Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice

[14C4]GA53, [14C4]GA44, and [2H2/14C4]GA19 were injected separately into seedlings of rice (Oryza sativa) using a dwarf mutant (d35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography mass spectrometry (GC-MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA53-->GA44-->GA19-->GA20 and the presence of endogenous GA53, GA44, GA19, GA20 and GA1. Previous metabolic studies have shown the presence of the step, GA20-->GA1 in rice. Taken together, the data establish in vegetative shoots of rice the presence of the early 13-hydroxylation pathway, a pathway that originates from GA12 and leads to bioactive GA1.     

Metabolism of Gibberellin A(12) and A(12)-Aldehyde in Developing Seeds of Pisum sativum L

Metabolism of [¹⁴C]gibberellin (GA) A₁₂ (GA₁₂) and [¹⁴C]gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [¹⁴C]GA₁₂ was slower in seed coats than in cotyledons. [¹⁴C]GA₁₂-aldehyde was also metabolized to conjugates in seed coats. Seed coat [¹⁴C]-metabolites produced from [¹⁴C]GA₁₂-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [¹⁴C]GA₅₃-aldehyde, [¹⁴C]GA₁₂-aldehyde conjugate, and [¹⁴C]GA₅₃-aldehyde conjugate were major metabolites produced from [¹⁴C]GA₁₂-aldehyde by seed coats aged 20-22 days or older. The dilution of ¹⁴C in these compounds by ¹²C, as compared to the supplied [¹⁴C]GA₁₂-aldehyde, indicated that they are endogenous. Feeding [¹⁴C]GA₅₃-aldehyde led to the production of [¹⁴C]GA₅₃-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [¹⁴C]GA₁₂, [¹⁴C]GA₁₂-aldehyde, or [³H]GA₉, were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA₁₅, GA₂₄, GA₉, GA₅₁, GA₅₁-catabolite, GA₂₀, GA₂₉, and GA₂₉-catabolite were detected in seed coats, whereas GA₉, GA₅₃, GA₄₄, GA₁₉, GA₂₀, and GA₂₉ were found in cotyledons. The highest GA levels were for GA₂₀ and GA₂₉ in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA₂₉ and GA₂₉-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).     

The Metabolism of Gibberellin A20 to Gibberellin A1 by Tall and Dwarf Mutants of Oryza sativa and Arabidopsis thaliana

The purpose of this study was to demonstrate the metabolism of gibberellin A20 (GA20) to gibberellin A1 (GA1) by tall and mutant shoots of rice (Oryza sativa L.) and Arabidopsis thaliana (L.) Heynh. The data show that the tall and dx mutant of rice and the tall and ga5 mutant of Arabidopsis metabolize GA20 to GA1. The data also show that the dy mutant of rice and the ga4 mutant of Arabidopsis block the metabolism of GA20 to GA1. [17-13C,3H]GA20 was fed to tall and the dwarf mutants, dx and dy, of rice and tall and the dwarf mutants, ga5 and ga4, of Arabidopsis. The metabolites were analyzed by high-performance liquid chromatography and full-scan gas chromatography-mass spectrometry together with Kovats retention index data. For rice, the metabolite [13C]GA, was identified from tall and dx seedlings; [13C]GA1 was not identified from the dy seedlings. [13C]GA29 was identified from tall, dx, and dy seedlings. For Arabidopsis, the metabolite [13C]GA1 was identified from tall, ga5, and ga4 plants. The amount of [13C]GA1 from ga4 plants was less than 15% of that obtained from tall and ga5 plants. [13C]GA29 was identified from tall, ga5, and ga4 plants. [13C]GA5 and [13C]GA3 were not identified from any of the six types of plant material.     

Decapitation Reduces the Metabolism of Gibberellin A20 to A1 in Pisum sativum L., Decreasing the Le/le Difference

When the metabolism of [13C,3H]gibberellin (GA)20 in Pisum sativum L. was investigated using decapitated plants and stem sections, no evidence was obtained for the recently postulated inhibitor of GA20 3[beta]-hydroxylase (V.A. Smith [1992] Plant Physiol 99: 372-377). Instead, the results are consistent with the hypothesis that the mutation le reduces GA1 production by altering the structure or level of the 3[beta]-hydroxylase.     

