The synthesis of [3H]gibberellin A3 and [3H]gibberellin A1 by the palladium-catalyzed actions of carrier-free tritium on gibberellin A3

Reaction of gibberellin A₃ (GA₃) with carrier-free tritium gas and 5% palladium on calcium carbonate as catalyst gave a complex mixture of products, several of which were isolated and identified. Three of the purified products are the radioactive forms of naturally occurring gibberellins: [³H]GA₃ (1), [³H]GA₁ (2) and [³H]tetrahydro GA₃ (4). Another substance was isolated and tentatively identified as [³H]16,17-dihydro GA₃ (3). GLC was used to determine the specific activities of 1 and 2. [³H]GA₃ likely arises from palladium catalysed nonspecific exchange of GA₃ alkane hydrogen atoms with tritium. [³H]GA₁ is also exchange labeled but most of its radioactivity is due to tritium addition to the C-1,2 olefinic bond of GA₃.     

Metabolism of [3H]gibberellin A4 in somatic suspension cell cultures of carrot

The native gibberellin A₄ (GA₄), in radioactive form ([1,2-³H]GA₄, 1.06 Ci/mmol), was fed to carrot somatic cell cultures (suspension and immobilized cell systems) and its metabolism over a 48 hr period was investigated. It was found that the [³H]GA₄ was metabolized to at least two GAs, [³H]GA₁ and [³H]GA₈, six GA glucosyl conjugates, [³H]GA₁-0(3)-glucoside, [³H]GA₁-0(13)-glucoside, [³H]GA₁-glucosyl ester, [³H]GA₄-glucoside, [³H]GA₄-glucosyl ester, a [³H]GA₈ glucosyl conjugate(s) and a previously unknown [³H]GA₁ glucosyl conjugate ([³H]GA₁-0(3,13)-diglucoside-like compound). The GA₁-diglucoside-like compound was found only in extracts of cells and was present in significant amounts (33 % of total extractable radioactivity). All other metabolites were present in both cells and medium. For extracts of the medium, no differences between the suspension and immobilized cultures existed in types of [³H]GA₄ metabolites although quantitative differences were apparent.     

Interconversion of gibberellin A5 to gibberellin 3 in seedlings of dwarf Pisum sativum

[³H]-Gibberellin A₅ ([³H]-GA₅) applied to seedlings of dark-grown dwarf pea (Pisum sativum L. cv. Meteor), was converted to two acidic compounds, GA₃ and a chromatographically similar unknown. Identification of GA₃ was made by gas-liquid radiochromatography using three stationary phases.     

Metabolism of [3H]gibberellin A5 in developing pharbitis nil seeds

The native gibberellin A₅ (GA₅), as [1-³H]GA₅ (3.2 Ci/mmol) was fed to seed capsules (0.58 μCi/capsule) of Pharbitis nil cv Violet at the 2-week stage of development, and its metabolism in the seeds was investigated after 43 hr. Extractable radioactivity in free GA metabolites was 38%, with 56% in GA glucosyl conjugate-like substances. Only 2.5% of the extractable radioactivity remained as [³H]GA₅. Tentative identifications, based on comparisons with authentic standards after sequential chromatography on silica gel partition column → gradient-eluted C₁₈ HPLC → isocratic-eluted C₁₈ HPLC-radiocounting (RC), showed that [³H]GA₅ was converted to at least six free GAs, GA₁, GA₃, GA₆, GA₈, GA₂₂, GA₂₉, a GA₅ methyl ester-like metabolite, and at least twelve GA glucosyl conjugate-like substances, GA₅-glucoside (GA₅-G), GA₅-glucosyl ester (GA₅-GE), GA₁-O(3)-G, GA₁-O(13)-G, GA₁-GE, GA₃-O(3)-G, GA₃-O(13)-G, GA₃-GE, GA₆-G or GE, GA₈-O(2)-G, GA₂₂-G or GE and GA₂₉-O(2)-G. After lower specific activity feeds of [1,2-³H]GA₅ (74 mCi/mmol; 0.1 μCi/capsule) at approximately the same stage of development, the presence of GA₁, GA₃, GA₅, GA₆, GA₈ and GA₂₉ was further confirmed by sequential (after C₁₈ HPLC-RC) capillary gas chromatography-selected ion monitoring (GC-SIM), using six characteristic ions. However, for GA₂₂ only a trace of the parent ion was present at the appropriate retention time.     

