Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm

Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₅, [¹⁴C]GA₂₄ and [¹⁴C]GA₂₅, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [¹⁴C]Gibberellin A₅₃ was similarly converted to [¹⁴C]GA₄₄, [¹⁴C]GA₁₉, [¹⁴C]GA₁₇ and small amounts of a fourth product, which was preliminarily identified as [¹⁴C]GA₂₀, a C₁₉-gibberellin. All GAs except [¹⁴C]GA₂₀ were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et. al, Planta 195, 98–107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.     

The partial purification and characterisation of gibberellin 2β-hydroxylases from seeds of Pisum sativum

The gibberellin (GA) 2-hydroxylases in mature and immature seeds of Pisum sativum have been partially purified and characterised. The enzymes are unstable when stored below pH 7.0 or in the absence of a thiol reagent. The optimum assay pH is between 7.4 and 7.8 and activity is dependent upon the presence of -ketoglutarate, Fe²⁺ and ascorbate. The 2-hydroxylase activities for GA₁, GA₄, GA₉ and GA₂₀ are chromatographically inseparable and correspond to a protein of M_r 44000. The rate of GA 2-hydroxylation varies according to substrate and some evidence indicates that the 2-hydroxylase activities for GA₁ and GA₄ and for GA₉ and GA₂₀ may reside in different proteins. During pea seed maturation, the specific activity of the enzyme(s) increases dramatically and reaches a maximum at a time when endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ are also at their greatest concentration. This correlation is not the result of substrate induction of enzyme activity. Since the GA 2-hydroxylases operate at maximal rate at low substrate concentrations they are incapable of rapidly 2-hydroxylating excessive quantities of (exogenously applied) GA₁ or GA₂₀. On the basis of the kinetic parameters of the GA 2-hydroxylase activities, a generalised model is discussed for the control of the steady-state levels of bioactive hormone under normal physiological conditions.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GA_n gibberellin A_n - HPLC high-performance liquid chromatography - HSS high-speed supernatant - LSS low-speed supernatant - PMSF phenylmethane sulphonyl fluoride     

Metabolic formation and occurrence of gibberellin A1-3-0-β-D-glucopyranoside in immature fruits of Phaseolus coccineus L.

Endogenous pools of presumptive gibberellin (GA) glucosyl conjugates of Phaseolus coccineus were metabolically labelled by feeding of [³H]GA₁ to immature fruits. The [³H]GA₁ glucoside fraction was isolated and the main constituent tentatively identified by enzymic hydrolysis, ion exchange chromatography and elution volume on HPLC-RC as GA₁-3-0--D-glucopyranoside.     

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     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

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     

Growth and gibberellin-A1 metabolism in normal and gibberellin-insensitive (Rht3) wheat (Triticum aestivum L.) seedlings

Growth parameters were determined for tall (rht3) and dwarf (Rht3) seedlings of wheat (Triticum aestivum L.). Plant statures and leaf length were reduced by 50% in dwarfs but root and shoot dry weights were less affected. Leaves of dwarf seedlings had shorter epidermal cells and the numbers of cells per rank in talls and dwarfs matched the observed relationships in overall length. Talls grew at twice the rate of dwarfs (2.3 compared with 1.2 mm h^-1). [³H]Gibberellin A₁ ([³H]GA₁) was fed to seedlings via the third leaf and metabolism was followed over 12 h. Immature leaves of tall seedlings transferred radioactivity rapidly to compounds co-chromatographing with [³H]gibberellin A₈ ([³H]GA₈) and a conjugate of [³H]GA₈, whereas leaves of dwarf seedlings metabolised [³H]GA₁ more slowly. Roots of both genotypes produced [³H]GA₈-like material at similar rates. Isotopic dilution studies indicated a reduced 2-hydroxylation capacity in dwarfs, but parallel estimates of the endogenous GA pool size, obtained by radioimmunoassay, indicated a 12–15 times higher level of GA in the dwarf immature leaves. Dwarfing by the Rht3 gene does not appear to operate through enhanced, or abnormal metabolism of active gibberellins and the act of GA metabolism does not bear an obligate relationship to the growth response.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography     

