the first time in a higher plant (maize shoots).

Ten gibberellins (GAs) have been identified by Kovats retention indices and full mass spectra from GC-MS analysis of purified extracts of sporophytes of the tree-fern, Cyathea australis. These include the known GA(1), GA(4), GA(9), GA(15), GA(24), GA(35), and GA(58) and three new GAs, 12beta-hydroxyGA(9) (GA(69)), 12alpha-hydroxyGA(9) (GA(70)) and 12beta-hydroxyGA(4) (GA(71)). The structure of GA(71) was established by the preparation and characterization of its methyl ester (as a metabolite of GA(4) methyl ester in a culture of prothallia of Lygodium japonicum).

Gibberellin A(1) (GA(1)), 3-epi-GA(1), GA(4), GA(9), 11alpha-hydroxyGA(12), 12alpha-hydroxyGA(12), GA(15), GA(17), GA(19), GA(20), GA(25), GA(37), GA(40), GA(58), GA(69), GA(70), and GA(71) have been identified from Kovats retention indices and full scan mass spectra by capillary GC-MS analyses of purified extracts from sporophytes of the tree fern, Cibotium glaucum. Abscisic acid, dihydrophaseic acid, an epimer of 4'-dihydrophaseic acid, and the epimeric ent-6alpha, 7alpha, 16alpha, 17-(OH)(4) and ent-6alpha, 7alpha, 16beta, 17-(OH)(4) derivatives of ent16, 17-dihydrokaurenoic acid, in addition to the epimeric 16alpha, 17- and 16beta, 17-dihydroxy-16, 17-dihydro derivatives of GA(12), were also identified in extracts of C. glaucum. An oxodihydrophaseic acid and a hydroxydihydrophaseic acid were also detected. In extracts of sporophytes of Dicksonia antarctica, GA(4), GA(9), 12alpha- and 12beta-hydroxyGA(12), GA(15), GA(25), and GA(37) were identified by the same criteria, as well as abscisic acid, phaseic acid, 8'-hydroxymethylabscisic acid and dihydrophaseic acid. This is the first time that GA(40) has been identified in a higher plant; it is also the first report of the natural occurrence of the two gibberellins, 11alpha- and 12beta-hydroxyGA(12). The total gibberellin (GA) content in C. glaucum (tall) was at least one order of magnitude greater than that of D. antarctica (dwarf) based on total ion current response in GC-MS and bioassay data. Abscisic acid was a major component of D. antarctica and the oxodihydrophaseic acid was a major component of C. glaucum.

Gibberellins A(12) (GA(12)), GA(53), GA(44), GA(19), GA(17), GA(20), GA(29), GA(1), and GA(8) have been identified from extracts of vegetative shoots of normal (wild type) maize using full scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Seven of these gibberellins (GAs) have been quantified by capillary gas chromatography-selected ion monitoring using internal standards of [(14)C(4)]GA(53), [(14)C(4)]GA(44), [(2)H(2)] GA(19), [(13)C(1)]GA(20), [(13)C(1)]GA(29), [(13)C(1)]GA(1), and [(13)C(1)]GA(8). Quantitative data from extracts of normal, dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings support the operation of the early 13-hydroxylation pathway in vegetative shoots of Zea mays. These data support the positions in the pathway blocked by the mutants, previously assigned by bioassay data and metabolic studies. The GA levels in dwarf-2, dwarf-3, and dwarf-5 were equal to, or less than, 2.0 nanograms per 100 grams fresh weight, showing that these mutants are blocked for steps early in the pathway. In dwarf-1, the level of GA(1) was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA(20) and GA(29) accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA(20) to GA(1). Since the level of GAs beyond the blocked step for each mutant is greater than zero, each mutated gene probably codes for an altered gene product, thus leading to impaired enzyme activities.