Clive R. Spray

Internode length in Zea mays L.

Abstract[13C, 3H]Gibberellin A20 (GA20) has been fed to seedlings of normal (tall) and dwarf-5 and dwarf-1 mutants of maize (Zea mays L.). The metabolites from these feeds were identified by combined gas chromatography-mass spectrometry. [13C, 3H]Gibberellin A20 was metabolized to [13C, 3H]GA29-catabolite and [13C, 3H]GA1 by the normal, and to [13C, 3H]GA29 and [13C, 3H]GA1 by the dwarf-5 mutant. In the dwarf-1 mutant, [13C, 3H]GA20 was metabolized to [13C, 3H]GA29 and [13C, 3H]GA29-catabolite; no evidence was found for the metabolism of [13C, 3H]GA20 to [13C, 3H]GA1. [13C, 3H]Gibberellin A8 was not found in any of the feeds. In all feeds no dilution of 13C in recovered [13C, 3H]GA20 was observed. Also in the dwarf-5 mutant, the [13C]label in the metabolites was apparently undiluted by endogenous [13C]GAs. However, dilution of the [13C]label in metabolites from [13C, 3H]GA20 was observed in normal and dwarf-1 seedlings. The results from the feeding studies provide evidence that the dwarf-1 mutation of maize blocks the conversion of GA20 to GA1.

The metabolism of gibberellin A20 to gibberellin A1 by tall and dwarf mutants of Oryza sativa and Arabidopsis thaliana

The purpose of this study was to demonstrate the metabolism of gibberellin A20 (GA20) to gibberellin A1 (GA1) by tall and mutant shoots of rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. The data show that the tall and dx mutant of rice and the tall and ga5 mutant of Arabidopsis metabolize GA20 to GA1. The data also show that the dy mutant of rice and the ga4 mutant of Arabidopsis block the metabolism of GA20 to GA1. [17–13C,3H]GA20 was fed to tall and the dwarf mutants, dx and dy, of rice and tall and the dwarf mutants, ga5 and ga4, of Arabidopsis. The metabolites were analyzed by high-performance liquid chromatography and full-scan gas chromatography-mass spectrometry together with Kovats retention index data. For rice, the metabolite [13C]GA, was identified from tall and dx seedlings; [13C]GA1 was not identified from the dy seedlings. [13C]GA29 was identified from tall, dx, and dy seedlings. For Arabidopsis, the metabolite [13C]GA1 was identified from tall, ga5, and ga4 plants. The amount of [13C]GA1 from ga4 plants was less than 15% of that obtained from tall and ga5 plants. [13C]GA29 was identified from tall, ga5, and ga4 plants. [13C]GA5 and [13C]GA3 were not identified from any of the six types of plant material.

Metabolism and Biological Activity of Gibberellin A4 in Vegetative Shoots of Zea mays, Oryza sativa, and Arabidopsis thaliana

[17–13C,3H]Gibberellin A4 (GA4) was injected into the shoots of tall (W23/L317), dwarf-1 (d1), and dwarf-5 (d5) Zea mays L. (maize); tall (cv Nipponbare), dwarf-x (dx), and dwarf-y (dy) Oryza sativa L. (rice); and tall (ecotype Landsberg erecta), ga4, and ga5 Arabidopsis thaliana (L.) Heynh. [13C]GA4 and its metabolites were identified from the shoots by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA4 was metabolized to GA1 in all nine genotypes. GA4 was also metabolized in some of the genotypes to 3-epi-GA1, GA2, 2[beta]-OH-GA2, 3-epi-GA2, endo-GA4, 16[alpha], 17-H2–16, 17-(OH)2-GA4, GA34, endo-GA34, GA58, 15-epi-GA63, GA71, and 16-epi-GA82. No evidence was found for the metabolism of GA4 to GA7 or of GA4 to GA3. The bioactivities of GA4 and GA1 were determined using the six dwarf mutants for assay. GA4 and GA1 had similar activities for the maize and rice mutants. For the Arabidopsis mutants, GA4 was more active than GA1 at low dosages; GA4 was less active than GA1 at higher dosages.

Native gibberellins and the metabolism of [14C]gibberellin A53 and of [17-13C, 17-3H2]gibberellin A20 in tassels of Zea mays

Abstract The native hormones from tassels of maize (Zea mays) were re-investigated. The previous identification by GC/SIM of GA1, GA8 and GA29 in normal tassels was confirmed by full GC/MS scans at the correct Kovats retention indices. In tassels of dwarf-1 mutants, GA44,−GA19, GA17, GA20 and the 16,17-dihydro, 7β,16α,17-trihydroxy derivative of ent-kaurenoic acid were identified by GC/MS. Gibberellin A1 was not found in the mutant tassels. [14C]Gibberellin A53 was fed to tassels of the dwarf-5 mutant. In the ethyl acetate-soluble acidic fraction from the feeds, [14C]GA44 was identified by GC/MS; [14C]GA19 and [14C]GA29 were identified by GC/SIM. The GA29 is probably a metabolite of the feeds because the dwarf-5 mutant is known to control the step copalyl pyrophosphate to ent-kaurene in the maize GA-biosynthetic pathway and because GA29 was not identified in a control experiment. The n-butanol fractions obtained from the feeds were shown, by GC/MS, to contain [14C]GA53 after hydrolysis, suggesting that conjugated [14C]GA53 is a major metabolite from GA53 feeds. [17-13C, 17-3H2]Gibberellin A20 was fed to normal, dwarf-1 and dwarf-5 tassels. In each case, analysis of the purified ethyl acetate-soluble acidic extracts by GC/MS led to the identification of [13C]GA29 and unmetabolized [13C]GA20 in which no 13C-isotope dilution was observed.