Metabolism and Biological Activity of Gibberellin A4 in Vegetative Shoots of Zea mays,Oryza sativa,and Arabidopsis thaliana

[17-13C,3H]Gibberellin A4 (GA4) was injected into the shoots of tall (W23/L317), dwarf-1 (d1), and dwarf-5 (d5) Zea mays L. (maize); tall (cv Nipponbare), dwarf-x (dx), and dwarf-y (dy) Oryza sativa L. (rice); and tall (ecotype Landsberg erecta), ga4, and ga5 Arabidopsis thaliana (L.) Heynh. [13C]GA4 and its metabolites were identified from the shoots by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA4 was metabolized to GA1 in all nine genotypes. GA4 was also metabolized in some of the genotypes to 3-epi-GA1, GA2, 2[beta]-OH-GA2, 3-epi-GA2, endo-GA4, 16[alpha], 17-H2-16, 17-(OH)2-GA4, GA34, endo-GA34, GA58, 15-epi-GA63, GA71, and 16-epi-GA82. No evidence was found for the metabolism of GA4 to GA7 or of GA4 to GA3. The bioactivities of GA4 and GA1 were determined using the six dwarf mutants for assay. GA4 and GA1 had similar activities for the maize and rice mutants. For the Arabidopsis mutants, GA4 was more active than GA1 at low dosages; GA4 was less active than GA1 at higher dosages.     

Further identification of endogenous gibberellins in the shoots of pea, line g2

To interpret the metabolism of radiolabeled gibberellins A₁₂-aldehyde and A₁₂ in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [¹⁴C]GA₁₂ was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [¹⁴C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA₅₃, GA₄₄, GA₁₉ and GA₂₀ was demonstrated and their HPLC peak identity ascertained. The ¹⁴C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA₅₃ to 4% in GA₂₀. Calculated levels of GA₂₀, GA₁₉, GA₄₄, and GA₅₃ were 42, 8, 10, and 0.5 nanograms/gram, respectively.     

Photoperiod modification of [C]gibberellin a(12) aldehyde metabolism in shoots of pea, line g2

In G2 peas (Pisum sativum L.) apical senescence occurs only in long days (LD), and indeterminate growth is associated with elevated gibberellin (GA) levels in the shoot in short days (SD). Metabolism of GA₁₂ aldehyde was investigated by feeding shoots grown in SD or LD with [¹⁴C]GA₁₂ aldehyde through the cut end of the stem for 0.5 to 6 hours in the light and analyzing the tissue extract by high performance liquid chromatography. More radioactive products were detected than can be accounted for by the two GA metabolic pathways previously known to be present in peas. Three of the major products appear to be GA conjugates, but an additional pathway(s) of GA metabolism may be present. The levels of putative C₂₀ GAs, [¹⁴C]GA₅₃, [¹⁴C]GA₄₄, [¹⁴C]GA₁₉, and/or [¹⁴C] GA₁₇, were all elevated in SD as compared to LD. Putative [¹⁴C]GA, was slightly higher in LD than in SD. Putative [¹⁴C]GA₅₃ was a major metabolite after 30 minutes of treatment in SD but had declined after longer treatment times to be replaced by elevated levels of putative [¹⁴C] GA₄₄ and [¹⁴C]GA_19/17. Metabolism of GA₂₀ was slow in both photoperiods. Although GA₂₀ and GA₁₉ are the major endogenous GAs as determined by gas chromatography-mass spectrometry, putative [¹⁴C]GA₂₀ and [¹⁴C]GA₁₉ were never major products of [¹⁴C]GA₁₂ aldehyde metabolism. Thus, photoperiod acts in G2 peas to change the rate of GA₅₃ production from GA₁₂ aldehyde, with the levels of the subsequent GAs on the 13-OH pathway being determined by the amount of GA₅₃ being produced.     

