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.     

Developing Inflorescences of Male and Female <Emphasis Type="Italic">Rumex acetosa</Emphasis> L. Show Differences in Gibberellin Content

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Gibberellin Signal Transduction in Rice

Rumex acetosa L. (common sorrel) is a dioecious perennial in the family Polygonaceae. Gibberellins (GAs) of the early 13-hydroxylation pathway and the putative early 3, 13-hydroxylation pathway were previously identified in young R. acetosa inflorescences by GC-MS. In this investigation to examine the GA content of individual inflorescences ELISAs were used for quantitative analysis. Significant differences were revealed between the sexes in the GA content of young inflorescences, and GC-SRM was used to validate the observed trends. Males had higher levels of the 3, 13-hydroxylated C₂₀-GA GA₁₈ and the 2, 13-hydroxylated C₁₉-GA GA₂₉, whereas females had higher levels of the 13-hydroxylated C₂₀-GAs GA₅₃ and GA₁₉. It is suggested that the conversion from C₂₀-GAs to C₁₉-GAs is under tighter control in the inflorescences of females compared to male plants and therefore there is accumulation of the C₂₀-GAs in the females. Results from flowering bioassays using authentic GAs indicate that differences in GA content between the sexes are unlikely to be a consequence of sex determination.     

Convergent pathways of gibberellin A1 biosynthesis in Brassica

[²H, ³H]Gibberellin A₄ (GA₄) or [²H, ³H] GA₉ were applied to the shoot tips of seedlings of elongated internode (ein), a tall mutant of rapid cycling Brassica rapa. Following [²H]GA₉ application, [²H]GA₅₁, [²H]GA₂₀ and [²H]GA₄ were identified as products by GC-MS, while [²H]GA₃₄ and [²H]GA₁ were formed from [²H]GA₄. Other isotopically labelled products, including abundant putative conjugates, were also produced, but were not identified. Thus, in B. rapa, GA₁ biosynthesis involves the convergence of at least two metabolic pathways; it can be formed via GA₄ or GA₂₀, the latter of which can originate from GA₉ or from GA₁₉.     

Internode length in Zea mays L.

[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.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - RP reverse phase     

Internode length in Pisum

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 3-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.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthetic pathways from GA₁₂-aldehyde, GA₁₂ and GA₅₃ were investigated in cell-free systems from developing embryos of Cucurbita maxima L. Gibberellin A₁₂-aldehyde and GA₁₂ were converted to GA₂₅, putative 12α-hydroxyGA₂₅, GA₁₃ and GA₃₉ as main products. Minor products were GA₄, GA₃₄ and, when GA₁₂ was the substrate, putative 12α-hydroxyGA₁₂. The intermediates GA₁₅ and GA₂₄ accumulated at low protein concentrations. The influence of various factors on GA₁₂ metabolism was examined. At low 2-oxoglutarate and ascorbate concentrations, or at acid pH, 3β-hydroxylated products predominated, whereas with increasing 2-oxoglutarate and ascorbate concentrations, or at neutral pH, the yield of 12α-hydroxylated GAs increased. Gibberellin A₅₃ was metabolised mainly to the C₂₀-GAs GA₄₄, GA₁₉, GA₁₇, GA₂₃ and GA₂₈, with the C₁₉-GAs GA₂₀, GA₁ and GA₈ as minor products. Only C₁₉-GAs were 2β-hydroxylated, which is a main characteristic of the embryo systems. In addition to GA₁₃, GA₂₅, GA₃₉, GA₄₃, GA₄₉, GA₅₈, GA₇₄, 12α-hydroxyGA₂₅ and GA₃₉ 3-isovalerate, which were known previously from embryos of C. maxima, GA₁, GA₄, GA₁₇, GA₂₈, GA₃₇, GA₃₈, GA₄₈, GA₈₅, 12α-hydroxyGA₃₇ and putative 12α-hydroxyGA₄₃ were identified as endogenous components by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Evidence for putative 2β-hydroxyGA₂₈ and GA₂₃ was also obtained but it was less conclusive because of contamination.     

