Identification of Pea Gibberellins by Studying [C]GA(12)-Aldehyde Metabolism

Experiments were designed to test the hypothesis that the labeled products recovered from plant tissue incubated with [¹⁴C]GA₁₂-7-aldehyde ([¹⁴C]GA₁₂ald) would serve as appropriate [¹⁴C]markers for the recovery of naturally-occurring gibberellins (GAs). The [¹⁴C]GA₁₂ald (about 200 millicuries per millimole) was synthesized from pumpkin endosperm using [4,5-¹⁴C]mevalonic acid. It was added to the adaxial surface of isolated pea cotyledons at 22 days after flowering. Products recovered after 0.5 and 4.0 hour incubations yielded four major peaks which were separated by high performance liquid chromatography (HPLC). These products were purified by multiple-column HPLC using on-line radioactivity detection. They were then added as [¹⁴C]markers to two unlabeled pea extracts. In general, preparative HPLC followed by further HPLC purification resulted in a single UV-absorbing peak co-eluting with each [¹⁴C]marker. These [¹⁴C] and UV-absorbing peaks were shown to contain GA₅₃, GA₄₄, GA₂₀, GA₁₉, and GA₁₇ by GC-MS. The finding of GA₅₃ is novel; all others have previously been found in pea. Endogenous GAs of pea were thus readily detected using [¹⁴C]GA₁₂ald metabolites as [¹⁴C]markers to recover naturally occurring GAs suggesting that the method may be applicable in detecting naturally occurring GAs in other species.     

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).     

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.     

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.     

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.     

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.     

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.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

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     

Changes in Endogenous Gibberellins and the Metabolism of [H]GA(4) after Geostimulation in Shoots of the Oat Plant (Avena sativa)

The recovery from “lodging,” or bending over, by shoots of 42-day-old Avena sativa plants is controlled primarily by a negatively geotropic differential growth of the lower halves of the p-1 node-pulvinus and the base of the p-1 internode, relative to the upper halves. Although geostimulation causes a significant reduction in p-1 internode length, dry matter accumulation in the p-1 node-pulvinus is increased, apparently at the expense of the sheath. Recovery to an angle of 30° is associated with changes in endogenous gibberellin-like substances (GAs), and in differential metabolism of applied [³H]GA₄ (1.4 Curie per millimole). Although geostimulation depressed total GAs (relative to upright plant parts) to 0.40 and 0.13 for node-pulvini and sheaths, respectively, it increased them 2-fold for internodes. Within the plant part geostimulation increased GAs (relative to upper halves) 29- and 7-fold in lower halves of node-pulvini and internodes, respectively, but reduced GAs to 0.3 in lower halves of sheaths. At age 42 days a GA_4/7-like (nonpolar) substance predominates, with lesser amounts of a GA₃-like (polar) substance. Native GAs of Avena include GA₃, GA₄, and GA₇. Geostimulation enhanced the ratio of nonpolar to polar GAs for both halves of internodes, but tended to depress it for sheaths and nodepulvini.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

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.     

Effects of Chilling and ABA on [H]Gibberellin A(4) Metabolism in Somatic Embryos of Grape (Vitis vinifera L. x V. rupestris Scheele)

Previous work has indicated that changes in gibberellin (GA) metabolism may be involved in chilling-induced release from dormancy in somatic embryos of grape (Vitis vinifera L. × V. rupestris Scheele). We have chilled somatic embryos of grape for 2, 4, or 8 weeks, then incubated them with [³H]GA₄ (of high specific activity, 4.81 × 10¹⁰ becquerel per millimole) for 48 hours at 26°C. Chilling had little effect on the total amount of free [³H]GA-like metabolites formed during incubation at 26°C, but did change the relative proportions of individual metabolites. The amount of highly water-soluble [³H] metabolites formed at 26°C decreased in embryos chilled for 4 or 8 weeks. The concentration of endogenous GA precursors (e.g., GA₁₂ aldehyde-, kaurene-, and kaurenoic acid-like substances) increased in embryos chilled for 4 or 8 weeks. Treatment with abscisic acid (ABA) (known to inhibit germination in grape embryos) concurrent with [³H]GA₄ treatment at 26°C, reduced the uptake of [³H] GA₄ but had little effect on the qualitative spectrum of metabolites. However, in the embryos chilled for 8 weeks and then treated with ABA for 48 hours at 26°C, there was a higher concentration of GA precursors than in untreated control embryos. Chilled embryos thus have an enhanced potential for an increase in free GAs through synthesis from increased amounts of GA precursors, or through a reduced ability to form highly water-soluble GA metabolites (i.e., GA conjugates or polyhydroxylated free GAs).     