Kaurenolides and fujenoic acids are side products of the gibberellin P450-1 monooxygenase in Gibberella fujikuroi

The steps involved in kaurenolide and fujenoic acids biosynthesis, from ent-kauradienoic acid and ent-6alpha,7alpha-dihydroxykaurenoic acid, respectively, are demonstrated in the gibberellin (GA)-deficient Gibberella fujikuroi mutant SG139, which lacks the entire GA-biosynthesis gene cluster, complemented with the P450-1 gene of GA biosynthesis (SG139-P450-1). ent-[2H]Kauradienoic acid was efficiently converted into 7beta-hydroxy[2H]kaurenolide and 7beta,18-dihydroxy[2H]kaurenolide by the cultures while 7beta-hydroxy[2H]kaurenolide was transformed into 7beta,18-dihydroxy[2H]kaurenolide. The limiting step was found to be hydroxylation at C-18. In addition, SG139-P450-1 transformed ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid into [14C4]fujenoic acid and [14C4]fujenoic triacid. Fujenal was also converted into the same products but was demonstrated not to be an intermediate in this sequence. All the above reactions were absent in the mutant SG139 and were suppressed in the wild-type strain ACC917 by disruption of the P450-1 gene. Kaurenolide and fujenoic acids synthesis were associated with the microsomal fraction and showed an absolute requirement for NADPH or NADH, all properties of cytochrome P450 monooxygenases. Only 7beta-hydroxy[14C4]kaurenolide synthesis and not further 18-hydroxylation was detected in the microsomal fraction. The substrates for the P450-1 monooxygenase, ent-kaurenoic acid and [2H]GA12, efficiently inhibited kaurenolide synthesis with I50 values of 3 and 6 microM, respectively. Both substrates also inhibited ent-6alpha,7alpha-dihydroxy[14C4]kaurenoic acid metabolism by SG139-P450-1. Conversely, [14C4]GA14 synthesis from [14C4]GA12-aldehyde was inhibited by ent-[2H]kauradienoic acid and fujenal with I50 values of 10 and 30 microM, respectively. These results demonstrate that kaurenolides and seco-ring B kaurenoids are formed by the P450-1 monooxygenase (GA14 synthase) of G. fujikuroi and are thus side products that probably result from stabilization of radical intermediates involved in GA14 synthesis.     

Thermoinductive Regulation of Gibberellin Metabolism in Thlaspi arvense L. : I. Metabolism of [H]-ent-Kaurenoic Acid and [C]Gibberellin A(12)-Aldehyde

Field pennycress (Thlaspi arvense L.) is a winter annual crucifer with a cold requirement for stem elongation and flowering. In the present study, the metabolism of exogenous [²H]-ent-kaurenoic acid (KA) and [¹⁴C]-gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) was compared in thermo- and noninduced plants. Thermoinduction greatly altered both quantitative and qualitative aspects of [²H]-KA metabolism in the shoot tips. The rate of disappearance of the parent compound was much greater in thermoinduced shoot tips. Moreover, there was 47 times more endogenous KA in noninduced than in thermoinduced shoot tips as determined by combined gas chromatography-mass spectrometry (GC-MS). The major metabolite of [²H]-KA in thermoinduced shoot tips was a monohydroxylated derivative of KA, while in noninduced shoot tips, the glucose ester of the hydroxy KA metabolite was the main product. Gibberellin A₉ (GA₉) was the only GA in which the incorporation of deuterium was detected by GC-MS, and this was observed only in thermoinduced shoot tips. The amount of incorporation was small as indicated by the large dilution by endogenous GA₉. In contrast, thermo- and noninduced leaves metabolized exogenous [²H]-KA into GA₂₀ equally well, although the amount of conversion was also limited. These results are consistent with the suggestion (JD Metzger [1990] Plant Physiol 94: 000-000) that the conversion of KA in to GAs is under thermoinductive control only in the shoot tip, the site of perception for thermoinductive temperatures in field pennycress. There were essentially no differences in the qualitative or quantitative distribution of metabolites formed following the application of [¹⁴C]-GA₁₂-aldehyde to the shoot tips of thermo- or noninduced plants. Thus, the apparent thermoinductive regulation of the KA metabolism into GAs is probably limited to the two metabolic steps involved in converting KA to GA₁₂-aldehyde.     

