Metabolism of Tritiated Gibberellin A(9) by Shoots of Dark-grown Dwarf Pea, cv. Meteor

Tritium-labeled gibberellin A₉ (³H-GA₉) was metabolized by etiolated shoots of dwarf pea (Pisum sativum cv. Meteor) to GA₂₀, GA₁₀, 2,3-dihydro-GA₃₁, and a number of highly polar, acidic GA-like substances. Identifications were made by gasliquid radiochromatography and combined gas chromatography-mass spectrometry. Kinetic studies showed that GA₃₀ and 2,3-dihydro-GA₃₁ were produced within 5 hours following ³H-GA₉ application to pea shoots. The polar GA-like substances were produced between 5 and 10 hours after ³H-GA₉ application. Levels of GA₁₀ increased with time, and since no GA₁₀ was produced during the purification procedures, GA₁₀ was, in all probability, produced from ³H-GA₉ within the plant tissue. The radioactive interconversion products produced by pea from ³H-GA₉ have chromatographic properties similar to biologically active GA-like substances present in etiolated shoots of dwarf pea. Large scale applications of ³H-GA₉ with very low specific activity to etiolated pea shoots showed that the radioactivity of the interconversion products was correlated exactly with biological activity as assayed by dwarf rice (Oryza sativa cv. Tan-ginbozu).     

In vivo Binding of Gibberellin A(1) in Dwarf Pea Epicotyls

Binding of [(3)H]gibberellin A(1) (GA(1)) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [(3)H]GA(1) at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/-)abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 10(-6) molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively.     

Metabolism of [H]Gibberellin A(5) by Immature Seeds of Apricot (Prunus armeniaca L.)

Immature seeds of apricot (Prunus armeniaca L.) were fed the native gibberellin A₅ (GA₅) as 1- and 1,2-[³H]GA₅ (5.3 Curies per millimole to 16 milliCuries per millimole) at doses (42 nanograms to 10.6 micrograms per seed) 2 to 530 times the expected endogenous level. After 4 days of incubation, seeds were extracted and free [³H]GA-like metabolites were separated from the highly H₂O-soluble [³H]metabolites. For high specific activity feeds the retention times (Rts) of radioactive peaks were compared with Rts of authentic GAs on sequential gradient-eluted → isocratic eluted reversed-phase C₁₈ high performance liquid chromatography (HPLC) -radiocounting (RC). From high substrate feeds (530 and 230 × expected endogenous levels) HPLC-RC peak groupings were subjected to capillary gas chromatography-selected ion monitoring (GC-SIM), usually six characteristic ions. The major free GA metabolites of [³H] GA₅ were identified as GA₁, GA₃, and GA₆ by GC-SIM. The major highly water soluble metabolite of [³H]GA₅ at all levels of substrate GA₅ had chromatographic characteristics similar to authentic GA₁-glucosyl ester. Expressed as a percentage of recovered radioactivity, low substrate [³H]GA₅ feeds (2 × expected endogenous level) yielded a broad spectrum of metabolites eluting at the Rts where GA₁, GA₃, GA₅ methyl ester, GA₆, GA₂₂, GA₂₉ (17, 14, 1.6, 7, 1.1, 0.5%, respectively) and GA glucosyl conjugates of GA₁, GA₃, GA₅, and GA₈ (33, 11, 1, 0.1%, respectively) elute. Metabolites were also present at Rts where GA glucosyl conjugates of GA₆ and GA₂₉ would be expected to elute (8 and 0.1%, respectively). Only 5% of the radioactivity remained as GA₅. Increasing substrate GA₅ levels increased the proportion of metabolites with HPLC Rts similar to GA₁, GA₆, and especially GA₁ glucosyl ester, primarily at the expense of metabolites with HPLC Rts similar to GA₃, GA₃-glucosyl ester, and a postulated conjugate of GA₆. There was evidence that high doses of substrate GA₅ induced new metabolites which often, but not always, differed from GA₁, GA₃, and GA₆ in HPLC Rt. These same metabolites, when analyzed by GC-SIM yielded m/e ions the same as the M⁺ and other characteristic m/e ions of the above GAs, albeit at differing GC Rt and relative intensities.     

