Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L

[17-¹³C,³H]-Labeled gibberellin A₂₀ (GA₂₀), GA₅, and GA₁ were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). ¹³C-Labeled GA₂₉, GA₈, GA₅, GA₁, and 3-epi-GA₁, as well as unmetabolized [¹³C]GA₂₀, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-¹³C, ³H]GA₂₀ to all three genotypes. ¹³C-Labeled GA₈ and 3-epi-G₁, as well as unmetabolized [¹³C]GA₁, were identified by GC-SIM from feeds of [17-¹³C, ³H]GA₁ to all three genotypes. From feeds of [17-¹³C, ³H]GA₅, ¹³C-labeled GA₃ and the GA₃-isolactone, as well as unmetabolized [¹³C]GA₅, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-¹³C, ³H]GA₅ to [¹³C]GA₁, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA₂₀, as follows: (a) GA₂₀ → GA₁ → GA₈; (b) GA₂₀ → GA₅ → GA₃; and (c) GA₂₀ → GA₂₉. The in vivo biogenesis of GA₃ from GA₅, as well as the origin of GA₅ from GA₂₀, are conclusively established for the first time in a higher plant (maize shoots).     

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.     

Identification of six endogenous gibberellins in spinach shoots

Analysis of highly purified extracts from spinach shoots by combined gas chromatography-mass spectrometry has demonstrated the presence of six 13-C-hydroxylated gibberellins (GAs): GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, and GA₂₉. The major GAs were GA₁₇, GA₁₉, and GA₂₀, whereas the other three GAs occurred in trace amounts. Structural considerations suggest that the six GAs identified in spinach are related in the following metabolic sequence: GA₅₃ → GA₄₄ → GA₁₉ → GA₁₇ → GA₂₀ → GA₂₉.     

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

Cell-free extracts capable of converting [¹⁴C]-labeled gibberellins (GAs) were prepared from spinach (Spinacia oleracea L.) leaves. [¹⁴C]-labeled GAs, prepared enzymically from [¹⁴C]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: GA₁₂ → GA₅₃ → GA₄₄ → GA₁₉ → GA₂₀. The pH optima for the enzymic conversions of [¹⁴C]GA₅₃, [¹⁴C]GA₄₄ and [¹⁴C]GA₁₉ were approximately 7.0, 8.0, and 6.5, respectively. These three enzyme activities required Fe²⁺, α-ketoglutarate and O₂ for activity, and ascorbate stimulated the conversion of [¹⁴C]GA₅₃ and [¹⁴C]GA₁₉. 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 GA₅₃ and GA₁₉ are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing GA₄₄ 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 GA₂₀ content of spinach plants in LD than in SD.     

Characterization of the oligosaccharides from lipid-linked oligosaccharides of mung bean seedlings

Lipid-linked oligosaccharides were synthesized with the particulate enzyme preparation from mung bean (Phaseolus aureus) seedlings in the presence of GDP-[¹⁴C] mannose. The oligosaccharides were released from the lipids by mild acid hydrolysis and purified by several passages on Biogel P-4 columns. Five different oligosaccharides were purified in this way. Based on their relative elution constants (K_d) compared to a variety of standard oligosaccharides, they were sized as (mannose-acetylglucosamine) Man₇GlcNAc₂, Man₅GlcNAc₂, Man₃GlcNAc₂, Man₂GlcNAc₂, and ManGlcNAc₂. These oligosaccharides were treated with endoglucosaminidase H and α- and β-mannosidase, and the products were examined on Biogel P-4 columns. They also were subjected to a number of chemical treatments including analysis of the reducing sugar by NaB³H₄ reduction, methylation analysis, and in some cases acetolysis. From these data, the likely structures of these oligosaccharides are as follows: E, Manβ-GlcNAc-GlcNAc; D, Manα1→3Manβ-GlcNAc-GlcNAc; C, Manα1→2Manα1→3Manβ-GlcNAc-GlcNAc; B, Manα1→2Manα1→2Manα1→ 3(Manα1→6)Manβ-GlcNAc-GlcNAc; and A, Manα1→2Manα1→ 2Manα1→3(Manα1→ [Manα1→6]Manα1→6) Manβ-GlcNAc-GlcNAc. The synthesis of the Man₇GlcNAc₂ was greatly diminished when tunicamycin (10 μg/ml) was added to the incubation mixtures.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

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.     

Expression of gibberellin mutations in fruits of Pisum sativum L.