Daylength and spatial expression of a gibberellin 20-oxidase isolated from hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Physiologically active gibberellins (GAs) are key regulators of shoot growth in trees. To investigate this mechanism of GA-controlled growth in hybrid aspen, we cloned cDNAs encoding gibberellin 20-oxidase (GA 20-oxidase), a key, highly regulated enzyme in the biosynthesis of GAs. Clones were isolated from leaf and cambium cDNA libraries using probes generated by polymerase chain reaction, based on conserved domains of GA 20-oxidases. Upon expression in Escherichia coli, the GST-fusion protein was shown to oxidise GA12 as well as oxidising the 13-hydroxylated substrate GA53, successively to GA9 and GA20, respectively. The gene PttGA20ox1 was expressed in meristematic cells and growing tissues such as expanding internodes, leaves and roots. The expression was negatively regulated by both GA4 and overexpression of phytochrome A. RNA analysis also showed that the expression was down-regulated in late-expanding leaf tissue in response to short days (SDs). Actively growing tissues such as early elongating internodes, petioles and leaf blades had the highest levels of C19-GAs. Upon transfer to SDs an accumulation of GA19 was observed in early elongating internodes and leaf blades. The levels of C19-GAs were also to some extent changed upon transfer to SDs. The levels of GA20 were down-regulated in internodes, and those of GA1 were significantly reduced in early expanding leaf blades. In roots the metabolites GA19 and GA8 decreased upon shifts to SDs, while GA20 accumulated slightly. The down-regulation of GA 20-oxidase activity in response to SDs was further indicated by studies of [14C]GA12 metabolism in shoots, demonstrating that the substrate for GA 20-oxidase, [14C]GA53, accumulates in SDs.     

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     

Internode Length in Pisum: Gene na May Block Gibberellin Synthesis between ent-7alpha-Hydroxykaurenoic Acid and Gibberellin A(12)-Aldehyde

The elongation response of the gibberellin (GA) deficient genotypes na, ls, and lh of peas (Pisum sativum L.) to a range of GA-precursors was examined. Plants possessing gene na did not respond to precursors in the GA biosynthetic pathway prior to GA₁₂-aldehyde. In contrast, plants possessing lh and ls responded as well as wild-type plants (dwarfed with AMO-1618) to these compounds. The results suggest that GA biosynthesis is blocked prior to ent-kaurene in the lh and ls mutants and between ent-7α-hydroxykaurenoic acid and GA₁₂-aldehyde in the na mutant. Feeds of ent-[³H]kaurenoic acid and [²H]GA₁₂-aldehyde to a range of genotypes supported the above conclusions. The na line WL1766 was shown by gas chromatography-mass spectrometry (GC-MS) to metabolize [²H]GA₁₂-aldehyde to a number of[²H]C₁₉-GAs including GA₁. However, there was no indication in na genotypes for the metabolism of ent-[³H]kaurenoic acid to these GAs. In contrast, the expanding shoot tissue of all Na genotypes examined metabolised ent-[³H]kaurenoic acid to radioactive compounds that co-chromatographed with GA₁, GA₈, GA₂₀, and GA₂₉. However, insufficient material was present for unequivocal identification of the metabolites. The radioactive profiles from HPLC of extracts of the node treated with ent-[³H]kaurenoic acid were similar for both Na and na plants and contained ent-16α,17-dihydroxykaurenoic acid and ent-6α,7α,16β,17-tetrahydroxykaurenoic acid (both characterized by GC-MS), suggesting that the metabolites arose from side branches of the main GA-biosynthetic pathway. Thus, both Na and na plants appear capable of ent-7α-hydroxylation.     