Biosynthesis of (-)-Kaurene in Cell-free Extracts of Immature Pea Seeds

Mevalonate-¹⁴C was incorporated into (—)-kaurene-¹⁴C in cell-free extracts of immature pea (Pisum sativum L.) seeds. The identification of ¹⁴C-product as (—)-kaurene was based on: A) comparison with authentic (—)-kaurene on thin-layer and gas-liquid chromatography; and B) oxidation of ¹⁴C-product and (—)-kaurene with osmium tetroxide to form the common derivative kaurane-16,17-diol. The enzyme system is heat labile and is dependent upon ATP and Mg²⁺ or Mn^2-, with Mn²⁺ being a more effective activator than Mg²⁺. The reaction rate was proportional to enzyme concentration in reaction mixtures containing 0.45 to 1.8 mg protein n/ml, and was linear with time through 120 minutes in standard reaction mixtures. Enzyme preparations from immature seeds of tall and dwarf peas appeared to synthesize (—)-kaurene at the same rate. Synthesis of (—)-kaurene was readily inhibited by Amo-1618. (2-Chloroethyl)-trimethylammonium chloride (CCC) also inhibited (—)-kaurene synthesis; however, approximately 1000-fold higher concentrations of CCC were required to evoke the same percentages of inhibition as Amo-1618.     

Gibberellin Metabolism in Maize (The Stepwise Conversion of Gibberellin A12-Aldehyde to Gibberellin A20

The stepwise metabolism of gibberellin A12-aldehyde (GA12-aldehyde) to GA20 is demonstrated from seedling shoots of maize (Zea mays L.). The labeled substrates [13C,3H]GA12-aldehyde, [13C,3H]GA12, [14C4]GA53, [14C4/2H2]GA44, and [14C4/2H2]GA19 were fed individually to dwarf-5 vegetative shoots. Both [13C,3H]GA12-aldehyde and [13C,3H]GA12 were also added individually to normal shoots. The labeled metabolites were identified by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA12-aldehyde was metabolized to GA53-aldehyde, GA12, GA53, GA44, and GA19; GA12 was metabolized to 2[beta]-hydroxy-GA12, GA53, 2[beta]-hydroxyGA53, GA44, 2[beta]-hydroxyGA44, and GA19; GA53 was metabolized to GA44, GA19, GA20, and GA1; GA44 was metabolized to GA19; and GA19 was metabolized to GA20. These results, together with previously published data from this laboratory, document the most completely defined gibberellin pathway for the vegetative tissues of higher plants.     

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.     

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).     

Effects of Light, Abscisic Acid, and N-Benzyladenine on the Metabolism of [H]Gibberellin A(4) in Seeds and Seedlings of Lettuce, cv. Grand Rapids

Gibberellin A₄ (GA₄) can substitute for light in the germination of Grand Rapids lettuce seeds. Seeds imbibed in [³H]GA₄ do not convert this to other GAs prior to, or immediately following, visible germination: thus GA₄ alone can promote radicle expansion. Abscisic acid inhibited [³H]GA₄-induced germination, but did not significantly affect [³H]GA₄ uptake or metabolism during germination. ⁶N-benzyladenine overcame the inhibitory effect of abscisic acid and increased [³H]GA₄ uptake, although radicle emergence was delayed somewhat.     

Gibberellin A20 in seed of Pisum sativum L., cv. Alaska

Summary The main gibberellin in immature seed of Pisum sativum L., cv. Alaska, is identified as GA₂₀ by GC-MS. GA₉ may also be present.     

Isolation and Structure of Gibberellin A18 from Immature Seeds of Lupinus luteus

A new gibberellin, tentatively called Lupinus gibberellin I, was isolated from young yellow lupin seeds. It has been shown to have structure (X), and now named gibberellin A₁₈.     