Growth physics and water relations of red-light-induced germination in lettuce seeds

Red light (R) and gibberellins (GA) each induce a water potential decrease in the axes of lettuce (Lactuca sativa L.) embryos resulting in germination of intact seeds (achenes) or an increase in growth of the axes of isolated embryos. The fruit coat and endosperm are a substantial barrier to the penetration of exogeneous GA. Isolated embryos take up 35 times as much [³H]GA₁ as the embryos of intact seeds and respond to less than 1·10^-10 M GA₃ or GA₄₊₇. We calculated that only 1·10^-8 M of either GA₃ or GA₄₊₇ would result in 50% germination if the GA were able freely to penetrate the fruit coat. Exogenous GA₃ or GA₄₊₇, at concentrations insufficient to cause germination, result in an apparent synergistic promotion of germination when suboptimal R is applied. Yet suboptimal concentrations of exogenous GA₃ or GA₄₊₇ and suboptimal R result in only additive increases in the growth response in axes of isolated embryos. Dose-response curves demonstrate quantitative increases in the growth response of the isolated axes after R or GA treatments insufficient to induce germination in intact seeds, indicating that a threshold potential must be achieved by the embryonic axes before germination can occur.Abbreviations FR far=red light - GA gibberellin - PEG poly-ethylene glycol 4000 - P_fr far-red-absorbing phytochrome - R red light III.=Carpita et al. 1979b; IV.=Carpita et al. 1979c     

Separation and characterisation of three 2-oxoglutarate-dependent dioxygenases from Cucurbita maxima L. endosperm involved in gibberellin biosynthesis

Three enzymes of the gibberellin (GA) biosynthetic pathway, a 7-oxidase, a 20-oxidase and a 3-hydroxylase, were partially purified from Cucurbita maxima endosperm by ammonium sulfate precipitation, gel-filtration and anion-exchange chromatography. The enzyme activities, which were assayed by the oxidation of GA₁₂-aldehyde to GA₁₂, of GA₁₂ to GA₁₅ (and GA₂₄) and of GA₁₅ to GA₃₇, respectively, were completely separated from each other. The apparent molecular masses as estimated by gel-filtration high-performance liquid chromatography were 34.5 kDa for the 7-oxidase, 44.5 kDa for the 20-oxidase and 58 kDa for the 3-hydroxylase. The Michaelis-Menten constants (K _m) were 8.6 M, 0.15M and 8.7 M for the respective substrates. All three enzymes had properties typical of 2-oxoglutarate dependent dioxygenases. 2-Oxoglutarate was essential for activity and served as a co-substrate, giving K _m values of 6.1 M, 91 M and 41 M with the 7-oxidase, 20-oxidase and 3-hydroxylase, respectively. Furthermore, 2 oxo[5-¹⁴C]glutarate was oxidised stoichiometrically to [¹⁴C]succinate when the GA-substrates were oxidised to their respective products, and the 11 ratio was maintained under different oxygen concentrations. Approximately equimolar amounts of ¹⁴CO₂ were released from 2-oxo[1-¹⁴C]glutarate when GA₁₂ was oxidised to GA_15/24 by the 20-oxidase. A crude enzyme preparation containing all three enzyme activities (and a 2-hydroxylase) converted GA₁₂-aldehyde to [¹⁸O₂]GA₄ and [¹⁸O₅]GA₄₃ under ¹⁸O₂, showing that all O-atoms incorporated after GA₁₂-aldehyde originate from O₂. Accordingly, the reaction rates were near zero under anaerobic conditions, although very low concentrations of O₂ sufficed to sustain the reactions. Both Fe²⁺ and dithiothreitol stimulated the enzyme activities strongly, but if they were added together, catalase was needed to prevent inhibition. The pH dependence showed two opposite trends; the 7-oxidase was most active at pH 6 and below, whereas the other enzymes were maximally active above pH 6.5.Abbreviations BSA bovine serum albumin - GA_n gibberellin A_n - DTT dithiothreitol - GC-MS combined gas chromatography-mass spectrometry - MeTMSi methyl ester trimethylsilyl ether We thank Mr. Keith Hall (Long Ashton) for assistance with the oxygen concentration measurements and Mrs. Gudrun Bodtke (Göttingen) and Mrs. Brigitte Schattenberg (Göttingen) for able technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft, Germany, and the Agricultural and Food Research Council, UK, and by an Academic Research Collaboration award jointly from the Deutsche Akademische Austauschdienst (DAAD) and the British Council.     