Monooxygenases involved in GA12 and GA14 synthesis in Gibberella fujikuroi

A microsomal preparation from mycelia of the gibberellin (GA)-producing fungus Gibberella fujikuroi catalyzed the first two steps in the conversion of the biosynthetic intermediate GA12-aldehyde to gibberellic acid (GA3). [14C]GA12-Aldehyde was converted to radiolabelled GA14, the major product, together with smaller amounts of non-hydroxylated GA12. The microsomal activities required reduced pyridine nucleotides and molecular oxygen. However, GA12 and GA14 synthesis differed markedly in the preferred electron source. Formation of GA12 required NADH or NADPH, while GA14 synthesis from GA12-aldehyde occurred only with NADPH. Marked differences were also found in the activating effect of FAD. When NADPH was the reductant, the rate of GA14 synthesis was enhanced 3.5 times by 5 microM FAD while this flavin nucleotide did not alter the synthesis of GA12. In contrast, GA12 synthesis was activated 3.8 times by 50 microM FAD in the presence of NADH. Both activities were inhibited by carbon monoxide and cytochrome c. These properties suggest that the 3beta-hydroxylation of GA12-aldehyde and further oxidation of carbon 7 are catalyzed by cytochrome P-450 monooxygenases in Gibberella fujikuroi.     

Gibberellin Biosynthesis in Maize. Metabolic Studies with GA(15), GA(24), GA(25), GA(7), and 2,3-Dehydro-GA(9)

[17-(14)C]-Labeled GA(15), GA(24), GA(25), GA(7), and 2,3-dehydro-GA(9) were separately injected into normal, dwarf-1 (d1), and dwarf-5 (d5) seedlings of maize (Zea mays L.). Purified radioactive metabolites from the plant tissues were identified by full-scan gas chromatography-mass spectrometry and Kovats retention index data. The metabolites from GA(15) were GA(44), GA(19), GA(20), GA(113), and GA(15)-15,16-ene (artifact?). GA(24) was metabolized to GA(19), GA(20), and GA(17). The metabolites from GA(25) were GA(17), GA(25) 16alpha,17-H(2)-17-OH, and HO-GA(25) (hydroxyl position not determined). GA(7) was metabolized to GA(30), GA(3), isoGA(3) (artifact?), and trace amounts of GA(7)-diene-diacid (artifact?). 2,3-Dehydro-GA(9) was metabolized to GA(5), GA(7) (trace amounts), 2,3-dehydro-GA(10) (artifact?), GA(31), and GA(62). Our results provide additional in vivo evidence of a metabolic grid in maize (i.e. pathway convergence). The grid connects members of a putative, non-early 3,13-hydroxylation branch pathway to the corresponding members of the previously documented early 13-hydroxylation branch pathway. The inability to detect the sequence GA(12) --> GA(15) --> GA(24) --> GA(9) indicates that the non-early 3,13-hydroxylation pathway probably plays a minor role in the origin of bioactive gibberellins in maize.     

Seed and 4-chloroindole-3-acetic acid regulation of gibberellin metabolism in pea pericarp.

In this study, we investigated seed and auxin regulation of gibberellin (GA) biosynthesis in pea (Pisum sativum L.) pericarp tissue in situ, specifically the conversion of [14C]GA19 to [14C]GA20. [14C]GA19 metabolism was monitored in pericarp with seeds, deseeded pericarp, and deseeded pericarp treated with 4-chloroindole-3-acetic acid (4-CI-IAA). Pericarp with seeds and deseeded pericarp treated with 4-CI-IAA continued to convert [14C]GA19 to [14C]GA20 throughout the incubation period (2-24 h). However, seed removal resulted in minimal or no accumulation of [14C]GA20 in pericarp tissue. [14C]GA29 was also identified as a product of [14C]GA19 metabolism in pea pericarp. The ratio of [14C]GA29 to [14C]GA20 was significantly higher in deseeded pericarp (with or without exogenous 4-CI-IAA) than in pericarp with seeds. Therefore, conversion of [14C]GA20 to [14C]GA29 may also be seed regulated in pea fruit. These data support the hypothesis that the conversion of GA19 to GA20 in pea pericarp is seed regulated and that the auxin 4-CI-IAA can substitute for the seeds in the stimulation of pericarp growth and the conversion of GA19 to GA20.     

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.     

