Jake MacMillan

Internode length in Pisum: The Le gene controls the 3?-hydroxylation of gibberellin A20 to gibberellin A1

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [13C, 3H]GA20. The substrate was metabolised to [13C, 3H]GA1, [13C, 3H]GA8 and [13C, 3H]GA29 in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [13C, 3H]GA29 and, in one line, [13C, 3H]GA29-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3β-hydroxylation of GA20 to GA1. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [13C, 3H]GA20 metabolites were considerably diluted by endogenous [12C]GAs, implying that the metabolism of [13C, 3H]GA20 mirrored that of endogenous [12C]GA20. In contrast, the [13C, 3H]GA20 metabolites in na lines showed no dilution with [12C]GAs, confirming that the na mutation prevents the production of C19-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the 12C:13C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [13C, 3H]GA20 and extraction. No [12C]GA1 and only traces of [12C]GA8 (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.

Occurrence of Gibberellins in Vascular Plants, Fungi, and Bacteria.

The occurrence of GA1 to GA126 in vascular plants, fungi, and bacteria is listed. The data are discussed with reference to criteria for identification and to the frequency of occurrence of GAs in vascular plants.

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.

Qualitative and quantitative analyses of gibberellins throughout seed maturation in Pisum sativum cv. Progress No. 9.

SummaryIn addition to the previously identified GA20 and GA29 in immature seeds of Pisum sativum L. cv. Progress No. 9, GA9, GA17, GA38, GA44, abscisic acid and dihydrophaseic acid have been identified. The levels of GA9, GA17, GA20 and GA29 have been determined throughout seed maturation by GC-MS. GA20 and GA29 are the major gibberellins in terms of quantity, the other gibberellins remain at very low levels throughout development of the seed.

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A20 (GA20) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A29 and GA29-catabolite were the major metabolites of GA20 in the genotype na le. However, low levels of GA1, GA8 and GA8-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a ‘leaky’ mutation. Gibberellin A20 was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3β-hydroxylated metabolite of GA20, GA8 (2β-hydroxy GA1), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA8 levels. A similar relationship was found on comparing GA20 metabolism in the three genotypes led, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3β-hydroxylation of GA20 to GA1). These results indicate that GA20 has negligible intrinsic activity and support the contention that GA1 is the only GA active per se in promoting stem growth in pea.

Proposed procedure for the allocation of trivial names to the gibberellins.

THE symbols A1, A2, A3 and A4 were originally used by Takahashi et al.1,2 to denote the first four gibberellins, isolated from the fungus Gibberella fujikuroi. This convenient trivial nomenclature was continued by MacMillan et al.3–5, Cross et al.6–8, Hanson9,10 and Galt11,12 to include gibberellins A5 to A17. Continuity of the sequence gibberellins A1 to A17 has been maintained, and confusion avoided, for the following two reasons. First, gibberellins A1 to A4 were named by one group at the University of Tokyo and gibberellins A5 to A17 by a second group, who kept in personal contact when the Akers Research Laboratories, ICI, Ltd., where they worked was closed down. Fortuitously, there was no overlap in time between the numbering of these two groups of gibberellins by these two groups of workers. Second, Tamura and his group and Mitsui and his colleagues have avoided possible confusion while waiting for an agreed procedure for the allocation of gibberellin A numbers, by using provisional names for their recently discovered plant gibberellins based on their source, namely, Bamboo13,14, Pharbitis15, Canavalia-I and -II16,17, Lupinus-I18 and Lupinus-II (personal communication from K. Koshimizu and T. Mitsui) gibberellins.

Gibberellin Biosynthesis from Gibberellin A12-Aldehyde in Endosperm and Embryos of Marah macrocarpus

