Isolation and expression of three gibberellin 20-oxidase cDNA clones from Arabidopsis.

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

Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA25 and of GA53 to GA17, as well as the formation of the C19-GAs, GA1, GA9, and GA20, from their respective aldehyde precursors, GA23, GA24, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted M(r) (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxygenases.

Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins.

Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA12 yielded GA13, GA43 and 12α-hydroxyGA43 as major products, GA4, GA37, GA39, GA46 and four unidentified compounds as minor products. The intermediates GA15, GA24 and GA25 accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA43 was inferred from its mass spectrum and by its formation from both GA39 and GA43. Gibberellin A39 and 12α-hydroxyGA43 were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A12-aldehyde was metabolised to essentially the same products as GA12 but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A53, formed from GA12 by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA1 GA23, GA28, GA44, and putative 2ß-hydroxyGA28. Minor products were GA19, GA20, GA38 and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA53) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553-558), GA1, GA8, GA25, GA28, GA36, GA48 and 12α-hydroxyGA43 were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA43 and GA1 (or GA8), respectively, as their end products. Evidence for endogenous GA20 and GA24 was also obtained but it was less conclusive due to interference.

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, 3ß-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 2ß-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 2ß-hydroxyGA28 and GA23 was also obtained but it was less conclusive because of contamination.

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. [(14)C]- or [17-(2)H2]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 α-amylase production in the aleurone layer. There was no evidence for 12ß- 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.

ent-Kaurene Biosynthesis in Germinating Barley (Hordeum vulgare L., cv Himalaya) Caryopses and Its Relation to alpha-Amylase Production.

ent-Kaurene biosynthesis as a prerequisite for gibberellin (GA) biosynthesis was studied in germinating Hordeum vulgare L., cv Himalaya caryopses and correlated, in time, with the appearance of alpha-amylase activity. The rate of ent-kaurene biosynthesis was estimated by inhibiting its further metabolism with plant growth retardants (triapenthenol or tetcyclacis) and measuring its accumulation by isotope dilution using combined gas chromatographymass spectrometry. In the inhibitor-treated caryopses, ent-kaurene accumulation began approximately 24 hours after imbibition and proceeded at a rate of about 1 to 2 picomoles per hour per caryopsis, depending on the batch of seeds. In the absence of inhibitor, ent-kaurene did not accumulate, indicating that it is normally turned over rapidly, presumably to further intermediates of the GA biosynthesis pathway and eventually to GAs. ent-Kaurene accumulation occurred almost exclusively in the shoot, which is, therefore, probably the site of biosynthesis. alpha-Amylase production began between 30 and 36 hours after imbibition and, thus, correlated well with de novo GA biosynthesis, as estimated from ent-kaurene accumulation. However, inhibition of ent-kaurene oxidation by plant growth retardants did not reduce the alpha-amylase production significantly, although it did reduce shoot elongation. We conclude that ent-kaurene is produced in the shoot and is continuously converted to GA, which is essential for normal shoot elongation, but not for the production of alpha-amylase in the aleurone layer.

The partial purification and characterization of a gibberellin C-20 hydroxylase from immature Pisum sativum L. seeds.

A gibberellin (GA) C-20 hydroxylase that catalyses the conversion of GA53 to GA44 was purified from developing pea embryos by ammonium-sulfate precipitation, gel filtration and anion-exchange column chromatography. The purification was about 270-fold and 15% of the enzymic activity was recovered. The relative molecular mass was 44000 by Sephadex G-200 gel filtration. The apparent Michaelis constant was 0.7 µM and the isoelectric point was 5.6-5.9. The enzymic activity was optimal at pH 7.0 2-Oxoglutarate and ascorbate were required for activity. Low concentrations of Fe(2+) stimulated the reaction, but externally added Fe(2+) was not essential, even in the most purified preparation. Catalase and bovine serum albumin also stimulated. Dithiothreitol preserved the activity during purification but was not needed during incubation. In fact, the simultaneous presence of dithiothreitol and Fe(2+) in the incubation mixture was inhibitory to the purified enzyme. The cofactor requirements are typical for those of 2-oxoglutarate-dependent dioxygenases.When the incubation time was long enough, GA53 was converted to both GA44 and GA19. The proportions of these two products remained constant throughout the purification, but this does not necessarily mean that their formations is catalysed by a single enzyme. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the final preparation contained several proteins. Although the most prominent protein band was located within the range expected for the enzyme on the grounds of its molecular weight, this band did not represent the enzyme, since it separated from the GA C-20 hydroxylase activity on ultrathin-layer isoeletric focusing.