C]GA(12)-Aldehyde, [C]GA(12), and [H]- and [C]GA(53) Metabolism by Elongating Pea Pericarp

Gibberellins (GAs) are either required for, or at least promote, the growth of the pea (Pisum sativum L.) fruit. Whether the pericarp of the pea fruit produces GAs in situ and/or whether GAs are transported into the pericarp from the developing seeds or maternal plant is currently unknown. The objective of this research was to investigate whether the pericarp tissue contains enzymes capable of metabolizing GAs from [¹⁴C]GA₁₂-7-aldehyde ([¹⁴C]GA₁₂ald) to biologically active GAs. The metabolism of GAs early in the biosynthetic pathway, [¹⁴C]GA₁₂ and [¹⁴C]GA₁₂ald, was investigated in pericarp tissue isolated from 4-day-old pea fruits. [¹⁴C]GA₁₂ald was metabolized primarily to [¹⁴C]GA₁₂ald-conjugate, [¹⁴C]GA₁₂, [¹⁴C]GA₅₃, and polar conjugate-like products by isolated pericarp. In contrast, [¹⁴C]GA₁₂ was converted primarily to [¹⁴C]GA₅₃ and polar conjugate-like products. Upon further investigations with intact 4-day-old fruits on the plant, [¹⁴C]GA₁₂ was found to be converted to a product which copurified with endogenous GA₂₀. Lastly, [²H]GA₂₀ and [²H]GA₁ were recovered 48 hours after application of [²H]- and [¹⁴C]GA₅₃ to pericarp tissue of intact 3-day-old pea fruits. These results demonstrate that pericarp tissue metabolizes GAs and suggests a function for pericarp GA metabolism during fruit growth.     

Metabolism of gibberellins A1 and A3 in fruits and shoots of Prunus avium

Isotope-labelled GA metabolites were identified by GC--MS, following HPLC fractionation of extracts derived from fruits or shoots, that had been incubated with [2H]- and [3H]- GA1 or [2H]- and [3H]- GA3. GA1 (1) was converted into GA8 (10) by developing fruits and vegetative shoots of sweet cherry (Prunus avium cv. 'Stella'), while GA3 (4) was converted into GA3-isolactone (17). Other metabolites of each GA were detected but were not identified unequivocally. These included a metabolite of GA1 (1) in fruitlets that was more polar (by reverse phase HPLC) than GA8 (10) and a metabolite of similar polarity to GA87 (6), was obtained after incubating fruitlets with GA3 (4). However, no evidence was obtained to suggest that GA87 (6) was a metabolite of GA3 (4) or that GA85 (2) was a metabolite of GA1 (1) in these tissues, under the conditions used. The pattern of metabolites obtained from vegetative tissues was similar to that from fruitlets. However, the results suggested that GA1 (1) and GA3 (4) were metabolised at a greater rate in shoots from mature trees than in shoots from seedlings, and that GA1 (1) was metabolised more rapidly than GA3 (4) in juvenile and mature shoots. We conclude from these observations that GA3 (4) is not a precursor of GA87 (6) and GA32 (5), also, that GA1 (1) is not a precursor of GA85 (2) and GA86 (3) in developing fruits or in vegetative shoots of sweet cherry.     

Pollination Increases Gibberellin Levels in Developing Ovaries of Seeded Varieties of Citrus

Reproductive and vegetative tissues of the seeded Pineapple cultivars of sweet orange (Citrus sinensis L.) contained the following C-13 hydroxylated gibberellins (GAs): GA53, GA17, GA19, GA20, GA1, GA29, and GA8, as well as GA97, 3-epi-GA1, and several uncharacterized GAs. The inclusion of 3-epi-GA1 as an endogenous substance was based on measurements of the isomerization rates of previously added [2H2]GA1. Pollination enhanced amounts of GA19, GA20, GA29, and GA8 in developing ovaries. Levels of GA1 increased from 5.0 to 9.5 ng/g dry weight during anthesis and were reduced thereafter. The amount of GA in mature pollen was very low. Emasculation reduced GA levels and caused a rapid 100% ovary abscission. This effect was partially counteracted by either pollination or application of GA3. In pollinated ovaries, repeated paclobutrazol applications decreased the amount of GA and increased ovary abscission, although the pattern of continuous decline was different from the sudden abscission induced by emasculation. The above results indicate that, in citrus, pollination increases GA levels and reduces ovary abscission and that the presence of exogenous GA3 in unpollinated ovaries also suppresses abscission. Evidence is also presented that pollination and GAs do not, as is generally assumed, suppress ovary abscission through the reactivation of cell division.