Jan E. Graebe

Isolation and Expression of Three Gibberellin 20-Oxidase cDNA Clones from Arabidopsis

Using degenerate oligonucleotide primers based on a pumpkin (Cucurbita maxima) gibberellin (GA) 20-oxidase sequence, six different fragments of dioxygenase genes were amplified by polymerase chain reaction from Arabidopsis thaliana genomic DNA. One of these was used to isolate two different full-length cDNA clones, At2301 and At2353, from shoots of the GA-deficient Arabidopsis mutant ga1–2. A third, related clone, YAP169, was identified in the Database of Expressed Sequence Tags. The cDNA clones were expressed in Escherichia coli as fusion proteins, each of which oxidized GA12 at C-20 to GA15, GA24, and the C19 compound GA9, a precursor of bioactive GAs; the C20 tricarboxylic acid compound GA25 was formed as a minor product. The expression products also oxidized the 13-hydroxylated substrate GA53, but less effectively than GA12. The three cDNAs hybridized to mRNA species with tissue-specific patterns of accumulation, with At2301 being expressed in stems and inflorescences, At2353 in inflorescences and developing siliques, and YAP169 in siliques only. In the floral shoots of the ga1–2 mutant, transcript levels corresponding to each cDNA decreased dramatically after GA3 application, suggesting that GA biosynthesis may be controlled, at least in part, through down-regulation of the expression of the 20-oxidase genes.

The biosynthesis of all major pea gibberellins in a cell-free system from Pisum sativum

Abstract The soluble fraction of a cell-free system from immature pea embryos converts gibberellin A 12 (GA 12 ) sequentially to GA 15 , GA 24 , GA 9 and GA 51 , which are all not hydroxylated at C-13. GA 12 can also be 13-hydroxylated by the microsomal fraction to GA 53 , which is converted further by the soluble fraction sequentially to GA 44 , GA 19 , GA 20 and GA 29 , the same series as before but 13-hydroxylated. The microsomal 13-hydroxylation requires oxygen and NADPH, the conversions by the soluble fraction require oxygen, Fe 2+ and α-ketoglutarate and are stimulated by ascorbate. GA 15 and GA 44 serve as substrates in the above sequences after alkaline hydrolysis of the lactone functions only, but such hydrolysis is not required for the conversion of GA 9 to GA 51 and GA 20 to GA 29 . The products of the in vitro system are identical with the major GAs found endogenously in immature pea seeds and the sequences obtained are considered representative of the natural biosynthetic pathway of GAs in many plant species.

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.

Gibberellin A4 produced by Sphacelomamanihoticola, the cause of the superelongation disease of cassava (Manihotesculenta)

Gibberellin A4 was identified by combined gas chromatography-mass spectrometry in the culture medium of Sphacelomamanihoticola, a fungus known to cause the “superelongation disease” of cassava (Manihotesculenta). Gibberellin A4 was synthesized in ageing cultures and reached concentrations as high as 400 μg/l nutrient broth. After Gibberellafujikuroi, Sphacelomamanihoticola is the second phytopathogenic fungus known with certainty to produce an active gibberellin in considerable amounts.

Hormones of young tassels of Zea mays

Abstract The ethyl acetate-soluble acids from an aqueous methanolic extract of young tassels from Zea mays plants were fractionated by treatment with PVP, then by chromatography on a column of celite-charcoal. Methylated and trimethylsilylated fractions were analysed by GC/MS and the following compounds were identified by comparison with reference spectra: GA17, GA19, GA20, GA44, GA53, ABA, phaseic acid and dihydrophaseic acid. Evidence is also presented for the presence of metabolise C of ABA and of a 16,17-dihydro-17-hydroxy-derivative of GA53. In addition, the presence of small amounts of GA1, GA8 and GA29, was indicated from a derivatized fraction analysed by capillary GC/SICM.