The gibberellin (GA) economy of young pea (Pisum sativum L.) fruits was investigated using a range of mutants with altered GA biosynthesis or deactivation. The synthesis mutation lh-2 substantially reduced the content of both GA₄ and GA₁ in young seeds. Among the other synthesis mutations, ls-1, le-1 and le-3, the largest reduction in seed GA₁ content was only 1.7-fold (le-1), while GA₄ was not reduced in these mutants, and in fact accumulated in some experiments (compared with the wild type). Mutation sln appeared to block the step GA₂₀ to GA₂₉ in young pods and seeds, but not as strongly as in older seeds. Mutations ls-1, le-1 and le-3 markedly reduced pod GA₁ levels, but pod elongation was not affected. After feeds of [¹³C,³H]GA₂₀ to leaves, the pods contained ¹³C,³H-labelled GA₂₀, GA₁, GA₂₉ and GA₈₁, and the seeds, [¹³C,³H]GA₂₀ and [¹³C,³H]GA₂₉. These findings are discussed in relation to recent suggestions regarding the role and origin of GA₁ in pea fruits. Received: 6 June 1997 / Accepted: 15 July 1997     

Reversible conjugation of gibberellins in situ in maize

Gibberellins [³H]GA₄ (1.33 Curies per millimole) and [³H]GA₂₀ (2.36 Curies per millimole) were injected into the shanks of maize (Zea mays L.) cobs during rapid grain filling and mature seeds were subsequently harvested. Extracts of mature, dry seeds from 1980 feeds yielded only 20 to 30% of the ³H radioactivity in acidic, ethyl acetate-soluble form, and this was principally associated with the precursor, with lesser amounts of the major metabolite, [³H]GA₁ (putative identification based on sequential SiO₂ partition, and gradient-eluted reverse-phase C₁₈ high performance liquid chromatography [HPLC]). Most of the radioactivity in the dry seeds was associated with compounds having partition characteristics of, and co-chromatographing on, sequential SiO₂ partition and reverse-phase HPLC with glucosyl conjugates of the precursors (GA₄ or GA₂₀) and their probable major metabolite (GA₁). The majority of conjugate associated with the precursor GA₄ eluted coincidental with GA₄ glucoside. Subsequent acid or enzymic hydrolysis (β-glucosidase or cellulase) yielded the free GAs, putative identification being based on isocratic HPLC of each ³H-labeled conjugate → hydrolysis → isocratic HPLC of the ³H-labeled hydrolysate. Upon imbibition of the seeds, radioactivity associated with the conjugate fraction decreased; concomitantly, statistically significant increases in levels of free [³H]GA-like compounds were observed. Although the specific ratios of GA-like and GA-glucosyl conjugate-like substances varied substantially across years, hybrids, and even, in different plants from the same hybrid, this `reversible conjugation' (i.e. apparent conjugation during seed maturation followed by release of the GA moiety during germination), was reproducible for [³H]GA₂₀ in seed from two maize hybrids produced over 2 years.     

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 identification and metabolism of GA9 and its metabolites in seed coats and shoots of pea,line G2

[³H]gibberellin A₉ was applied to shoots or seed parts of G2 pea to produce radiolabeled metabolites. These were used as markers during purification for the recovery of endogenous GA₉ and its naturally occurring metabolites. GA₉ and its metabolites were purified by HPLC, derivatized and examined by GC-MS. Endogenous GA₉, GA₂₀, GA₂₉ and GA₅₁ were identified in pea shoots and seed coats. GA₅₁-catabolite and GA₂₉-catabolite were also detected in seed coats. GA₇₀ was detected in seed coats following the application of 1 g of GA₉. Applied [³H]GA₉ was metabolized through both the 13-hydroxylation and 2-hydroxylation pathways. Labeled metabolites were tentatively identified on the basis of co-chromatography on HPLC with endogenous compounds identified by GC-MS. In shoots [³H]GA₅₁ and [³H]GA₅₁-catabolite were the predominant metabolites after 6 hrs, but by 24 hrs there was little of these metabolites remaining, while [³H]GA₂₉-catabolite and an unidentified metabolite predominated. In seed coats [³H]GA₅₁ was the initial product, later followed by [³H]GA₅₁-catabolite and an unidentified metabolite (different from that in shoots), with lesser amounts of [³H]GA₂₀, [³H]GA₂₉ and [³H]GA₂₉-catabolite. [³H]GA₇₀ was a very minor product in both cases. [³H]GA₉ was not metabolized by pea cotyledons.Edited by T.J. Gianfagna.Author for correspondence     

Polyamine metabolism and its relation to response of the aleurone layers of barley seeds to gibberellic Acid

Polyamine metabolism and its relation to the induction of α-amylase formation in the aleurone layers of barley seeds (Hordeum vulgare cv Himalaya) in response to gibberellic acid (GA₃) has been investigated. A high-performance liquid chromatographic system has been employed for qualitative and quantitative analyses of putrescine (Put), cadaverine (Cad), spermidine (Spd), spermine (Spm), and agmatine (Agm).