Gibberellins in dark- and red-light-grown shoots of dwarf and tall cultivars of Pisum sativum: The quantification,metabolism and biological activity of gibberellins in Progress no. 9 and Alaska

The stem growth in darkness or in continuous red light of two pea cultivars, Alaska (Le Le, tall) and Progress No. 9 (le le, dwarf), was measured for 13 d. The lengths of the first three internodes in dark-grown seedlings of the two cultivars were similar, substantiating previous literature reports that Progress No. 9 has a tall phenotype in the dark. The biological activity of gibberellin A₂₀ (GA₂₀), which is normally inactive in le le geno-types, was compared in darkness and in red light. Alaska seedlings, regardless of growing conditions, responded to GA₂₀. Dark-grown seedlings of Progress No. 9 also responded to GA₂₀, although red-light-grown seedlings did not. Gibberellin A₁ was active in both cultivars, in both darkness and red light. The metabolism of [¹³C³H]GA₂₀ has also been studied. In dark-grown shoots of Alaska and Progress No. 9 [¹³C³H]GA₂₀ is converted to [¹³C³H]GA₁, [¹³C³H]GA₈, [¹³C]GA₂₉, its 2-epimer, and [¹³C³H]GA₂₉-catabolite. [¹³C³H] Gibberellin A₁ was a minor product which appeared to be rapidly turned over, so that in some feeds only its metabolite, [¹³C³H]GA₈, was detected. However results do indicate that the tall growth habit of Progress No. 9 in the dark, and its ability to respond to GA₂₀ in the dark may be related to its capacity to 3-hydroxylate GA₂₀ to give GA₁. In red light the overall metabolism of [¹³C³H]GA₂₀ was reduced in both cultivars. There is some evidence that 3-hydroxylation of [¹³C³H]GA₂₀ can occur in red light-grown Alaska seedlings, but no 3-hydroxylated metabolites of [¹³C³H]GA₂₀ were observed in red light-grown Progress. Thus the dwarf habit of Progress No. 9 in red light and its inability to respond to GA₂₀ may be related, as in other dwarf genotypes, to its inability to 3-hydroxylate GA₂₀ to GA₁. However identification and quantification of native GAs in both cultivars showed that red-light-grown Progress does contain native GA₁. Thus the inability of red light-grown Progress No. 9 seedlings to respond to, and to 3-hydroxylate, applied GA₂₀ may be due to an effect of red light on uptake and compartmentation of GAs.Abbreviations AMO-1618 2-isopropyl-4-(trimethylammonium chloride)-5-methylphenyl piperidine-1-carboxylate - cv. cultivar - GC-MS gas chromatography-mass spectrometry - GA_(n) gibberellin A_(n) - HPLC high-pressure liquid 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     

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.     

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.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

Effect of exo-16,17-dihydro-gibberellin A5 on gibberellin A20 metabolism in seedlings of dwarf rice (Oryza sativa L. cv. Tan-ginbozu)

Several of the 16,17-dihydro gibberellins (GAs) inhibit elongation in a variety of species. In a study of their mechanism of action we have investigated the effect of exo-16,17-dihydro-Ga₅ (diHGA₅) on the metabolism of GA₂₀ in dwarf rice (Oryza sativa cv. Tan-ginbozu). A mixture of [³H]- and [³H]-GA₂₀ (100 ng per plant) was applied in microdrops to 4 d old seedlings which were harvested 72 h later. Concurrent treatment with diHGA₅ at 100 ng or 333 ng per plant reduced GA₂₀-induced elongation of the second leaf sheath by 41–66%. There was a concomitant reduction in the amount of [²H₂]GA₁ present at harvest, measured by gas chromatography-mass spectrometry-selected ion monitoring. The [²H₂]GA₂₉ content was also reduced. There was no clear effect of diHGA₅ on the total radioactivity recovered, or on conversion of the [³H]GA₂₀ to putative [³H]GA conjugates, or on the amount of [²H₂]GA₂₀ found. No free [²H₂]GA₈ was detected. In other experiments there was little effect of diHGA₅ on elongation induced by treatment with GA₁. We conclude that diHGA₅ inhibited GA₂₀-induced elongation in dwarf rice shoots at least partly by reducing the 3-hydroxylation of GA₂₀ to GA₁.Abbreviations diHGA₅ = exo- 16, 17-dihydro-gibberellin A₅ - GA = gibberellin - GC-MS-SIM = gas chromatography-mass spectrometry-selected ion monitoring     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutantgai ofArabidopsis thaliana (L.) Heynh