Evidence for Phytochrome Regulation of Gibberellin A(20) 3beta-Hydroxylation in Shoots of Dwarf (lele) Pisum sativum L

The effect of light on the dwarfing allele, le, in Pisum sativum L. was tested as the growth response to gibberellins prior to or beyond the presumed block in the gibberellin biosynthetic pathway. The response to the substrate (GA₂₀), the product (GA₁), and a nonendogenous early precursor (steviol) was compared in plants bearing the normal Le and the deficient lele genotypes in plants made low in gibberellin content genetically (nana lines) or by paclobutrazol treatment to tall (cv Alaska) and dwarf (cv Progress) peas. Both genotypes responded to GA₁ under red irradiation and in darkness. The lele plants grew in response to GA₂₀ and steviol in darkness but showed a much smaller response when red irradiated. The Le plants responded to GA₂₀ and steviol in both light and darkness. The red effects on lele plants were largely reversible by far-red irradiation. It is concluded that the deficiency in 3β-hydroxylation of GA₂₀ to GA₁ in genotype lele is due to a Pfr-induced blockage in the expression of that activity.     

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).     

Metabolism of Tritiated Gibberellins in d-5 Dward Maize: II. [H]Gibberellin A(1), [H]Gibberellin A(3), and Related Compounds

After 30 minutes of incubation of young leaf sections of d-5 maize (Zea mays L.) in [³H]gibberellin A₁ ([³H]GA₁), the metabolite [³H]GA₈ was present in significant amounts, with a second metabolite, [³H]GA₈-glucose ([³H]GA₈-glu), appearing soon after. A third [³H]GA₁ metabolite, the polar uncharacterized conjugate [³H]GA₁-X, took more than 1 hour to appear. The protein synthesis inhibitor cycloheximide inhibited the production of all [³H]GA₁ metabolites, indicating a possible protein synthesis requirement for [³H]GA₁ metabolism.     

Interaction of Gibberellin A3 and Ancymidol in the Growth and Cell-Wall Extensibility of Dwarf Pea Roots

Effects of ancymidol (Anc) and gibberellin A₃ (GA₃) on rootgrowth, osmotic concentration and cell-wall extensibility ofthe root were investigated in the gibberellin-sensitive cultivarof dwarf pea, Little Marvel. Anc strongly suppressed elongationof both shoots and roots in darkness. Although the elongationof shoots of this dwarf cultivar was severely retarded in thelight, it was repressed still further by Anc. GA₃ promoted elongationof shoots both in the presence and in the absence of Anc, whereasit reversed suppression of root elongation by Anc. The concentrationof GA₃ required for the recovery of root elongation was lowerthan that required for the promotion of shoot elongation. Treatmentwith Anc led to increased thickening of roots with increasednumbers of cells per cross section and lateral expansion ofcells in the cortex. GA₃ had little effect on the osmotic concentration of cell sapobtained from root segments. Anc-treated roots did not respondto acid solutions by elongation, whereas GA₃-treated roots respondednormally to such solutions. Anc suppressed but GA₃ enhancedthe cell-wall extensibility of roots as measured in vivo andin vitro. These results indicate that a low concentration of gibberellinplays a role in normal elongation of roots by maintaining theextensibility of the cell wall in this gibberellin-sensitivedwarf pea. (Received January 17, 1994; Accepted July 15, 1994)     

Metabolism of Tritiated Gibberellins in d-5 Dwarf Maize: I. In Excised Tissues and Intact Dwarf and Normal Plants

Metabolism of [³H]gibberellin A₁ ([³H]GA₁) was followed in intact seedlings and excised apices and leaf tissue of both dwarf and normal (tall) plants of d-5 maize (Zea mays L.). The three metabolites produced were tentatively identified as [³H]GA_s, [³H]GA_s-glucoside ([³H]GA_s-glu), and [³H]GA₁-X, an unknown.     

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.     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.