Soluble enzyme preparations from embryos and endosperm of Marah macrocarpus (previously Echinocystis macrocarpa) were incubated with [14C4]gibberellin(GA)12-aldehyde,[14C4]GA12, [14C1] GA9, 2,3-didehydro[14C1]GA9, [14C1]GA20, and [17–13C,3H]GA5. Embryo preparations converted GA12-aldehyde, GA12, and GA9 to GA4 and GA7; 2,3-didehydroGA9 to GA7; GA5 to GA3; and GA20 (incompletely) to GA1 and GA60, but not to GA3. Endosperm preparations converted GA12-aldehyde and GA12 to GA15, GA24, GA25, and GA9, but, unlike embryo preparations, not to GA4 or GA7. However, GA4 and GA7 were formed from GA9 and GA7 was formed from 2,3-didehydroGA9. Metabolism of GA5 to GA3 and GA20 to GA1 was low. 2,3-DidehydroGA9 accumulated when GA9 was incubated with a desalted endosperm preparation. A cDNA clone (M3–8), selected from an embryo-derived cDNA library using a DNA fragment generated by reverse transcriptase polymerase chain reaction, was expressed in Escherichia coli. The fusion protein converted GA12 to GA9 (major) and GA25 (minor); GA53 was metabolized less effectively and only to GA44. Thus, the M3–8 protein is functionally similar to GA 20-oxidases from Arabidopsis thaliana, Spinacia oleracea, and Pisum sativum, but different from that from Cucurbita maxima seeds, to which its amino acid sequence is most closely related. mRNA hybridizing to M3–8 accumulated in embryos and endosperm of M. macrocarpus, but was absent in vegetative tissues.

Biosynthesis of gibberellins A12, A15, A24, A36, and A37 by a cell-free system from Cucurbita maxima

Abstract GA12-aldehyde obtained from mevalonate via ent-kaurene, ent-kaurenol, ent-kaurenoic acid and ent-7α-hydroxykaurenoic acid in a cell-free system from immature seeds of Cucurbita maxima was converted to GA12 by the same system. When Mn2+ was omitted from the system GA12-aldehyde and GA12 were converted further to several products. Among these GA15, GA24, GA36 and GA37 were conclusively identified by GC-MS. With the exception of GA37 these GAs have not previously been found in higher plants. Another biosynthetic pathway led from ent-7α-hydroxykaurenoic acid to very polar products via what was tentatively identified as ent-6α, 7α-dihydroxykaurenoic acid. An unidentified component with an MS resembling that of a dihydroxykaurenolide was also obtained from incubations with mevalonate.

Further studies on the metabolism of gibberellins (GAs) A9, A20 and A29 in immature seeds of Pisum sativum cv. progress No. 9

Seed maturation of Pisum sativum cv. Progress No. 9 proceeds more slowly in winter than in summer even when the parent plants are grown in greenhouse conditions with light-and heat-supplementation. For parent plants grown under “summer” and “winter” conditions the metabolism of [3H]GA9 in cultured seeds is qualitatively different in seeds of equivalent age and qualitatively the same in seeds of equivalent weight. 13-Hydroxylation of [3H]GA9→[3H]GA20 is restricted to early stages of seed development. 2β-Hydroxylation of [3H]GA9→2β-OH-[3H]GA9 has only been observed at a stage of development after endogenous GA9 has accumulated. 2β-OH-GA9 has been shown to be endogenous to pea and is named GA51. H2-GA31 and its conjugate have not been shown to be present in pea and may be induced metabolites of [3H]GA9. The metabolism of GA20→GA29 is used to illustrate a technique of feeding [2H][3H]GAs in order to distinguish a metabolite from the same endogenous compound. The in vitro conversion of [3H]GA20→[3H]GA29, and the virtual non-metabolism of [3H]GA29 have been confirmed for seeds in intact fruits. These results are discussed in relation to the apparent absence of conjugated GAs in mature pea seeds.

Position of the metabolic block for gibberellin biosynthesis in mutant b1-41a of Gibberella fujikuroi☆

Abstract Mutant B1-41a, obtained by UV-irradiation of Gibberella fujikuroi strain GF-1a, does not metabolise mevalonic acid lactone (MVL), ent -kaur-16-ene, ent -kaurenol, and ent -kaurenal to gibberellins. ent -Kaur-16-ene-19-oic acid is completely metabolised to give the same gibberellins in similar concentration as unsupplemented cultures of the parent strain. It is concluded that this mutant is blocked for gibberellin synthesis at the step from ent -kaurenal to ent -kaurenoic acid. Comparison of the incorporation of MVL into GA 3 by the mutant and the parent strains indicate that the metabolic block is 97·5% effective. A method of preparing ent -kaur-16-ene, labelled at C-15 and C-17 by [ 2 H] and [ 3 H] is described.