A mutant gene that increases gibberellin production in brassica

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

Enhanced production of gibberellin A4 (GA4) by a mutant of Gibberella fujikuroi in wheat gluten medium

Mutants of Gibberella fujikuroi with different colony characteristics, morphology and pigmentation were generated by exposure to UV radiation. A mutant, Mor-189, was selected based on its short filament length, relatively high gibberellin A₄ (GA₄) and gibberellin A₃ (GA₃) production, as well as its lack of pigmentation. Production of GA₄ by Mor-189 was studied using different inorganic and organic nitrogen sources, carbon sources and by maintaining the pH of the fermentation medium using calcium carbonate. Analysis of GA₄ and GA₃ was done by reversed-phase high-performance liquid chromatography and LC-MS. The mutants of G. fujikuroi produced more GA₄ when the pH of the medium was maintained above 5. During shake flask studies, the mutant Mor-189 produced 210 mg l^?1 GA₄ in media containing wheat gluten as the nitrogen source and glucose as the carbon source. Fed-batch fermentation in a 14 l agitated fermenter was performed to evaluate the applicability of the mutant Mor-189 for the production of GA₄. In 7-day fed-batch fermentation, 600 mg l^?1 GA₄ were obtained in the culture filtrate. The concentration of GA₄ and GA₃ combined was 713 mg l^?1, of which GA₄ accounted for 84% of the total gibberellin. These values are substantially higher than those published previously. The present study indicated that, along with maintenance of pH and controlled glucose feeding, the use of wheat gluten as the sole nitrogen source considerably enhances GA₄ production by the mutant Mor-189.     

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

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

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.     

Metabolism of tritiated gibberellin a(20) in maize

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

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.     

Biosynthetic Origin of Gibberellins A(3) and A(7) in Cell-Free Preparations from Seeds of Marah macrocarpus and Malus domestica

Cell-free preparations from seeds of Marah macrocarpus L. and Malus domestica L. catalyzed the conversion of gibberellin A₉ (GA₉) and 2,3-dehydroGA₉ to GA₇; GA₉ was also metabolized to GA₄ in a branch pathway. The preparation from Marah seeds also metabolized GA₅ to GA₃ in high yield; GA₆ was a minor product and was not metabolized to GA₃. Using substrates stereospecifically labeled with deuterium, it was shown that the metabolism of GA₅ to GA₃ and of 2,3-dehydroGA₉ to GA₇ occurs with the loss of the 1β-hydrogen. In cultures of Gibberella fujikuroi, mutant B1-41a, [1β,2β-²H₂]GA₄, was metabolized to [1,2-²H₂]GA₃ with the loss of the 1α- and 2α-hydrogens. These results provide further evidence that the biosynthetic origin of GA₃ and GA₇ in higher plants is different from that in the fungus Gibberella fujikuroi.     

Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin

The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill—about 10 nanograms GA₃ equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [¹⁴C]-GA₁₂-7-aldehyde (GA₁₂ald)—the universal GA precursor—by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA₁₂ald to two products—[¹⁴C]GA₁₂ (42% yield) and [¹⁴C]GA₁₅ (7%). Within 5 minutes, intact embryos converted GA₁₂ald to [¹⁴C]GA₁₂ and [¹⁴C]GA₁₅ in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [¹⁴C]GA₁₂ was identified as a product of [¹⁴C]GA₁₂ald metabolism on the basis of co-chromatography with authentic GA₁₂ on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [¹⁴C]GA₁₂ and authentic [¹⁴C]GA₁₂ to cotyledons of both peas and soybeans. The [¹⁴C]GA₁₅ was identified as a metabolite of [¹⁴C]GA₁₂ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [¹⁴C] metabolites of [¹⁴C]GA₁₂ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA₇ by GC-mass spectrometry. Since [¹⁴C]GA₉ was not found as a [¹⁴C]metabolite of [¹⁴C]GA₁₂ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA₁₂ald → GA₁₂ → GA₁₅ → GA₂₄ → GA₃₆ → GA₄ → GA₇.     

Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA₃, GA_4, and GA₇) but also pure GA₄ or GA₇. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA₃, GA₄, and GA₇ explaining the varying GA levels of genetically almost identical mutant strains.     