The endogenous gibberellins (GAs) from shoots of the GA-insensitive mutant,gai, ofArabidopsis thaliana were analyzed and compared with the GAs from the Landsberg erecta (Ler) line. Twenty GAs were identified in Ler plants by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention indices (KRI's). These GAs are members of the early-13-hydroxylation pathway (GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, GA₂₉, and GA₈), the non-3,13-hydroxylation pathway (GA₁₂, GA₁₅, GA₂₄, GA₂₅, GA₉, and GA₅₁), and the early-3-hydroxylation pathway (GA₃₇, GA₂₇, GA₃₆, GA₁₃, GA₄, and GA₃₄). The same GAs, except GA₅₃, GA₄₄, GA₃₇, and GA₂₉ were detected in thegai mutant by the same methods. In addition, extracts fromgai plants contained GA₄₁ and GA₇₁. Both lines also contained several unknown GAs. In Ler plants these were mainly hydroxy-GA₁₂ derivatives, whereas in thegai mutant hydroxy-GA₂₄, hydroxy-GA₂₅, and hydroxy-GA₉ compounds were detected. Quantification of seven GAs by GC-selected ion monitoring (SIM), using internal standards, and comparisons of the ion intensities in the SIM chromatograms of the other thirteen GAs, demonstrated that thegai mutant had reduced levels of all C₂₀-dicarboxylic acids (GA₅₃, GA₄₄, GA₁₉, GA₁₂, GA₁₅, GA₂₄, GA₃₇, GA₂₇, and GA₃₆). In contrast,gai plants had increased levels of C₂₀-tricarboxylic acid GAs (GA₁₇, GA₂₅, and GA₄₁) and of all C₁₉-GAs (GA₂₀, GA₁, GA₈, GA₉, GA₅₁, GA₄, GA₃₄, and GA₇₁) except GA₂₉. The 3β-hydroxylated GAs, GA₁ and GA₄, and their respective 2β-hydroxylated derivatives, GA₈ and GA₃₄, were the most abundant GAs found in shoots of thegai mutant. Thus, thegai mutation inArabidopsis results in a phenotype that resembles GA-deficient mutants, is insensitive to both applied and endogenous GAs, and contains low levels of C₂₀-dicarboxylic acid GAs and high levels of C₁₉-GAs. This indicates that theGAI gene controls a step beyond the synthesis of an active GA. Thegai mutant is presumably a GA-receptor mutant or a mutant with a block in the transduction pathway between the receptor and stem elongation. We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA for [¹³C]GA₈, and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility (grant No. DRR00480), for advice with mass spectrometry. This work was supported by a fellowship from the Spanish Ministry of Agriculture (I.N.I.A.) to M.T., by the U.S. Department of Energy under Contract DE-ACO2-76ERO-1338, and by U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Co-function of C3-and C4-photosynthetic pathways in C3, C4 and C3-C4 intermediate Flaveria species

