Persistence of [14C] gibberellin A3 and [3H] gibberellin A1 in senescing,ethylene treated Citrus and tomato fruit

The fate of [¹⁴C] gibberellin A₃ and [³H] gibberellin A₁ was examined in senescing fruit of Shamouti orange (Citrus sinensis L. Osbeck) and tomato (Lycopersicon esculentum Mill.). Gibberellin A₃ was highly persistent in Citrus peel (t 1/2=18 days) and to a lesser degree in tomato (t 1/2=5.5 days). Ethylene and ethephon caused a slight enhancement of gibberellin A₃ metabolism in Citrus and tomato fruit, respectively. Gibberellin A₁ was metabolized by Citrus peel at a relatively high rate (t 1/2 < 24 h) and ethylene slightly reduced this rate. It is concluded that the ethylene-induced enhancement of senescence does not involve major effects on the deactivation of applied gibberellins.Abbreviations GA₃ gibberellin A₃ - GA₁ gibberellin A₁     

Activity of gibberellins A24 and A25 in five bioassay systems

Summary The growth-promoting activity of two C₂₀ gibberellins, gibberellins A₂₄ and A₂₅, was determined in five bioassay systems and compared with that of gibberellic acid in four of the bioassays and with gibberellin A₄ in the other. Gibberellin A₂₄ was highly active in the oat mesocotyl, oat 1st leaf and cucumber hypocotyl assays. Gibberellin A₂₅ was consistently less active than gibberellin A₂₄ and was anactive over the tested range in the oat 1st leaf assay.     

Biochemical Studies on “Bakanae” Fungus. Part 51–53

The outline of this report has already been published in this Journal as a communication to the Editors. The relation among four isomers, gibberellin A₁, gibberellin C, pseudogibberellin A₁ and isogibberellin A₁ was clarified. The partial structure of gibberellin C is presented.     

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

A cell-free system from immature pea seeds converts ¹⁴C-labelled $\text{ent}$-kaurene to $\text{ent}$-kaurenol, $\text{ent}$-kaurenal, $\text{ent}$-kaurenoic acid, $\text{ent}$-7α-hydroxykaurenoic acid, and gibberellin A₁₂-aldehyde. The latter becomes converted further to 13-hydroxygibberellin A₁₂, gibberellin A44, gibberellin A₁₂-alcohol, and several unidentified products. Thus the biosynthesis of gibberellins $\text{via ent}$-kaurene is now established for a member of the $\text{Leguminosae}$. It is the first time that 13-hydroxylation of gibberellins has been observed in a cell-free system and that gibberellin A₁₂-alcohol has been obtained in any biological system.     

The conversion of mevalonic acid into gibberellin A12-aldehyde in a cell-free system from Cucurbita pepo

Summary A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-¹⁴C into ent-kaur-16-en-19-oic acid (I), ent-7-hydroxy-kaur-16-en-19-oic acid (II), and ent-gibberell-16-en-7-al-19-oic acid (III) (gibberellin A₁₂-aldehyde). The products were identified by gas liquid chromatography and by combined gas chromatography-mass spectrometry of the methylated and trimethylsilylated fractions. The radioactivity of the compounds was established by recrystallisation to constant specific radioactivity. Gibberellin A₁₂ (IV), also detected in the system after incubation by combined gas chromatographymass spectrometry may be an artefact, derived from gibberellin A₁₂-aldehyde by a non-enzymatic conversion.With gibberellin A₁₂-aldehyde, the cell-free biosynthesis of an ent-gibberellane has been achieved for the first time.     

Regulation of Bud Rest in Tubers of Potato, Solanum tuberosum L: VIII. Early Effects of Gibberellin A(3) and Abscisic Acid on Ultrastructure

Using the electron microscope, we compared the effects of abscisic acid and gibberellin A₃ on excised buds from resting potato (Solanum tuberosum L.) tubers. Cells of abscisic acid-treated buds became progressively more vacuolated during a 12-hour time course study as compared with control (water) and gibberellin A₃-treated buds. Concentric configurations of endoplasmic reticulum were present in apical cells of freshly excised buds. After about 6 hours these configurations began to open and disperse, and after 12 hours, intact concentric configurations were no longer evident. Both abscisic acid and gibberellin A₃ induced opening and dispersal of the concentric configurations, sometimes as early as 0.5 hour after excision and treatment with hormones.     