Metabolism of [3H]gibberellin A5 in developing pharbitis nil seeds

The native gibberellin A₅ (GA₅), as [1-³H]GA₅ (3.2 Ci/mmol) was fed to seed capsules (0.58 μCi/capsule) of Pharbitis nil cv Violet at the 2-week stage of development, and its metabolism in the seeds was investigated after 43 hr. Extractable radioactivity in free GA metabolites was 38%, with 56% in GA glucosyl conjugate-like substances. Only 2.5% of the extractable radioactivity remained as [³H]GA₅. Tentative identifications, based on comparisons with authentic standards after sequential chromatography on silica gel partition column → gradient-eluted C₁₈ HPLC → isocratic-eluted C₁₈ HPLC-radiocounting (RC), showed that [³H]GA₅ was converted to at least six free GAs, GA₁, GA₃, GA₆, GA₈, GA₂₂, GA₂₉, a GA₅ methyl ester-like metabolite, and at least twelve GA glucosyl conjugate-like substances, GA₅-glucoside (GA₅-G), GA₅-glucosyl ester (GA₅-GE), GA₁-O(3)-G, GA₁-O(13)-G, GA₁-GE, GA₃-O(3)-G, GA₃-O(13)-G, GA₃-GE, GA₆-G or GE, GA₈-O(2)-G, GA₂₂-G or GE and GA₂₉-O(2)-G. After lower specific activity feeds of [1,2-³H]GA₅ (74 mCi/mmol; 0.1 μCi/capsule) at approximately the same stage of development, the presence of GA₁, GA₃, GA₅, GA₆, GA₈ and GA₂₉ was further confirmed by sequential (after C₁₈ HPLC-RC) capillary gas chromatography-selected ion monitoring (GC-SIM), using six characteristic ions. However, for GA₂₂ only a trace of the parent ion was present at the appropriate retention time.     

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.     

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.     

Synthesis of [2H]gibberellins from steviol using the fungus Gibberella fujikuroi

[²H]Steviol (ent-13-hydroxykaur-16-en-19-oic acid) was synthesized from steviol acetate norketone (ent-13-acetoxy-16-oxo-17-norkauran-19-oic acid) by the Wittig reaction using (methyl-d₃)triphenylphosphonium bromide. A mixture of steviol analogs was produced containing from one to four ²H/molecule. [²H]Steviol was fed to strain LM-45-399 of the fungus Gibberella fujikuroi which was grown on synthetic medium (ICI, 0% N) in the presence of the growth retardant CCC. [²H]GA₁, [²H]GA₁₈, [²H]GA₂₃ and [²H]GA₅₃ were isolated from the fungal medium after 4 days. This strain converted steviol to 13-hydroxy GAs in the highest yields of the four Gibberella strains tested, and in amounts suitable for metabolic studies with higher plants.     

Metabolism of ent-kaurenol-[17-14C], ent-kaurenal-[17-14C] and ent-kaurenoic acid-[17-14C] by germinating Hordeum distichon grains

Subcellular fractions from germinated barley embryos, chloroplast preparations and whole germinating barley grains are able to carry out the conversions ent-kaurenol → ent-kaurenal → ent-kaurenoic acid → ent-hydroxykaurenoic acid, the initial steps of the biosynthetic pathway to gibberellins. Whole grains, and chloroplasts to a slight extent, incorporate radioactivity from ent-kaurenol-[17-¹⁴C] and ent-kaurenoic acid-[17-¹⁴C] into materials with similar but distinct properties from the gibberellins GA₁, GA₃, GA₄ and GA₇.     

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport,in stelar tissues of Phaseolus coccineus roots

Summary Movement of both [³H]GA₁ and [¹⁴C]GA₃ through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [³H]GA₁ in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [¹⁴C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [³H]GA₁ injected into cotyledons moved into and acropetally along the seedling root.     

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.