Identification of Gibberellins in Norway Spruce (Picea abies [L.] Karst.) by Combined Gas Chromatography-Mass Spectrometry.

Gibberellins A(1) (GA(1)), A(3) and A(9) were identified from extracts of shoots of 6-month old Norway spruce (Picea abies) seedlings by the use of sequential reverse and normal phase high performance liquid chromatography (HPLC), bioassay, radioimmunoassay (RIA) and combined gas chromatography-mass spectrometry (GC-MS). The bioassay and RIA were used after fractionation by HPLC to detect the GA-containing fractions, which were then examined by GC-MS. The GAs identified are considered to be endogenous.

Comparison of gas chromatography-mass spectrometry, radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.).

Quantitative estimates of gibberellin A9 in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A9, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.

Gibberellin metabolism in cell-free extracts from spinach leaves in relation to photoperiod.

Cell-free extracts capable of converting [(14)C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [(14)C]-labeled GAs, prepared enzymically from [(14)C]mevalonic acid, were incubated with these extracts, and products were identified by gas chromatography-mass spectrometry. The following pathway was found to operate in extracts from spinach leaves grown under long day (LD) conditions: GA(12) --> GA(53) --> GA(44) --> GA(19) --> GA(20). The pH optima for the enzymic conversions of [(14)C]GA(53), [(14)C]GA(44) and [(14)C]GA(19) were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe(2+), alpha-ketoglutarate and O(2) for activity, and ascorbate stimulated the conversion of [(14)C]GA(53) and [(14)C]GA(19). Extracts from plants given LD or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA(53) and GA(19) are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA(44) remains high irrespective of light or dark treatment. This photoperiodic control of enzyme activity is not due to the presence of an inhibitor in plants grown in SD. These observations offer an explanation for the higher GA(20) content of spinach plants in LD than in SD.

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 2ß-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 2ß-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA8 and GA29, both 2ß-hydroxylated, were much higher in the testas than in the embryos.

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C20-GAs are converted to C19-GAs. Gibberellin A12 labeled with (14)C at C-20 was prepared from [3'-(14)C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that (14)CO2 was formed at the same rate and in nearly equivalent amounts as (14)C-labeled C19-GAs whereas [(14)C]formic acid and [(14)C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO2 was investigated by control incubations with [(14)C]formic acid. The rate of release of (14)CO2 from [(14)C]formic acid was only one fiftieth of the rate of (14)CO2 release from [(14)C]GA12 as the substrate. We conclude that in the formation of C19-GAs from C20-GAs, the C-20 is removed directly as CO2.

Conversion of [(14)C]gibberellin A 12-aldehyde to C 19- and C 20-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 [(14)C]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 (14)C-isotope peaks. GA8 and GA28 were also identified but contained no (14)C. All the [(14)C]GA12-aldehyde metabolites, except GA15, GA24 and GA53-aldehyde, are known endogenous GAs of P. coccineus.

Conversion of gibberellin A20 to gibberellins A 1 and A 5 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 (Δ(2) double-bond formation) require oxygen, Fe(2+) and α-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.

The biosynthesis of a C19-gibberellin from mevalonic acid in a cell-free system from a higher plant.

Incubation of a cell-free system from immature seeds of Cucurbita maxima Duch. with [(14)C]GA12-aldehyde derived from [(14)C]mevalonic acid in the same system yielded the C19-gibberellin, [(14)C]GA4, in addition to the C20-gibberellins, [(14)C]GA37, [(14)C]GA13 and [(14)C]GA43. (GA-gibberellin).

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

A cell-free system prepared from immature seed of Cucurbita pepo incorporates the label from mevalonate-2-(14)C into ent-kaur-16-en-19-oic acid (I), ent-7α-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.

The enzymic preparation of (14)C-kaurene.

Endosperm from immature seeds of Cucurbita pepo L. converts 2-(14)C-DL-mevalonate to (14)C-(-)-kaurene with a yield of nearly 40% of the active isomer. Kaurene is the main product and the only diterpene hydrocarbon which is formed from mevalonate in the system and is therefore easily obtained radiochemically pure. The product was identified by thin-layer chromatography and recrystallization with authentic (-)-kaurene to constant specific radioactivity.

Isoprenoid biosynthesis in a cell-free system from pea shoots.

A cell-free system consisting of a soluble fraction and plastid fragments from pea shoots incorporates 2-(14)C-mevalonate very actively into farnesol, squalene, geranylgeraniol, and other isoprenoids of different carbon-chain lengths. The products have different cofactor requirements, which makes it possible to channel the pathway into different products by varying the incubation mixture.