Qualitative and Quantitative Analyses of Gibberellins in Vegetative Shoots of Normal, dwarf -1, dwarf -2, dwarf -3, and dwarf -5 Seedlings of Zea mays L.

Abstract

Gibberellins A12 (GAI2), GA53, GA44, GA19, GA,7, GA20, GA29, GA1, and GAB 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 114C41GA53, I'4C41GA44, 12H21 GA1I, 113C,IGA2o, 1'3C,IGA29, 113C,IGAI, and 1'3C,JGA8. 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, was very low (0.23 nanograms per 100 grams fresh weight) and less than 2% of that in normal shoots, while GA20 and GA29 accumulated to levels over 10 times those in normals; these results confirm that the dwarf-1 mutant blocks the conversion of GA20 to GA,. 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. (8, 19), and GA20 to GA, (19); third, bioassay data, using the dwarf-l (dl), dwarf-2 (d2), dwarf-3 (d3) and dwarf-5 (d5) mutants indicate that these single-gene, nonallelic mutants block specific steps in the early 13-hydroxylation pathway. The pattern of response suggests the position in the pathway blocked by each mutant. For example, GA20 and GA, are both highly active when assayed on d5 whereas GA20 is 1% as active as GA, when assayed on dl. Thus, the d5 mutant blocks a step before GA20, and the dl mutant the late step, GA20 to GA,. In addition, the GA precursor ent-kaurene is active on d5, suggesting that the d5 block is before ent-kaurene (Fig. 1); fourth, feeding studies with [17-'3C,3H2]GA20 show that GA20 is metabolized to GA, in shoots of normal and d5 seedlings, but not in dl seedlings (19). Also feeding studies with ['4C]mevalonic acid, ['4C]geranylgeranyl-pyrophosphate and [3H]copalylpyrophosphate, show these precursors to be metabolized to ent-kaurene by normal seedlings and at a reduced level by d5 seedlings (6). In this paper, evidence is presented for the presence of GA12, and GA8 in the seedling shoots of normal maize (Fig. 1). The data are based on full scan mass spectra and Kovats retention indices (14) from capillary GC-MS. In addition, quantitative data are presented for the levels of specific endogenous GAs in normal, dl, d2, d3, and d5 seedlings using labeled internal standards and GC-SIM.3 These data provide additional support for the positions of the steps in the metabolic pathway blocked by the mutant genes (Fig.