Active polyamine metabolism occurs in the aleurone cells of deembryonate barley half seeds during imbibition. The aleurone layers isolated from fully imbibed half seeds contain about 880 nanomoles of Put, 920 nanomoles of Spd, and 610 nanomoles of Spm as free form per gram tissue dry weight while the levels of Cad and Agm are relatively low. The polyamine levels do not change significantly in the aleurone layers in response to added GA₃ (1.5 micromolar) during the 8-hour lag period of the growth substance-induced formation of α-amylase. Also, the polyamine levels are not altered by the presence of abscisic acid (3 micromolar) which inhibits the enzyme induction by GA₃. Kinetic studies show that both applied [U-¹⁴C]ornithine and [U-¹⁴C]arginine are primarily incorporated into Put during 2 hours of incubation, but the incorporation is not significantly affected by added GA₃. Additionally, added GA₃ does not affect the uptake and turnover of [1,4-¹⁴C]Put, nor does it affect the conversion of Put → Spd or Spd → Spm. Treatment of the aleurone layers with GA₃ for 2 hours results in no significant changes in the total activities or the specific activities of ornithine decarboxylase and arginine decarboxylase.

Experiments with polyamine synthesis inhibitors demonstrate that the level of Spd in the aleurone layers could be substantially reduced by the presence of methylglyoxal-bis(guanylhydrazone) (MGBG) during imbibition. MGBG treatment does not affect in vivo incorporation of [8-¹⁴C] adenosine into ATP. The lower the level of Spd the less α-amylase formation is induced by added GA₃. The reduction of GA₃-induced α-amylase formation by MGBG treatment can be either completely or partially overcome by added Spd, depending upon the concentration of MGBG used in the imbibition medium. The results indicate that the early action of GA₃, with respect to induction of α-amylase formation in barley aleurone layers, appears to be not on polyamine metabolism. However, polyamines, particularly Spd, may be involved in regulation of the growth substance-dependent enzyme induction.

     

Partial Purification and Characterization of the Gibberellin A(20) 3beta-Hydroxylase from Seeds of Phaseolus vulgaris

The GA₂₀ 3β-hydroxylase present in immature seeds of Phaseolus vulgaris has been partially purified and characterized. The physical characteristics of the enzyme are similar to those of the GA 2β-hydroxylases present in mature and immature seeds of Pisum sativum. It is acid-labile, hydrophobic, and of M_r 45,000. The enzyme catalyzes the synthesis of GA₁, GA₅, and GA₂₉ from GA₂₀. Activity is dependent upon the presence of Fe²⁺, ascorbate, 2-oxoglutarate, and oxygen. 2-Oxoglutarate does not function as a cosubstrate; in the presence of the enzyme, succinate is not a reaction product.     

Comparison of the levels of six endogenous gibberellins in roots and shoots of spinach in relation to photoperiod

This communication describes the distribution of gibberellins (GAs) in roots and shoots of spinach in relation to photoperiod. From previous work (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626) shoots were known to contain GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, and GA₂₉. We now show by combined gas chromatography—mass spectrometry that roots contain GA₄₄, GA₁₉, and GA₂₉. Trace amounts of GA₅₃ were detected by combined gas chromatography—selected ion current monitoring. Neither GA₁₇ nor GA₂₀ were detected in root extracts. Analysis by the d-5 corn bioassay also showed no effect of photoperiodic treatment on the levels of GA-like substances in root extracts. Both phloem and xylem exudates had patterns of GA-like activity similar to those found in shoots and roots, respectively. Moreover, foliar application of [³H]GA₂₀ resulted in the transport of label from the shoot to the roots. Over half of the label in the roots represented unmetabolized [³H]GA₂₀, indicating that part of the GA₂₀ in the phloem is transported to the roots. Consequently, if GA₂₀ is made in, or transported to the roots, it is rapidly metabolized in that organ. This is a clear indication that regulation of GA metabolism is greatly different in roots and shoots.     

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA₁ or its immediate precursors. The primary site of synthesis of GA₂₀ from GA₁₉ was immature leaflets and tendrils, and the synthesis of bioactive GA₁ and its inactive catabolite GA₈ occurred predominantly in stem tissue. GA₂₉, the inactive catabolite of GA₂₀, was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA₁, GA₁₉, or even GA₂₀. During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [³H]GA₁ (and [³H]GA₂₉) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA₂₀ 3β-hydroxylase protein.     