Dwarf mutants ofBrassica: Responses to applied gibberellins and gibberellin content

Eight rapid-cyclingBrassica genotypes differing in height were treated with gibberellins (GAs) by syringe application to the shoot tip. The height of two genotypes ofBrassica napus, Bn5-2 and Bn5-8, andB. rapa mutants,dwarf 1 (dwf1) anddwarf 2 (dwf2), was unaffected by exogenous GA₃ at dosages up to 0.1 μg/plant, a level which increased shoot elongation of normal genotypes. Thus, these dwarf mutants are “GA-insensitive.” In contrast to theB. napus dwarfs, twoB. rapa mutants,rosette (ros), anddormant (dor), elongated following GA₃ application. The dwarfros was most sensitive, responding to applications as low as 1 ng GA₃/plant. Furthermore,ros also responded to GA₁ and some of its precursors with decreasing efficacy: GA₃>ent-kaurenoic acid ≥GA₁>GA₂₀≥GA₁₉=GA₄₄≥GA₅₃. Endogenous GAs were measured by gas chromatography-selected ion monitoring using [²H₂]GA internal standards for calibration, from shoots of the GA-insensitive genotypes Bn5-2, Bn5-8 which contained theB. napus mutantdwarf 1, and from a normal genotype Bn5-1. Concentrations of GA₁ and GA₂₀ averaged 3.2- and 4.6-fold higher, respectively, and GA₁₉ levels also tended to be higher in the dwarfs than in the normal genotype.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

The levels of the biologically active gibberellin (GA), GA₁, and of its precursor, GA₂₀, were monitored at several stages during ontogeny in the apical portions of isogenic tall (Le) and dwarf (le) peas (Pisum sativum L.) using deuterated internal standards and gas chromatography-selected ion monitoring. The levels of both GAs were relatively low on emergence and on impending apical arrest. At these early and late stages of development the internodes were substantially shorter than at intermediate stages, but were capable of large responses to applied GA₃. Tall plants generally contained 10–18 times more GA₁ and possessed internodes 2–3 times longer than dwarf plants. Further, dwarf plants contained 3–5 times more GA₂₀ than tall plants. No conclusive evidence for the presence of GA₃ or GA₅ could be obtained, even with the aid of [²H₂]GA₃ and [²H₂]GA₅ internal standards. If GA₃ and GA₅ were present in tall plants, their levels were less than 0.5% and 1.4% of the level of GA₁, respectively. Comparison of the effects of gene le on GA₁ levels and internode length with the effects of ontogeny on these variables shows that the ontogenetic variation in GA₁ content was sufficient to account for much of the observed variation in internode length within the wild-type. However, evidence was also obtained for substantial differences in the potential length of different internodes even when saturating levels of exogenous GA₃ were present.Abreviations GA_n gibberellin A_n We thank Noel Davies, Omar Hasan, Leigh Johnson, Katherine McPherson and Naomi Lawrence for technical help, Professor L. Mander (Australian National University, Canberra) for deuterated GA standards and the Australian Research Council for financial assistance.     

Heterosis and the metabolism of gibberellin A20 in sorghum

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

Interconversion of gibberellin A1 to gibberellin A8 in seedlings of dwarf Oryza sativa

[³H]Gibberellin A₁ ([³H]GA₁)applied to seedlings of dwarf rice (Oryza sativa L. cv. Tanginbozu) was metabolized to GA₈. Identification of GA₈, was made by gas-liquid radiochromatography using three liquid stationary phases.     

The influence of dwarfing interstocks on the distribution and metabolism of xylem-applied [h]gibberellin a(4) in apple

The influence of an interstock of the dwarfing cultivar M9 and the nondwarfing cultivar MM115 on the distribution and metabolism of labeled gibberellic acid A₄ ([³H]GA₄) of high specific radioactivity (5.18 × 10¹⁰ becquerel per millimole) applied to the xylem of the rootstock in grafted apple (Malus × domestica Borkh.) trees was compared. Free [³H] GA-like metabolites of [³H]GA₄, including putative GA₁, GA₂, GA₃, and GA₃₄, as well as various ³H-putative GA glucosyl conjugates were detected in stem segments from both cultivars. M9 interstocks reduced the total uptake of [³H]GA₄ and decreased the proportion of ³H metabolites transported to the shoots and leaves of scions. The M9 interstock tissue and adjacent rootstock and scion tissue retained a much greater amount and a higher proportion of the label than did comparable tissue of the nondwarfing MM115 interstock. In addition, the amount and proportion of free [³H]GAs was higher, and the proportion of putative [³H]GA glucosyl conjugates lower, in M9 interstocks compared to MM115. These effects of the dwarfing interstock on GA distribution and metabolism indicate a significant role for GAs in any satisfactory explanation of the dwarfing mechanism in apple.     