Conversion of gibberellin A14 to other gibberellins in seedlings of dwarf Pisum sativum

Gibberellin A₁₄-[17-³H] applied to seedlings of dark grown dwarf pea (Pisum sativum L. cy. Meteor) was converted to GA₁, GA₈, GA₁₈, GA₂₃, GA₂₈, and GA₃₈. The sequence of interconversion of GA₁₄→ GA₁₈ → GA₃₈ → GA₂₃ → GA₁ → GA₈ is indicated. Identifications were made by gas-liquid radiochromatography using three liquid stationary phases.     

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

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.     

Metabolism of [3H]gibberellin A4 in somatic suspension cultures of anise

The native gibberellin A₄ (GA₄) was fed as [1, 2-³H]GA₄ (1.3 Ci/mmol) to anise somatic cultures maintained either at a proembryo-like stage with 2,4-dichlorophenoxyacetic acid (2,4-D), or allowed to undergo embryogenic development on a - 2,4-D medium. Proembryos, although only 20% of the dry wt of embryos, absorbed 1.4-times more [³H]GA₄/g dry wt than embryos. The [³H]GA₄ was metabolized to GA₁ and GA₈, and at least six conjugates [GA₄-glucoside (GA₄-G), GA₄ glucosyl ester (GA₄-GE), GA₁-0(3)-G, GA₁-0(13)-G, GA₁-GE and a GA₈-glucosyl conjugate]. The major metabolite was GA₄-G at each of two, 204 and 348 hr harvests (56–71 %), with GA₈-G increasing from < 1 % to 13 % with harvest time. The percentage and amount of GA₄-GE was highest at 204 hr (2% and 8 %, for embryos and proembryos, respectively), dropping to < 1 % at 348 hr, thereby indicating hydrolysis (e.g. reversible conjugation). Embryos had reduced amounts and percentages of biologically active GA₄ and GA₁, and most of their conjugates, but increased amounts and percentages of GA₈ and its conjugate(s). This finding is consistent with the hypothesis (based on present and past work) that high levels of biologically active GAs, especially GA₁, inhibit somatic embryogenesis in anise and carrot. The auxin, 2,4-D, may thus derive, at least in part, its ability to maintain the proembryo-like stage by inhibiting oxidative metabolism and conjugation of biologically active GAs.     

The biological activity of sixteen gibberellin A4 and gibberellin A9 derivatives using seven bioassays

C₂- and C₃-derivatives of GA₄ and GA₉ were tested for biological activity in a range of plant assays. The activity of most of these derivatives was equal to, or less than, that of the parent GAs. However, 2β-methylGA₄ and 2,2-dimethylGA₄ had a higher activity than GA₄ in some assays and the latter derivative was shown to be the most active GA known to date in the Forward oat first leaf, Tan-ginbozu dwarf rice and d₅-maize assays. Two other derivatives, 12,16-cycloGA₉ and 19-desoxyGA₉ had less activity than GA₉.     

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

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.     

Characterization of an enzyme from Phaseolus vulgaris seeds which hydroxylates GAi to GA8

Hydroxylation of gibberellin-[³H] A₁ (GA₁-[³H]) to GA₈-[³H] by the 95000 g supernatant fluid from imbibed bean seeds required Fe²⁺ or Fe³⁺ and O₂ but was insensitive to CO. The hydroxylating enzyme has a sedimentation coefficient of 4·5 S, and was precipitated by (NH₄)₂SO₄ at 35–60% saturation. This hydroxylase was specific for GA₁ and did not hydroxylate either pseudo-GA₁-[³H] or 16-ketoGA₁-[³H]. Virtually all hydroxylase activity was localized in the cotyledons.     