Reversing the inhibitory effect of paclobutrazol on seed germination of Amaranthus paniculatus by GA3, ethephon or ACC

The germination of Amaranthus paniculatus seeds was inhibited by applying paclobutrazol, a specific inhibitor of gibberellin biosynthesis. This inhibition was markedly counteracted by gibberellin A₃ (GA₃), suggesting that endogenous gibberellins are required for germination in this species. The inhibitory effect of paclobutrazol was also overcome by ethephon (2-chloroethylphosphonic acid) or the precursor of ethylene biosynthesis, ACC (1-aminocyclopropane-l-carboxylic acid). Thus the physiological effect of gibberellin can be mimicked by ethylene released from ethephon or synthesised from exogenous ACC. It is suggested, that endogenous gibberellins are involved in germination of Amaranthus paniculatus seeds and that action of GA₃ can be substituted by ethylene.Abbreviations ACC 1-aminocyclopropane-l-carboxylic acid - AMO-1618 (2-isopropyl-5methyl-4-trimethylammoniumchloride)-phenyl-l-piperidinium-carboxylate - ancymidol -cyclopropyl--(4-methoxyphenyl)-5-pyrimidine methanol - chloromequat chloride (2-chloroethyl)trimethylammoniumchloride - ethephon 2-chloroethylphosphonic acid - GA gibberellin A₃ - paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-lyl)pentan-3-ol - Phosphon D 2,4,dichlorobenzyl-tributhylphosphoniumchloride - tetcyclacis 5,(4-chlorophenyl)-3,4,5,9,10-pentaaza-tetracyclo)5,4,1,0,Z,6,0^8,11 dodeca-3,9-diene     

Hydrolysis of [17,17-2H2]gibberellin A20-glucoside and [17,17-2H2]gibberellin A20-glucosyl ester by Azospirillum lipoferum cultured in a nitrogen-free biotin-Based chemically-defined medium

Azospirillum lipoferum strain USA 5b, a gibberellin producing bacterium, was cultured in a nitrogen-free biotin-based chemically-defined medium in the presence of the glucosyl ester or the 13-O-glucoside of [17,17-²H₂]-gibberellin A₂₀. The [17,17-²H₂]-gibberellin A₂₀ conjugates were added at both the stationary phase of the cultures and at the beginning of the growth curve. Metabolism of the conjugates was examined after 72 h of incubation using capillary gas chromatography-mass spectrometry, with identification by full scan mass spectra. Metabolites identified were [17,17-²H₂]-gibberellin A₂₀, [17,17-²H₂]-gibberellin A₁ and [17,17-²H₂]-gibberellin A₃. Also, in the Azospirillum cultures fed at the beginning of the growth curve, gibberellin A₅ and gibberellin A₂₀ were characterized as endogenous by mass spectrometry/full spectrum. These results support the concept that the growth promotion in plants that is induced by Azospirillum infection may occur by a combination of both gibberellin production and gibberellin-glucoside/glucosyl ester deconjugation by the bacterium.     

Persistence of 14C-gibberellin A3 on the surface of Citrus fruit peel and on inert glass surfaces

The persistence of gibberellin A₃ on plant surfaces was examined using fruit of Marsh seedless grapefruit (Citrus paradisi Macf.) and an inert glass model system. ¹⁴C-gibberellin A₃ was applied to surfaces in aqueous treatment solutions or in waxing solutions. Dried-out treatment residues were removed by washing and analyzed for total and GA₃-like radioactivity. Gibberellin A₃ persisted without significant loss for at least 7 d in aqueous treatment solutions (pH 4.0 or 6.2) but was less persistent in the pH 10.4 waxing solution (t1/2=7 d).Loss of total peel surface radioactivity was fast during the first 3 days, slowing down afterwards. After 14 days 73% of the initial radioactivity could still be recovered from fruit peel surface and 70% of the recovered radioactivity was still in the form of gibberellin A₃. Gibberellin A₃ was somewhat more persistent in residues from pH 4 than pH 7 treatment solutions. Light had a slight enhancing effect on gibberellin A₃ decomposition on fruit peel under growth chamber conditions. After 12 d at 100% relative humidity, 88% of the radioactivity on glass surfaces was still in the form of gibberellin A₃, as against 45% at a relative humidity of 50%. Simulated field conditions, combining daily fluctuations in light, temperature and relative humidity, markedly enhanced gibberellin A₃ decomposition on glass surfaces (t1/2=2 d). Gibberellin A₃ was very persistent (90% after 9 d) in the waxing residues on fruit peel surface.Abbreviations GA₃ gibberellin A₃ - RH relative humidity     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Gibberellins in developing fruits of Pisum sativum cv. Alaska: Studies on their role in pod growth and seed development