The potential for C₄ photosynthesis was investigated in five C₃-C₄ intermediate species, one C₃ species, and one C₄ species in the genus Flaveria, using ¹⁴CO₂ pulse-¹²CO₂ chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating ¹⁴CO₂ into the C₄ acids malate and aspartate, following an 8-s pulse. The proportion of ¹⁴C label in these C₄ products ranged from 50–55% to 20–26% in the C₃-C₄ intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, ¹⁴CO₂ into aspartate as into malate. Generally, about 5–15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C₄ photosynthesis among the intermediate species based on the apparent rate of conversion of ¹⁴C label from the C₄ cycle to the C₃ cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C₄-cycle products and an increase in percentage label in C₃-cycle products during chase periods with ¹²CO₂, although the rate of change was slower than in the C₄ species, F. palmeri. In these C₃-C₄ intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C₃-C₄ intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C₄-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C₄ and C₃ cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C₃ species, 7–18% of the initial label was in malate+aspartate. However, only 40–50% of this label was in the C-4 position, indicating C₄-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O₂, the absorbed quantum yields for CO₂ uptake (in mol CO₂·[mol quanta]^-1) averaged 0.053 in F. cronquistii (C₃), 0.051 in F. trinervia (Spreng.) Mohr (C₄), 0.052 in F. ramosissima (C₃-C₄), 0.051 in F. anomala (C₃-C₄), 0.050 in F. linearis (C₃-C₄), 0.046 in F. floridana (C₃-C₄), and 0.044 in F. pubescens (C₃-C₄). In 2% O₂ an enhancement of the quantum yield was observed in all of the C₃-C₄ intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O₂ were intermediate in value to the C₃ and C₄ species, indicating a co-function of the C₃ and C₄ cycles in CO₂ assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O₂ presumably reflect an ineffcient transfer of carbon from the C₄ to the C₃ cycle. The response of the quantum yield to four increasing O₂ concentrations (2–35%) showed lower levels of O₂ inhibition in the C₃-C₄ intermediate F. ramosissima, relative to the C₃ species. This indicates that the co-function of the C₃ and C₄ cycles in this intermediate species leads to an increased CO₂ concentration at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase and a concomitant decrease in the competitive inhibition by O₂.Abbreviations PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - RuBP ribulose-1,5-bisphosphate     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Total synthesis of acetyl coenzyme a involved in autotrophic CO2 fixation inAcetobacterium woodii

The Gram positive anaerobeAcetobacterium woodii is able to grow autotrophically with a mixture of H₂ and CO₂ as the energy and carbon source. The question, by which pathway CO₂ is assimilated, was studied using long term isotope labeling.Autotrophically growing cultures produced acetate parallel to cell proliferation, and, when U-[¹⁴C]acetate was present as tracer, incorporated radioactivity into all cell fractions. The specific radioactivity and the label positions were determined for those representative cell compounds which biosynthetically originated directly from acetyl CoA (N-acetyl groups), pyruvate (alanine), oxaloacetate (aspartate), -ketoglutarate (glutamate), and hexosephosphates (glucosamine). Per mol compound the same amount of labeled acetate was incorporated into N-acetyl groups, alanine (C-2, C-3), aspartate (C-2, C-3), and twice the amount into glutamate (C-2, C-3, C-4, C-5) and into glucosamine. Consequently, the unlabeled carbon atoms of the C₃–C₆ compounds must have been derived from CO₂ by carboxylation subsequent to acetyl CoA synthesis. When 0.2 mM 2-[¹⁴C]pyruvate was added to autotrophically growing cultures, also a substantial amount of radioactivity was incorporated. Two important differences in comparison to the acetate experiment were observed: The N-acetyl groups were almost unlabeled and glutamate contained the same specific radioactivity as alanine or aspartate.These data showed that acetyl CoA is the central intermediate for biosynthesis and excluded the operation of the Calvin cycle inA. woodii. The results were consistent with the operation of a different autotrophic CO₂ fixation pathway in which CO₂ is converted into acetyl CoA by total synthesis via methyltetrahydrofolate; acetyl CoA is then further reductively carboxylated to pyruvate.     

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.