Interconversion of gibberellin A20 to gibberellin A29 by etiolated seedlings and germinating seeds of dwarf Pisum sativum

Tritium labelled gibberellin A₂₀ ([³H]-GA₂₀) applied to etiolated shoots and germinating seeds of dwarf pea (Pisum sativum L. cv. Meteor) was converted to gibberellin A₂₉. Identifications were made by GLRC and GC-MS.     

Metabolism of tritiated gibberellin a(20) in maize

After the application of 2.36 Curies per millimole [2,3-³H]gibberellin A₂₀ (GA₂₀) to 21-day-old maize (Zea mays L., hybrid CM7 × CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of ³H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [³H]GA₁ on the basis of co-chromatography with standard [³H]GA₁ on SiO₂ partition, high resolution isocratic elution reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [³H]GA₈ and C/D ring-rearranged [³H]GA₂₀, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C₁₈ high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA₁ and GA₈, or with retention times where conjugates of GA₂₀ would be expected to elute. Conversion to [³H]GA₁ was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [³H]GA₂₀-like metabolite was the major acidic product, while conversion to [³H]GA₁ was low.     

Lack of influence of photoperiod on the metabolism of gibberellin A20 in Salix pentandra

The influence of photoperiod on the metabolism of GA₂₀ in Salix pentandra was studied by feeding [³H]-GA₂₀ to seedlings which had been grown previously under long day (LD) or short day (SD) conditions. After 48 h in LD or SD, metabolites were separated on sequential, silica gel partition columns and reversed-phase C₁₈ HPLC. The principal metabolite co-chromatographed with [³H]-GA₁ and this conversion was confirmed by feeding [²H]-GA₂₀, which was converted to [²H]-GA₁ as identified by gas chromatography-selected ion monitoring. Chromatographic evidence also indicated the minor conversion of [³H]-GA₂₀ to [³H]-GA₈ (via [³H]-GA₁) and trace conversion to [³H]-GA₂₉ (GAs A_1.8,20.29 are native in Salix). Ethyl acetate-insoluble [³H] metabolites were formed and could be cleaved by cellulase to release putative [³H]-GA₂₀ and [³H]-GA₁ suggesting the conversion to glucosyl conjugates of these GAs. Metabolism of [³H]-GA₂₀ was slightly more rapid in plants previously grown under LD than SD, an effect which reflected the generally increased shoot growth under LD. However, altering the photoperiod after [³H]-GA₂₀ addition had only a slight effect on the metabolism of [³H]-GA₂₀ in Salix seedlings. This indicates that the conversion of GA₂₀ to GA₁ is not a controlling step in the photoperiodic regulation of growth cessation in Salix.     

Heterosis and the metabolism of gibberellin A20 in sorghum

The correlation between gibberellin (GA) metabolism and growth rate was investigated using two Sorghum bicolor inbred lines, Hegari and AT×623, and their heterotic F₁ hybrid. Previous studies have demonstrated that this hybrid is taller and has substantially greater shoot dry weights and leaf areas than either parental inbred. [³H]GA₂₀ was applied to the leaf whorl of seedlings and after 24 hours, plants were harvested and separated into roots, shoot cylinders containing the apical meristems, and leaf blades. Chromatographic analyses of metabolites indicated the conversions of [³H]GA₂₀ to [³H]GA_{1,8 and 29}. The conversion of [²H]GA₂₀ to [²H]GA₁ was demonstrated by gas chromatography-selected ion monitoring (GC-SIM). Putative glucosyl conjugates of all of the [³H]GAs were also produced and GA₈ was identified by GC-SIM following enzymic cleavage of the putative [³H]GA₈ glucosyl conjugate fraction. Comparing the genotypes, [³H]GA₂₀ metabolism was more rapid in the shoot cylinders of the hybrid than in the shoot cylinders from inbreds. In the hybrid samples, there was a three-fold increase in the putative conjugate(s) of [³H]GA₁ which was the principal metabolite, and increased production of [³H]GA₈ and the putative conjugates of [³H]GA₂₉ and [³H]GA₈. Conversely, levels of the remaining precursor, [³H]GA₂₀, and its putative conjugate(s) were reduced in the hybrid. The rate of GA₂₀ metabolism was thus positively correlated with growth rate across these sorghum genotypes. This correlation supports a promotive role of GA in the regulation of shoot growth and in the expression of heterosis (hybrid vigor) in sorghum.     