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos

Gibberellin (GA) metabolism from GA₁₂-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [¹⁴C]- or [17-²H₂]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA₁₂-aldehyde via the 13-deoxy pathway to GA₅₁ and via the 13-hydroxylation pathway to GA₂₉, GA₁ and GA₈. In addition, GA₃ was formed from GA₂₀ via GA₅. We conclude that the embryo is capable of producing gibberellins that can induce -amylase production in the aleurone layer. There was no evidence for 12- or 18-hydroxylation and GA₄ was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA₃, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography We thank Mrs. G. Bodtke and Mrs. B. Schattenberg for preparing the barley embryos and the Deutsche Forschungsgemeinschaft for supporting this work.     

Identification of gibberellins in the rice plant and quantitative changes of gibberellin A19 throughout its life cycle

The major endogenous gibberellin (GA) in shoots, roots and ears of the rice plant, Oryza sativa L. japonica cv. Nihonbare, was identified as GA₁₉ by combined gas liquid chromatography-mass spectrometry (GC-MS) and GC-selected ion current monitoring (GC-SICM). Another GA present in these tissues in small quantity was tentatively identified as GA₁ by GC-SICM, and GA₄ may be present in the seeds (kernels) of 3rd-leaf-stage seedlings. Using GC-SICM, the GA₁₉ content was quantified throughout the life cycle of rice plants. It was found to reach high levels (ca. 10–15 g/kg fresh weight) in 3rd-leaf seedlings, at panicle initiation (shoots), and during heading and anthesis (ears). The levels of GA₁₉ in Oryza sativa indica cv. T-136 underwent changes closely similar to those found in Nihonbare. The growth-promoting activity in rice of exogenous GA₁₉ is generally considerably less than that of GA₁. It therefore seems possible that GA₁₉ functions as a pool GA. The level of active GAs such as GA₁ may be regulated by the rate of biosynthesis of GA₁₉ or its metabolic conversions.Abbreviations GA(s) gibberellin(s) - GA_n gibberellin A_n - GA_n-MeTMS trimethylsilyl ether of GA_n methyl ester - GC-MS combined gas liquid chromatography-mass spectrometry - GC-SICM combined gas liquid chromatography-selected ion current monitoring - TLC thin-layer chromatography     

Gibberellin translocation in Pisum sativum L.

The physiological and biochemical consequences of treating Le (tall) and le (dwarf) pea seedlings with varying quantities of the gibberellins [³H]GA₂₀ and GA₁ have been investigated. Although the percentage uptake of these compounds from the site of application on the 3 stipules was low and most of the applied GA remained unmetabolised in situ, the quantitative relationship between GA translocation and GA dosage was found to be linear for GA₁ but saturating for GA₂₀. The movement of the GAs and their subsequently produced metabolites was mainly acropetal. They accumulated in greatest quantity in the apical extremities of the shoot. Overall, the extent to which GA₂₀ was metabolished in le seedlings was considerably less than in Le pea seedlings. Although all le tissues contained significantly less [³H]GA₁ than their Le counterparts, phenotypic effects of the le mutation were apparent only on internode and tendril development. Increased tissue growth, consequent upon GA treatment, was also apparent only in the internodes and tendrils of le plants. For internodes, GA₁ content determined the mid-logarithmic-phase growth rate and, consequently, final length. For tendrils, GA₂₀ rather than GA₁ may be the primary stimulatory agent.Abbreviations GA gibberellin - HPLC high-performance liquid chromatography - 1–6 consecutive developmental numbering system for plant tissues/organs as shown in Fig. 1 The author gratefully acknowledges financial support from Imperial Chemical Industries, Plant Protection, Jealott's Hill, Bracknell, Berks., UK and the Science and Engineering Research Council.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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     