Genetic regulation of gibberellin deactivation in Pisum

The regulation of gibberellin (GA) deactivation was examined using the sin (slender) mutation in the garden pea (Pisum sativum L.). This mutation blocks the deactivation of GA₂₀, the precursor of the bioactive GA₁. Firstly, crosses were made to combine sin with the GA biosynthesis mutations na, lhⁱ and le-3. The combination sin na produced a novel phenotype, with long (‘slender’) basal internodes and extremely short (‘nana’) upper internodes. In contrast, the double mutant sin lhⁱ was phenotypically dwarf. The mutation sin causes an accumulation of GA₂₀ in maturing seeds, and this was unaffected by na, since the na mutation is not expressed in seeds. In contrast, lhⁱ seeds did not accumulate GA₂₀, since lhⁱ imposes an early block on GA biosynthesis. Secondly, the effects of sin on several steps in GA deactivation were investigated. In maturing seeds, the mutation sin blocks two steps in GA₂₀ metabolism, namely, GA₂₀ to GA₂₉, and GA₂₉ to GA₂₉-catabolite. In the vegetative plant, on the other hand, sin blocked the step GA₂₀ to GA₂₉, but not GA₂₉ to GA₂₉-catabolite; the steps GA₂₀ to GA₈₁ and GA₂₀ to GA₁ were also not impaired in this mutant. It is clear that the effects of sin, like those of na, are strongly organ-specific. The presence of separate enzymes for the steps GA₂₀ to GA₂₉ and GA₂₉ to GA₂₉-catabolite was suggested by the observation that GA₈ inhibited the latter step, but not the former, and by the inability of GA₂₀ and GA₂₉ to inhibit each other's metabolism. It is suggested that the Sin gene may be a regulatory gene controlling the expression of two structural genes involved in GA deactivation.     

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     

A Gibberellin-Deficient Brassica Mutant-rosette

A single-gene mutant (rosette [ros/ros]) in which shoot growth and development are inhibited was identified from a rapid cycling line of Brassica rapa (syn campestris). Relative to normal plants, the mutant germinated slowly, had delayed or incomplete floral development, and reduced leaf, petiole, and internode growth. The exogenous application of GA₃ by foliar spray or directly to the shoot tip of rosette resulted in rapid flowering, bolting (shoot elongation), and viable seed production. Shoots of rosette contained endogenous levels of total gibberellin (GA)-like substances (`Tan-ginbozu' dwarf rice assay) of about one-tenth of that of the normal rapid-cycling line of B. rapa which consisted almost entirely of a very nonpolar, GA-like substance which yielded GA₁ and GA₃ upon mild acid hydrolysis. In a normal rapid-cycling B. rapa line, the nonpolar putative GA₁ and GA₃ conjugates were present, but additionally, free GA₁ and GA₃ were abundant and identified by gas chromatography-mass spectrometry-selected ion monitoring. The quantities of free GA₁ and GA₃ in the normal line and in rosette were quantified by GC-MS-SIM using [²H₂]GA₁ as an internal standard. Fourteen-day-old rosette and normal seedlings contained 5.3 and 23.2 ng GA₁ per plant, respectively. At day 21 the rosette plants contained 7.7 and 26.1 nanograms per plant of GA₁ and GA₃, while normal plants contained 31.1 and 251.5 nanograms per plant, respectively. Thus, normal plants contained from four to ten times higher levels of total GA-like substances, GA₁, or GA₃, than rosette. The ros allele results in reduced GA level, yielding the rosette phenotype whose delayed germination and flowering, and reduced shoot growth responses indicate a probable role for endogenous GA₁ and GA₃ in the regulation of these processes in Brassica.     

Interconversion of gibberellin A4 to gibberellins A1 and A34 by dwarf rice,cultivar Tan-ginbozu

Summary Interconversion of GA₄ to GA₁ and GA₃₄ occurred within 24 h of application of 1,2-[³H]-GA₄ to seedlings of dwarf rice, cv. Tan-ginbozu. Identification was made by direct comparison of the trimethylsilyl ether derivatives of the methyl esters of Silica-gel partition-column fractions on gas-liquid radiochromatography with derivatized GA₁ and GA₃₄ standards on three columns: 2% QF-1, 2% SE-30, and 1% XE-60. GA₂, an artifact of the purification and chromatography system, may also be formed by the plant. The conversions from GA₄ to GA₁ and GA₃₄ are single hydroxylations. At least two unidentified radioactive products were also formed by the plant. Interconversions were in the order of 0.3 to 0.8% of applied [³H]-GA₄.Abbreviations GA gibberellin - GLC gas-liquid chromatography - GLRC gasliquid radiochromatography - TMSMe trimethylsilyl ether of methyl ester - TLC thin-layer chromatography     

Seed effects on gibberellin metabolism in pea pericarp

Pea fruit (Pisum sativum L.) is a model system for studying the effect of seeds on fruit growth in order to understand coordination of organ development. The metabolism of ¹⁴C-labeled gibberellin A₁₂ (GA₁₂) by pea pericarp was followed using a method that allows access to the seeds while maintaining pericarp growth in situ. Identification and quantitation of GAs in pea pericarp was accomplished by combined gas chromatography-mass spectrometry following extensive purification of the putative GAs. Here we report for the first time that the metabolism of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₉ and [¹⁴C]GA₂₀ occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA₁₉ to GA₂₀ and caused the accumulation of radiolabeled and endogenous GA₁₉. Deseeded pericarp contained no detectable GA₂₀, GA₁, or GA₈, whereas pericarp with seeds contained endogenous and radiolabeled GA₂₀ and endogenous GA₁. These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA₁₉ to GA₂₀.