Gibberellin Metabolism in Maize (The Stepwise Conversion of Gibberellin A12-Aldehyde to Gibberellin A20.

The stepwise metabolism of gibberellin A12-aldehyde (GA12-aldehyde) to GA20 is demonstrated from seedling shoots of maize (Zea mays L.). The labeled substrates [13C,3H]GA12-aldehyde, [13C,3H]GA12, [14C4]GA53, [14C4/2H2]GA44, and [14C4/2H2]GA19 were fed individually to dwarf-5 vegetative shoots. Both [13C,3H]GA12-aldehyde and [13C,3H]GA12 were also added individually to normal shoots. The labeled metabolites were identified by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA12-aldehyde was metabolized to GA53-aldehyde, GA12, GA53, GA44, and GA19; GA12 was metabolized to 2[beta]-hydroxy-GA12, GA53, 2[beta]-hydroxyGA53, GA44, 2[beta]-hydroxyGA44, and GA19; GA53 was metabolized to GA44, GA19, GA20, and GA1; GA44 was metabolized to GA19; and GA19 was metabolized to GA20. These results, together with previously published data from this laboratory, document the most completely defined gibberellin pathway for the vegetative tissues of higher plants.

Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice

[14C4]GA53, [14C4]GA44, and [2H2/14C4]GA19 were injected separately into seedlings of rice (Oryza sativa) using a dwarf mutant (d35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography mass spectrometry (GC-MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA53-->GA44-->GA19-->GA20 and the presence of endogenous GA53, GA44, GA19, GA20 and GA1. Previous metabolic studies have shown the presence of the step, GA20-->GA1 in rice. Taken together, the data establish in vegetative shoots of rice the presence of the early 13-hydroxylation pathway, a pathway that originates from GA12 and leads to bioactive GA1.

3-epigibberellin A1: Natural occurrence in plants and artefactual formation from gibberellin A1

Abstract Whether 3-epigibberellin(3-epiGA1) is endogenous in plants or is formed from GA1 as an artefact, can be determined by adding [13C]GA1 to the plant material as an internal reference and measuring the 12C:13C ratio of 3-epiGA1 and GA1, recovered from the plant extract. We use this method to show that 3-epiGA1 is not a natural constituent of vegetative tissues of Zea mays (maize) but is endogenous in Lactuca sativa (lettuce).

Endogenous gibberellins from callus cultures of maize

Abstract This paper reports the identification of gibberellin A 1 (GA 1 ), GA 17 , GA 19 , GA 20 , GA 29 , and GA 53 from extracts of normal (wild-type) maize callus using full-scan GC-Mass Spectrometry and Kovats retention indices (KRIs). In addition, evidence is presented for the presence of GA 8 based on its molecular ion at the appropriate KRI. In a preliminary experiment, the presence of GA 3 , GA 5 , and GA 44 was suggested, based on GC-selected ion monitoring (SIM); however, the presence of these GAs was not confirmed by full-scan GC-Mass Spectrometry. The information represents the first identification of GAs from maize callus. All of the identified GAs are members of the early-13-hydroxylation pathway, a biosynthetic pathway that leads to bioactive GA 1 . GA 1 , GA 8 , GA 19 , GA 20 , and GA 29 were quantified from GC-SIM isotopic dilution data. GA 17 and GA 53 were quantified from GC-SIM data by comparing the ion intensity from each gibberellin to a known amount of a GA 19 standard. The levels of the identified GAs ranged from 6 pg g −1 fr. wt to 210 pg g −1 fr. wt.

GIBBERELLIN BIOSYNTHESIS IN ZEA MAYS: THE 3-HYDROXYLATION STEP GA20 TO GA1

Elongation growth in Zea mays (maize) is dependent on the presence of the gibberellin, GA 1 , which originates biosynthetically via an early-13-hydroxylation pathway. GA 1 is apparently the only biologically active gibberellin, per se, in this pathway. Other gibberellins in the pathway are active through their metabolism to GA 1 . The four gibberellin mutants of maize, d 5 , d 3 , d 2 and d 1 control specific and different steps in the pathway. The d 5 gene blocks an early step, CPP to ent -kaurene, the d 3 and d 2 genes control intermediate oxidation steps and the d 1 gene controls the terminal step, GA 20 to GA 1 . The data presented here show that 3-chloro-substitution of GA 20 resulted in the loss of activity when assayed on the d 5 mutant. At low levels no bioactivity was observed. At high levels (greater than 0.1 μg per plant) the bioactivity was 10% that of GA 1 . The bioassay data for 3-chloro-GA 20 support the position that hydroxylation at carbon-3 is necessary for the bioactivity of GA 20