Formation of GA20 glucosyl conjugates in seedlings ofVicia faba

[³H]GA₂₀ (1)¹, fed toVicia faba seedlings, was converted to [³H]GA₂₀ glucosyl ester (5) and [³H]GA₂₀-13-0-glucoside (6). The GA₂₀ glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA₂₀-Me formed by transesterification of (5). The [³H]GA₂₀-Me was crystallized to constant specific radioactivity with authentic GA₂₀-Me. On HPLC-RC the GA₂₀-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA₂₀ (1).     

HPLC-based methods for the identification of gibberellin conjugates: Metabolism of [3H]gibberellin A4 in seedlings of Phaseolus coccineus

A series of chromatographic and derivatization techniques has been developed for the identification of radiolabelled gibberellin (GA) conjugates. The methods are based on reversed-phase HPLC, gel permeation chromatography, anion-exchange chromatography, enzymatic hydrolysis and transesterification of conjugates, and derivatization of free GAs to methoxycoumaryl esters. The procedures have been used to identify GA₄-glucosyl ester, GA₄-3-O-glucoside, a GA₃₄-O-glucoside and GA₈-2-O-glucoside, in addition to GA₁ and GA₈, as products of [1,2-³H]GA₄ metabolism in shoots of light-grown Phaseolus coccineus seedlings.     

Lack of effect of photoperiod on metabolism of exogenous GA19 and GA1 in Salix pentandra seedlings

The effect of photoperiod on metabolism of 16,17-[³H₂]GA₁₉, and 1.2-[³H₂]GA₁ applied to intact seedlings of Salix pentandra, was investigated. No difference was found in conversion of 16,17-[³H₂]GA₁₉ to 16,17-[³H₂]GA₂₀, and 16,17-[³H₂]GA₁, or in metabolism of 1,2-[³H₂]GA₁ to [³H]GA₈ between plants grown in continuous light and plants exposed for 14 days to a 12-h photoperiod. Also, leaf discs from plants grown in long or short days, converted 16,17-[³H₂]GA₁₉ both in light and darkness. These data on metabolism of 16,17-[³H₂]GA₁₉, contrast with previous results, which have indicated a photoperiodic control of the metabolism of GA₁₉ to GA₂₀ in S. pentandra. Presence of these applied labelled GAs and their metabolites in different parts of seedlings was recorded, after application to intact seedlings as well as to isolated plant parts. When 16,17-[³H₂]GA₁₉ was applied through the roots of intact plants, the relative amounts of 16,17-[³H₂]GA₁ present in leaves and shoot apices were higher than in roots and stems. In corresponding experiments with 1,2-[³H₂]GA₁, relatively higher amounts of [³H₂]GA₈ were found in roots and stems than in leaves and shoot apices. Twenty-four hours after application of 16,17-[³H₂]GA₁₉ to isolated plant parts, 16,17-[³H₂]GA₂₀ and 16,17-[³H₂]GA₁ were found in leaves and roots, but not in internodes. Incubation of isolated plant parts with 1,2-[³H₂]GA₁ for 24 h resulted in presence of [³H]GA₈ in all parts. The results mentioned above were obtained by monitoring metabolites by HPLC with on-line radio counting. The conversions of 17-[²H₂]GA₁₉ to 17-[²H₂]GA₂₀ and 17-[²H₂]GA₁ in shoot apices and whole seedlings, and of 17-[²H₂]GA₈ in whole seedlings, were confirmed by GC-MS.     