Gibberellins and the procera mutant of tomato

The procera mutant of tomato (Lycopersicon esculentum L.) has a phenotype which is remarkably similar to that of normal tomatoes treated with exogenous gibberellin (GA), indicating that it might be a GA over-producer. However, analysis of endogenous GAs by gas chromatography-mass spectrometry showed that Procera actually has lower levels of GA₂₀ and GA₁ than normal. The reason for these anomalously low GA levels is not clear, as there was no difference between procera and normal plants in their ability to metabolize [³H]GA₂₀. The procera mutant responded to exogenous gibberellic acid with increased extension growth, but the proportional response for a given dose of GA was the same in procera and normal plants. It therefore appears that the procera mutation does not directly affect either the GA status of the plant, or its ability to respond to GA.Abbreviations GA gibberellin - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MeTMSi methyl trimethylsilyl - SIM selected ion monitoring     

Quantitative and qualitative changes in the endoplasmic reticulum of barley aleurone layers

Changes in the level of the endoplasmicreticulum (ER) marker enzyme cytochrome-c reductase (EC 1.6.2.1) were followed with time of imbibition of de-embryonated half-seeds of barley (Hordeum vulgare L.) and the subsequent incubation of their aleurone layers in gibberellic acid (GA₃) and H₂O. During imbibition there is an increase in the level of cytochrome-c-reductase activity and in the amount of 280-nm absorbance associated with this enzyme. When aleurone layers are incubated for a further 42 h in water, there is a doubling of the cytochrome-c-reductase activity. In GA₃, the activity of cytochrome-c reductase reaches a maximum at 24 h of incubation and thereafter falls to below 70% of its level at the beginning of the incubation period. Changes in the cytochrome-c-reductase activity correlate with changes in the fine structure of the aleurone cell. The ER isolated in low Mg²⁺ from aleurone layers incubated in buffer for up to 18 h has buoyant density of 1.13–1.14 g cc^-1 while that from layers incubated in GA₃ for 7.5–18 h has a density of 1.11–1.12 g cc^-1. The -amylase (EC3.2.1.1) isolated with the organelle fraction by Sepharose gel filtration is associated with the ER on isopycnic and rate-zonal density gradients, and its activity can be enhanced by Triton X-100. The soluble -amylase fraction from Separose-4B columns, on the other hand, is not Triton-activated but is acid-labile. Acid phosphatase (EC3.1.3.2) is distributed in at least three peaks on isopycnic gradients. In low Mg²⁺ the second peak of activity has a density of 1.12 g cc^-1 in GA₃-treated tissue and 1.13–1.14 g cc^-1 in H₂O-treated tissue. With high-Mg²⁺ buffers, this peak of phosphatase activity disappears. Acid-phosphatase activity is not enhanced by Triton X-100 nor is it acid-labile.Abbreviations EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - GA gibberellin - GA₃ gibberellic acid     

Internode length in Pisum sativum L. The kinetics of growth and [3H]gibberellin A20 metabolism in genotype na Le