Effect of Photoperiod on Metabolism of [H] Gibberellins A(1), 3-epi-A(1), and A(20) in Agrostemma githago L

To determine whether daylength influences the rate of metabolism of gibberellins (GAs) in the long-day (LD) rosette plant Agrostemma githago L., [³H]GA₂₀ and [³H]GA₁ were applied under short day (SD) and LD. Both were metabolized faster under LD than under SD. [³H]GA₂₀ was metabolized to a compound chromatographically identical to 3-epi-GA₁. [³H]GA₁ was metabolized to two acidic compounds, the major metabolite having chromatographic properties similar to, but not identical with GA₈. [³H]3-epi-GA₁ applied to plants under LD was metabolized much more slowly than was [³H]GA₁, and formed a very polar metabolite which did not partition into ethyl acetate at pH 2.5. Very polar metabolites were also formed after the feeds of [³H]GA₂₀ and [³H]GA₁. It was not possible to characterize these very polar compounds further because of their apparent instability. The results obtained suggest that in Agrostemma GA₂₀ is the precursor of 3-epi-GA₁, but there is at present no evidence indicating the precursor of GA₁.     

The role of gibberellins A1 and A3 in fruit growth of Pisum sativum L. and the identification of gibberellins A4 and A7 in young seeds

Gibberellins A₁ and A₃ are the major physiologically active gibberellins (GAs) present in young fruit of pea (Pisum sativum L.). The relative importance of these GAs in controlling fruit growth and their biosynthetic origins were investigated in cv. Alaska. In addition, the non-13-hydroxylated active GAs, GA₄ and GA₇, were identified for the first time in young seeds harvested 4 d after anthesis, although they are minor components and are not expected to play major physiological roles. The GA₁ content is maximal in seeds and pods at 6 d after anthesis, the time of highest growth-rate of the pod (Garcia-Martinez et al. 1991, Planta 184: 53–60), whereas gibberellic acid (GA₃), which is present at high levels in seeds 4–8 d after anthesis, has very low abundance in pods. Gibberellins A₁₉, A₂₀ and A₂₉ are most concentrated in seeds at, or shortly after, anthesis and their abundance declines rapidly with development, concomitant with the sharp increase in GA₁ and GA₃ content. Application of GA₁ or GA₃ to the leaf subtending an emasculated flower stimulated parthenocarpic fruit development. Measurement of the GA content of the pods at 4 d after anthesis indicated that only 0.002–0.5% of the applied GA was transported to the fruit, depending on dose. There was a linear relationship between GA₁ content and pod weight up to about 2 ng · (g FW)⁻¹, whereas no such correlation existed for GA₃ content. The concentration of endogenous GA₁ in pods from pollinated ovaries is just sufficient to give the maximum growth response. It is concluded that GA₁, but not GA₃, controls pod growth in pea; GA₃ may be involved in early seed development. The distribution of GAs within the seeds at 4 d post anthesis was also investigated. Most of the GA₁, GA₈, GA₁₉, GA₂₀ and GA₂₉ was present in the testa, whereas GA₃ was distributed equally between testa and endosperm and GA₄ was localised mainly in the endosperm. Of the GAs analysed, only GA₃ and GA₂₀ were detected in the embryo. Metabolism experiments with intact tissues and cell-free fractions indicated compartmentation of GA biosynthesis within the seed. Using ¹⁴C-labelled GA₁₂, GA₉, 2,3-didehydroGA₉ and GA₂₀ as substrates, the testa was shown to contain 13-hydroxylase and 20-oxidase activities, the endosperm, 3β-hydroxylase and 20-oxidase activities. Both tissues also produced 16,17-dihydrodiols. However, GA₁ and GA₃ were not obtained as products and it is unlikely that they are formed via the early 13-hydroxylation pathway. [¹⁴C]gibberellin A₁₂, applied to the inside surface of pods in situ, was metabolised to GA₁₉, GA₂₀, GA₂₉, GA₂₉-catabolite, GA₈₁ and GA₉₇, but GA₁ was not detected. Gibberellin A₂₀ was metabolised by this tissue to GA₂₉ and GA₂₉-catabolite. Received: 23 July 1996 / Accepted: 2 September 1996     

Influence of photoperiod on seed development in the genetic line of peas G2 and its relation to changes in endogenous gibberellins measured by combined gas chromatography — Mass spectrometry

When apical senescence in the genetic line of peas G2 was prevented by short days fruit development was also found to be retarded. The levels of GA₂₀ and GA₂₉ in cotyledons and pods grown under long or short days were measured by gas chromatography — mass spectrometry multiple ion monitoring using extracts derivatised with deuterated trimethylsilyl groups as internal standards. The levels of GA₂₀ but not GA₂₉, were increased by short days. Conventional gas chromatography — mass spectrometry showed that relative to GA₂₉ the levels of GA₁₉, the other GA identified in G2 cotyledons, were also increased in short days. The levels of GA₂₀ in the pods were highest during the main phase of pod growth early in fruit development.Abbreviations GA_n gibberellin A_n - GC/MS gas chromatography — mass spectrometry - MIM multiple ion monitoring - Me methyl ester - SIM single ion monitoring - TIC total ion current - TMS trimethylsilyl ether - TLC thin layer chromatography - TTLC instant thin layer chromatography     

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.