Gibberellins A₁, A₈, A₂₀ and A₂₉ were identified by capillary gas chromatography-mass spectrometry in the pods and seeds from 5-d-old pollinated ovaries of pea (Pisum sativum cv. Alaska). These gibberellins were also identified in 4-d-old non-developing, parthenocarpic and pollinated ovaries. The level of gibberellin A₁ within these ovary types was correlated with pod size. Gibberellin A₁, applied to emasculated ovaries cultured in vitro, was three to five times more active than gibberellin A₂₀. Using pollinated ovary explants cultured in vitro, the effects of inhibitors of gibberellin biosynthesis on pod growth and seed development were examined. The inhibitors retarded pod growth during the first 7 d after anthesis, and this inhibition was reversed by simultaneous application of gibberellin A₃. In contrast, the inhibitors, when supplied to 4-d-old pollinated ovaries for 16 d, had little effect on seed fresh weight although they reduced the levels of endogenous gibberellins A₂₀ and A₂₉ in the enlarging seeds to almost zero. Paclobutrazol, which was one of the inhibitors used, is xylem-mobile and it efficiently reduced the level of seed gibberellins without being taken up into the seed. In intact fruits the pod may therefore be a source of precursors for gibberellin biosynthesis in the seed. Overall, the results indicate that gibberellin A₁, present in parthenocarpic and pollinated fruits early in development, regulates pod growth. In contrast the high levels of gibberellins A₂₀ and A₂₉, which accumulate during seed enlargement, appear to be unnecessary for normal seed development or for subsequent germination.Abbreviations GA_(a) gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PFK perfluorokerosene - PVP polyvinylpyrrolidone     

The immunoassay of gibberellins

The development of sensitive and specific solid-phase enzyme immunoassays for gibberellic acid and gibberellins A₄ and A₇ is reported. The use of antisera of high apparent affinity (K_a over 10¹⁰ l mol^-1) in conjunction with alkaline phosphatase-labeled gibberellins allows, with minimum procedural effort, the quantitative determination of sub-picogram amounts of these gibberellins. The assays reported here are applicable to most gibberellins and can be set up with 1–1.5 mg of starting material. They represent the most sensitive methods for gibberellin determination known.Abbreviations GA gibberellin - GA₃ gibberellic acid - TLC thin-layer-chromatography     

Conversion of [14C]gibberellin A12-aldehyde to C19- and C20-gibberellins in a cell-free system from immature seed of Phaseolus coccineus L.

A cell-free system prepared from developing seed of runner bean (Phaseolus coccineus L.) converted [¹⁴C]gibberellin A₁₂-aldehyde to several products. Thirteen of these were identified by capillary gas chromatography-mass spectrometry as gibberellin A₁ (GA₁), GA₄, GA₅, GA₆, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₃₇, GA₃₈, GA₄₄ and GA₅₃-aldehyde, all giving mass spectra with ¹⁴C-isotope peaks. GA₈ and GA₂₈ were also identified but contained no ¹⁴C. All the [¹⁴C]GA₁₂-aldehyde metabolites, except GA₁₅, GA₂₄ and GA₅₃-aldehyde, are known endogenous GAs of P. coccineus.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC highperformance liquid chromatography - MVA mevalonic acid - S-2 2000-g supernatant     

The role of endogenous gibberellins in the formation of α-amylase by aleurone layers of germinating barley caryopses

Using sensitive and selective immunological assays we have shown that in germinating caryopses of Hordeum vulgare L. cv. Himalaya, the level of gibberellin A₄ (GA₄) rises approximately 18-to 20-fold shortly (2–4 h) before -amylase activity increases. Gibberellin A₄ is the predominant immunoreactive gibberelin during these developmental stages and reaches a peak amount of approximately 9 pmol per caryopsis about 48 h after imbibition. Isolated aleurone layers produce GA₄ in the presence of an exogenous gibberellin, such as GA₁, which is not a biosynthetic precursor for GA₄. Experiments with inhibitors of gibberellin biosynthesis indicate that gibberellin synthesis is required in this tissue for the induction of -amylase. The inductive effect of exogenously applied GA₁ is indirect and appears to be mediated by GA₄. Embryos form predominantly GA₁; however, very little of this material is released by isolated embryos into the incubation medium. The results presented make it unlikely that the role of the embryo in the process of -amylase induction in aleurone layers is to provide gibberellins or gibberellin precursors.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - RIA radioimmunoassay - TLC thin-layer chromatography     

The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) heynh.