GC/MS analysis of the plant hormones in seeds of Cucurbita maxima

Abstract The GC/MS detection is reported of over 30 compounds, in extracts of the endosperm and embryos from seeds of Cucurbita maxima. The compounds which were identified from reference spectra include: cis,trans-ABA; trans,trans-ABA; dihydrophaseic acid; IAA; GA4; GA12; GA13; GA25; GA39; GA43; GA49; ent-13-hydroxy-, ent-6α,7α-and ent-7α,13-dihydroxy-, and ent-6α,7α,13-trihydroxykaur-16-en-19-oic acids; ent-7α,16,17-trihydroxy- and ent-6α,7α,16,17-tetrahydroxy-kauran-19-oic acids, ent-6,7-seco-7-oxokauren-6,19-dioic acid and/or ent-6,7-secokauren-6,7,19-trioic acid, and 7β,12α-dihydroxykaurenolide. New compounds, the structures of which were deduced from GC/MS data, include: the 12α-hydroxy-derivatives of GA12, GA14, GA37 and GA4, and the 12β-hydroxy-derivatives of ent-7α-hydroxy- and ent-6α,7α-dihydroxykaurenoic acids.

Kaurenolide biosynthesis in a cell-free system from Cucurbita maxima seeds

Abstract A new product obtained by incubation of [2- 14 C ]-mevalonic acid with a cell-free system from Cucurbita maxima endosperm was identified by GC-MS as ent -kaura-6,16-dien-19-oic acid. When this compound was reincubated with the microsomal fraction it was converted to 7β-hydroxykaurenolide and hence to 7β,12α-dihydroxykaurenolide. The dienoic acid was also obtained by incubation of ent -kaurene, ent 1-kaurenol, ent -kaurenal and ent -kaurenoic acid, but not ent -7α-hydroxykaurenoic acid, with the microsomal fraction. Thus, in the C. maxima cell-free system, the kaurenolides are formed by a pathway which branches from the GA pathway at ent -kaurenoic acid and proceeds via the dienoic acid.

Gibberellin biosynthesis in cell-free system from immature seeds of Pisum sativum☆

Abstract A cell-free system from immature pea seeds converts 14 C-labelled ent -kaurene to ent -kaurenol, ent -kaurenal, ent -kaurenoic acid, ent -7α-hydroxykaurenoic acid, and gibberellin A 12 -aldehyde. The latter becomes converted further to 13-hydroxygibberellin A 12 , gibberellin A44, gibberellin A 12 -alcohol, and several unidentified products. Thus the biosynthesis of gibberellins via ent -kaurene is now established for a member of the Leguminosae . It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A 12 -alcohol has been obtained in any biological system.

The biosynthesis of 12α-hydroxylated gibberellins in a cell-free system from cucurbita maxima endosperm

Abstract A previously unknown pathway for the biosynthesis of 12α-hydroxylated gibberellins was found in a cell-free system from Cucurbita maxima endosperm. The microsome fraction converts the gibberellin precursor GA12-aldehyde simultaneously to GA12 and 12α-hydroxy-GA12-aldehyde. The ratio of these products is pH-dependent: above pH 6.5, the production of GA12 is favoured, whilst below pH 6.5, 12α-hydroxy-GA12-aldehyde is the predominant product. 12α-Hydroxy-GA12-aldehyde is converted further by soluble enzymes to 12α-hydroxy-GA14, 12α-hydroxy-GA15, 12α-hydroxy-GA37 and several unidentified products. This conversion is optimal between pH 6.0 and 6.5 in contrast to the previously known conversion of GA12-aldehyde to GA43 by soluble enzymes, which is optimal at pH 7.5. GA58, a major 12α-hydroxylated endogenous constituent of C. maxima endosperm, was not obtained when 12α-hydroxy-GA12-aldehyde was used as a substrate, but it was obtained together with GA4 when GA9 was incubated with a preparation containing both microsomal and soluble enzymes.

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.