A mutant gene that increases gibberellin production in brassica

A single gene mutant (elongated internode [ein/ein]) with accelerated shoot elongation was identified from a rapid cycling line of Brassica rapa. Relative to normal plants, mutant plants had slightly accelerated floral development, greater stem dry weights, and particularly, increased internode and inflorescence elongation. The application of the triazole plant growth retardant, paclobutrazol, inhibited shoot elongation, returning ein to a more normal phenotype. Conversely, exogenous gibberellin A₃ (GA₃) can convert normal genotypes to a phenotype resembling ein. The content of endogenous GA₁ and GA₃ were estimated by gas chromatography-selected ion monitoring using [²H]GA₁, as a quantitative internal standard and at day 14 were 1.5- and 12.1-fold higher per stem, respectively, in ein than in normal plants, although GA concentrations were more similar. The endogenous levels of GA₂₀ and GA₁, and the rate of GA₁₉ metabolism were simultaneously analyzed at day 7 by feeding [²H₂]GA₁₉ and measuring metabolites [²H₂]GA₂₀ and [²H₂]GA₁ and endogenous GA₂₀ and GA₁, with [²H₅]GA₂₀ and [²H₅]GA₁ as quantitative internal standards. Levels of GA₁ and GA₂₀ were 4.6- and 12.9-fold higher, respectively, and conversions to GA₂₀ and GA₁ were 8.3 and 1.3 times faster in ein than normal plants. Confirming the enhanced rate of GA₁ biosynthesis in ein, the conversion of [³H]GA₂₀ to [³H]GA₁ was also faster in ein than in the normal genotype. Thus, the ein allele results in accelerated GA₁ biosynthesis and an elevated content of endogenous GAs, including the dihydroxylated GAs A₁ and A₃. The enhanced GA production probably underlies the accelerated shoot growth and development, and particularly, the increased shoot elongation.     

Conversion of gibberellin A1 into gibberellin A3 by the mutant R-9 of Gibberella fujikuroi

A mutant R-9 of Gibberella fujikuroi has been isolated and shown to be blocked for GA₁ and GA₃ biosynthesis, but not for GA₄, G_A7 and other gibberellins. Cultures of this mutant convert low concentrations of [1,2-³H2]-GA₁ into GA₃ in a radiochemical yield of 2·7 %.     

Metabolism of [1,2-3H]gibberellin A4 by epicotyls and cell-free preparations from Phaseolus coccineus L. seedlings

Cell-free systems were prepared from germinating seed and seedlings of Phaseolus coccineus. Gibberellin A₄ (GA₄)-metabolising activity was detected in vitro using preparations from roots, shoots and cotyledons of germinating seed, but only up to 24 h after imbibition. Cell-free preparations from cotyledons converted [³H]GA₄ to GA₁, GA₃₄, GA₄-glucosyl ester and a putative O-glucoside of GA₃₄, and, in addition converted [³H]GA₁ to GA₈. Preparations from embryo tissues contained 2-hydroxylase activity, converting [³H]GA₄ to GA₃₄ and [³H]GA₁ to GA₈.The presence of GA-metabolising enzymes was also indicated by in-vivo feeds of [³H]GA₄ to epicotyls of intact 4-d-old seedlings, which resulted in the accumulation of GA₁, GA₈, GA₃-3-O-glucoside, GA₄-glucosyl ester, GA₈-2-O-glucoside and a putative O-glucoside of GA₃₄. Gibberellin A₁ was the first metabolite detected, 15 min after application of [³H]GA₄, but after 24 h most of the label was associated with GA₈-2-O-glucoside. Over 90% of the recovered radioactivity was found in the shoot. Within the shoot, movement was preferentially acropetal, and was not dependent upon metabolism of the applied [³H]GA₄.Abbreviations DEAE diethylaminoethyl - GA_n gibberellin A_n - GPC gel permeation chromatography - HPLC-RC high performance liquid chromatography-radio counting - S-1 1000·g supernatant - UDP uridine 5-diphosphate