Summary By selecting for germinating seeds in the progeny of mutagen-treated non-germinating gibberellin responsive dwarf mutants of the ga–1 locus in Arabidopsis thaliana, germinating lines (revertants) could be isolated. About half of the revertants were homozygous recessive for a gene (aba), which probably regulates the presence of abscisic acid (ABA). Arguments for the function of this gene were obtained from lines homozygous recessive for this locus only, obtained by selection from the F₂ progeny of revertant X wild-type crosses. These lines are characterized by a reduced seed dormancy, symptoms of withering, increased transpiration and a lowered ABA content in developing and ripe seeds and leaves.Abbreviations ABA Abscisic acid - GA₄₊₇ Mixture of gibberellin A₄ and A₇ - EMS Ethylmethanesulfonate - NG Non-germinating - G Germinating     

Characterization of gibberellins from light-grown Phaseolus coccineus seedlings by combined GC-MS

Following extensive purification of an extract from 5000 light-grown Phaseolus coccineus seedlings by procedures including countercurrent distribution, Sephadex G10, polyvinylpyrrolidone, charcoal-celite and silicic acid partition column chromatography, TLC preparative GLC, gibberellins A₁, A₄, A₅ and A₂₀ were characterized by combined GC-MS. In addition, an unknown compound isomeric with gibberellin A₁₇ but lacking an hydroxyl group, was also detected.     

Gibberellin metabolism in excised lettuce hypocotyls: Evidence for the formation of gibberellin A1 glucosyl conjugates

The properties of the water-soluble metabolites of [³H]gibberellin A₁ ([³H]GA₁) from lettuce (Lactuca sativa L.) hypocotyls were compared with those of authentic samples of gibberellin (GA) glucosyl esters and ethers. Partitioning against l-butanol at high and low pH was not an efficient method of differentiating between ester and ether conjugates of GA₁ or GA₃. Extraction into l-butanol at pH 2.5 was, however, useful as a group purification step. Gel-filtration on acrylamide indicated a mean molecular weight of ca. 600 for the polar material and high-voltage electrophoresis separated two compounds (LH 1 and LH 2) with differing charge properties. Both metabolites incorporated ¹⁴C from glucose and ³H from GA₁. Subsequent enzymatic hydrolysis of LH 1 released material with identical properties to [¹⁴C]glucose together with a second uncharacterised component. Feeding with [³H]GA₁ methyl ester greatly reduced the formation of LH 1 but not LH 2. The metabolites were provisionally identified as GA₁-glucosyl ester (LH 1) and GA₁-glucosyl ether (LH 2).Abbreviations GA gibberellin - LH1 GA₃-glucosyl ester - LH2 GA₁-glucosyl ether - HVE high voltage paper electrophoresis - TLC thin-layer chromatography     

Biochemical Studies on “Bakanae” Fungus

The production of gibberellin A₇ by Gibberella fujikuroi was studied by using newly devised assay method. Gibberellin A₇ increased at preferable temperature range between 32°C and 34°C at the controlled pH(6.0~7.5). The improved isolation process by using column chromatography composed of granular charcoal was found to be extremely convenient, because of its quick elution with satisfactory separation from gibberellic acid which is always accompanied by gibberellin A₇ in culture medium.     

Extractable and Diffusible Gibberellins From Light- and Dark-grown Pea Seedlings

Gibberellins were obtained from light- and dark-grown peas by solvent extraction and agar diffusion. Both A₅- and A₁-like gibberellins were obtained by extraction; however, by diffusion only the A₁-like gibberellin was found. There was no significant quantitative difference in the levels of diffusible or extractable gibberellin obtained from light- and dark-grown tall and dwarf peas. Several possible explanations for the discrepancy between diffusible and extractable gibberellin were investigated. Of these, only I was supported by experimental evidence, namely, that GA₅ can be converted to GA₁.     

Interaction of jasmonic acid with some plant growth regulators in the control of apple (Malus domestica) embryo germination

Embryos isolated from dormant apple seeds were treated with jasmonic acid (JA), gibberellin A₃ (GA₃), abscisic acid (ABA) and hydrogen cyanide in darkness and in light. The chemicals were present in the culture medium continuously and simultaneously or applied for 2 days and in different sequences. All treatments stimulated embryo germination except ABA, which was strongly inhibitory. Additive effects of JA with light and with GA₃ on embryo germination were observed, whereas ABA interacted synergically with JA, HCN and light. ABA and GA₃ were most effective when applied early during embryo incubation, but the late JA treatment was more stimulatory. It is concluded that JA does not act on the regulatory pathway that is initiated by light and which leads to embryo germination through gibberellin accumulation and alkaline lipase activation. ABA and HCN appear to be involved in the control of this pathway. JA and ABA may be involved in the control of alkaline lipase activity, independently of this regulatory chain.Abbreviations ABA abscisic acid - GA₃ gibberellin A₃ - JA jasmonic acid