The relationship between shoot growth and [³H]gibberellin A₂₀ (GA₂₀) metabolism was investigated in the GA-deficient genotype of peas, na Le. [17-¹³C, ³H₂]gibberellin A₂₀ was applied to the shoot apex and its metabolic fate examined by gas chromatographic-mass spectrometric analysis of extracts of the shoot and root tissues. As reported before, [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈ and [¹³C, ³H₂]GA₂₉ constituted the major metabolites of [¹³C, ³H₂]GA₂₀ present in the shoot. None of these GAs showed any dilution by endogenous ¹²C-material. [¹³C, ³H₂]GA₂₉-catabolite was also a prominent metabolite in the shoot tissue but showed pronounced isotope dilution probably due to carry-over of endogenous [¹²C]GA₂₉-catabolite from the mature seed. In marked contrast to the shoot tissue, the two major metabolites present in the roots were identified as [¹³C, ³H₂]GA₈-catabolite and [¹³C, ³H₂]GA₂₉-catabolite. Both of these compounds showed strong dilution by endogenous ¹²C-material. Only low levels of [¹³C, ³H₂]GA₁, [¹³C, ³H₂]GA₈, [¹³C, ³H₂]GA₂₀ and [¹³C, ³H₂]GA₂₉ accumulated in the roots. It is suggested that compartmentation of GA-catabolism may occur in the root tissue in an analogous manner to that shown in the testa of developing seeds. Changes in the levels of [1,3-³H₂]GA₂₀ metabolites over 10 d following application of the substrate to the shoot apex of genotype na Le confirmed the accumulation of [³H]GA-catabolites in the root tissues. No evidence was obtained for catabolic loss of [³H]GA₂₀ by complete oxidation or conversion to a methanol-inextractable form. The results indicate that the root system may play an important role in the regulation of biologically active GA levels in the developing shoot of Na genotypes of peas.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Inhibition of stem growth and gibberellin production in Agrostemma githago L. by the growth retardant tetcyclacis

The effects of the new growth retardant tetcyclacis (TCY) on stem growth and endogenous gibberellin (GA) levels were investigated in the long-day rosette plant Agrostemma githago. Application of TCY (10 ml of a 5·10^-5M solution daily) to the soil suppressed stem elongation in Agrostemma grown under long-day conditions. A total of 10 g GA₁ (1 g applied on alternate days) per plant overcame the growth retardation caused by TCY.Control plants and plants treated with TCY were analyzed for endogenous GAs after exposure to nine long days. The acidic extracts were fractionated by high-performance liquid chromatography. Part of each fraction was tested in the d-5 maize bioassay, while the remainder was analyzed by combined gas chromatography-selected ion monitoring. The bioassay results indicated that the GA content of plants treated with TCY was much lower than that of untreated plants. The data obtained by gas chromatography-selected ion monitoring confirmed that the levels of seven GAs present in Agrostemma were much reduced in TCY-treated plants when compared with the levels in control plants: GA₅₃ (13%), GA₄₄ (0%), GA₁₉ (1%), GA₁₇ (33%), GA₂₀ (15%), GA₁ (4%), and epi-GA₁ (13%). These results provide evidence that TCY inhibits stem growth in Agrostemma by blocking GA biosynthesis and thus lowering the levels of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - HPLC high-performance liquid chromatography - TCY Tetcyclacis (5-[4-chlorophenyl]-3,4,5,9,10-pentaaza-tetracyclo-5,4,1,0^2,6,0^8,11-dodeca-3,9-diene)     

Conversion of gibberellin A20 to gibberellins A1 and A5 in a cell-free system from Phaseolus vulgaris

The soluble fraction of a cell-free system from immature seeds of Phaseolus vulgaris L. converts gibberellin A₂₀ (GA₂₀) to GA₁ and GA₅. It does however not metabolize GA₁ and GA₂₉ to GA₅, showing that in this system GA₂₀ is converted directly to GA₅. The steps from GA₂₀ to GA₁ (3-hydroxylation) and from GA₂₀ to GA₅ (² double-bond formation) require oxygen, Fe²⁺ and -ketoglutarate, and are stimulated by ascorbate. The enzymes catalyzing these conversions bate. The enzymes catalyzing these conversions have properties similar to those of GA oxidases found in Cucurbita maxima and Pisum sativum.Abbreviations GA_n gibberellin A_n - HPLC high-performance liquid chromatography - GC-MS combined gas chromatography-mass spectrometry - TLC thin-layer chromatography - TMSi/TMSi trimethylsilyl ether/trimethylsilyl ester Graduate student, University of Tokyo