The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway.

In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.

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.

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.

Hydrolysis and reconjugation of gibberellin A20 glucosyl ester by seedlings of Zea mays L.

The [6-2H]glucosyl ester of [17-13C,3H]gibberellin A20 (GA20) was injected into light-grown 14-day-old seedlings of normal, dwarf-1, and dwarf-5 maize (Zea mays L.). The plant material was extracted 24 h later, and the extracts were purified by solvent partitioning, column chromatography, and HPLC. 13C-labeled metabolites were identified from the purified extracts by full-scan gas chromatography/mass spectrometry and selected ion current monitoring in conjunction with Kovats retention indices. The metabolites, [13C]GA20, [13C]GA29, [13C]GA20-13-O-glucoside, and [13C]GA29-2-O-glucoside, were identified from normal, dwarf-1, and dwarf-5 seedlings. [13C]GA8 and [13C]GA8-2-O-glucoside were also identified from normal and dwarf-5 seedlings but not from dwarf-1 seedlings. The data provide definitive evidence for the endogenous hydrolysis by the seedlings of the introduced conjugate and its reconjugation to three glucosides.

Metabolism of ent-Kaurene to Gibberellin A(12)-Aldehyde in Young Shoots of Normal Maize.

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.

Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L.

[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).

Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica.

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A(9) (GA(9)) and 2,3-dehydroGA(9) to GA(7); GA(9) was also metabolized to GA(4) in a branch pathway. The preparation from Marah seeds also metabolized GA(5) to GA(3) in high yield; GA(6) was a minor product and was not metabolized to GA(3). Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA(5) to GA(3) and of 2,3-dehydroGA(9) to GA(7) occurs with the loss of the 1beta-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1beta,2beta-(2)H(2)]GA(4), was metabolized to [1,2-(2)H(2)]GA(3) with the loss of the 1alpha- and 2alpha-hydrogens. These results provide further evidence that the biosynthetic origin of GA(3) and GA(7) in higher plants is different from that in the fungus Gibberella fujikuroi.

The dominant non-gibberellin-responding dwarf mutant (D8) of maize accumulates native gibberellins.

The endogenous gibberellins (GAs) were examined from young vegetative shoots of the dominant mutant, Dwarf-8, a GA-nonresponder, and normal maize; GA(44), GA(17), GA(19), GA(20), GA(29), GA(1), and GA(8), members of the early-13-hydroxylation pathway, were identified from both kinds of shoots by full-scan mass spectra and Kovats retention indices. In addition, we report the identification of 3-epi-GA(1), GA(3), GA(4), GA(5), GA(7), GA(9), GA(12), GA(15), GA(24), GA(34), and GA(53) by using the same criteria. [1,7,12,18-(14)C(4)]GA(53) and -GA(44), [17-(2)H(2)]GA(19), and [17-(13)C,(3)H(2)]GA(20), -GA(29), -GA(1), -GA(8), and -GA(5) were used as internal standards to determine the endogenous levels of these GAs by measurement of isotope dilution, using capillary gas chromatography and selected ion monitoring. Shoots of Dwarf-8 accumulate relatively high levels of GA(20), GA(1), and GA(8). The accumulation of GA(1) appears to be related to gene dosage. Since Dwarf-8 contains the same pattern of GAs as normals (including GA(1) and GA(3)), the genetic control point probably lies after GA(1) (and GA(3)). Thus Dwarf-8 may be a GA receptor mutant or a mutant that controls a product downstream from the binding of the bioactive GA to a receptor.

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.

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.

Endogenous Gibberellins from Sporophytes of Two Tree Ferns, Cibotium glaucum and Dicksonia antarctica.

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.

Identification of Ten Gibberellins from Sporophytes of the Tree fern, Cyathea australis.

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).

Internode length in Zea mays L. : The dwarf-1 mutation controls the 3ß-hydroxylation of gibberellin A20 to gibberellin A 1.

[(13)C, (3)H]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. [(13)C, (3)H]Gibberellin A20 was metabolized to [(13)C, (3)H]GA29-catabolite and [(13)C, (3)H]GA1 by the normal, and to [(13)C, (3)H]GA29 and [(13)C, (3)H]GA1 by the dwarf-5 mutant. In the dwarf-1 mutant, [(13)C, (3)H]GA20 was metabolized to [(13)C, (3)H]GA29 and [(13)C, (3)H]GA29-catabolite; no evidence was found for the metabolism of [(13)C, (3)H]GA20 to [(13)C, (3)H]GA1. [(13)C, (3)H]Gibberellin A8 was not found in any of the feeds. In all feeds no dilution of (13)C in recovered [(13)C, (3)H]GA20 was observed. Also in the dwarf-5 mutant, the [(13)C]label in the metabolites was apparently undiluted by endogenous [(13)C]GAs. However, dilution of the [(13)C]label in metabolites from [(13)C, (3)H]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.

Tannins as gibberellin antagonists.

Fourteen chemically defined hydrolyzable tannins and six impure mixtures of either condensed or hydrolyzable tannins were found to inhibit the gibberellin-induced growth of light-grown dwarf pea seedlings. The highest ratio of tannins to gibberellic acid tested (1000: 1 by weight) inhibited from 80 to 95% of the induced growth for all tannins tested except for two monogalloyl glucose tannins which inhibited only 50% of the induced growth. The lowest ratio tested (10: 1) inhibited the induced growth by less than 25% except for the case of terchebin where 50% inhibition was found. The inhibition of gibberellin-induced growth was found to be completely reversed by increasing the amount of gibberellin in three cases tested. Tannins alone did not inhibit endogenous growth of either dwarf or nondwarf pea seedlings. Eight compounds related to tannins, including coumarin, trans-cinnamic acid, and a number of phenolic compounds were also tested as gibberellin antagonists. Most of these compounds showed some inhibition of gibberellin-induced growth, but less than that of the tannins. At the highest ratio (1000: 1) the greatest inhibition was 55%; at the lowest ratio (10: 1) no more than 17% was observed. These compounds did not inhibit endogenous growth, and the inhibition of gibberellin-induced growth could be reversed by increasing the amount of gibberellin in two cases tested.Six chemically defined tannins were found to inhibit hypocotyl growth induced by gibberellic acid in cucumber seedlings. Growth induced by indoleacetic acid in the same test was not inhibited. The highest ratio of tannin to promotor tested gave strong inhibition of gibberellic acid-induced growth, but actually enhanced the growth induced by indoleacetic acid. This difference in action suggests a specificity between the tannins and gibberellic acid.

Gibberellin Production: Genetic Control in the Fungus Gibberelia fujikuroi.

The fungus Gibberella fujikuroi (Saw.) Wr. can be used in genetic studies of the production of gibberellins. A gene has been identified which controls a step in the biosynthetic pathway of gibberellin production. Apparently this step is early in the pathway for it affects the accumulation of all of the gibberellins produced by the fungus.