Gibberellins in Immature Seeds of Canavalia

Two novel gibberellins, GA₂₁ (I) and GA₂₂ (II), were isolated from immature seeds of sword bean, Canavalia gladiata DC. The isolation procedure of these substances as well as their growth-promoting effects on dwarf maize mutants d₁ and d₅, rice, cucumber and dwarf peas (Progress No. 9) are described.

The structures of two new gibberellins, GA₂₁ and GA₂₂, isolated from immature seeds of sword bean, were determined as 4a_α, 7_α-dihydroxy-8-methylenegibbane-1, 1, 10β-tricarboxylic acid 1→4a lactone (II) and 4a_α, 7_α-dihydroxy-l β-hydroxymethyl-8-methylenegibb-2-ene-1_α, 10β-dicaboxylic acid 1→4a lactone (IV), respectively, on the basis of chemical and physicochemical studies.     

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings

The levels of gibberellin A₁ (GA₁), GA₂₀, GA₁₉, GA₈, GA₂₉ and GA₈₁ (2-epiGA₂₉) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA₁; GA₈ and GA₁₉, but more GA₂₀, GA₂₉, and GA₈₁ than did those of tall plants. It is concluded that the partial block in 3β-hydroxylation of GA₂₀ to GA₁ is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA₁ levels. The reduction of the pool size of GA₁₉ in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA₂₀, GA₂₉, and GA₈₁ levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.     

Microbial Production of Plant Gibberellins and Related Compounds from ent-Kaurene Derivatives in Gibberella fujikuroi

ent-15α-Hydroxykaurenoic acid (8) was synthesized and fed to a mycelium suspension of Gibberella fujikuroi in the presence of 1-n-decylimidazole, a gibberellin biosynthesis inhibitor. The metabolites included 15β-hydroxy GA₂₄, GA₄₅ (GA of Pyrus communis), 15β-hydroxy GA₁₅ and 15β-hydroxy GA₂₅. Microbial production of 12α-hydroxy GAs from ent-12β-hydroxykaurene is also described.     

Effect of temperature regimes on gibberellin levels in thermosensitive dwarf apple trees

Orchard-grown dwarf apple (Malus domestica Borkh.) trees selected from a hybrid population were propagated by tissue culture but had a growth pattern similar to standard cv. Golden Delicious plants when grown at constant 27°C instead of the expected dwarf pattern of growth. Shoot elongation was markedly reduced, with or without gibberellin A₁ (GA₁) or GA₄ treatment, when trees were grown in an environment where day temperature was maintained at 35°C for 2 h in a ramped regime (night 20°C day ramped to 35°C, held for 2 h and ramped down to 20°C night over a 14-h photoperiod). Application of GA₁ or GA₄ partially overcame growth retardation resulting from prior paclobutrazol treatment of both standard and dwarf trees grown at constant 27°C and of standard trees grown in the ramped environment. However, these GAs had no effect on paclobutrazol-treated or untreated dwarfs grown in the ramped regime. Gas chromatography-mass spectrometry with labelled internal standards was used to quantify GA₁, GA₃, GA₈, GA₁₉, GA₂₀ and GA₂₉ in extracts from standard and dwarf plants grown either at a constant 27°C or in a 20-30-20°C ramped temperature regime. Standard plants, which elongate quite rapidly in either environment, had similar levels of these GAs in both temperature regimes. The slowly growing dwarfs in the ramped temperature environment contained three times more GA₁₉ than the rapidly elongating dwarfs grown at 27°C. The concentrations of the other GAs were reduced to ca 40% or less in plants grown in the ramped temperature regime compared with those grown at 27°C. These data suggest that shoot elongation of dwarf plants is sensitive to elevated temperatures both as a result of reduced responsiveness to GAs and because of a reduction in the concentration of GA₁, apparently as a result of a lower rate of conversion of GA₁₉ to GA₂₀. It is possible that the altered GA metabolism may be a consequence of the change in GA sensitivity.     

The quantitative relationship between gibberellin A1 and internode growth in Pisum sativum L.

The metabolism and growth-promoting activity of gibberellin A₂₀ (GA₂₀) were compared in the internode-length genotypes of pea, na le and na Le. Gibberellin A₂₉ and GA₂₉-catabolite were the major metabolites of GA₂₀ in the genotype na le. However, low levels of GA₁, GA₈ and GA₈-catabolite were also identified as metabolites in this genotype, confirming that the le allele is a leaky mutation. Gibberellin A₂₀ was approximately 20 to 30 times as active in promoting internode growth of genotype na Le as of genotype na le. However, the levels of the 3-hydroxylated metabolite of GA₂₀, GA₈ (2-hydroxy GA₁), were similar for a given growth response in both genotypes. In each case a close linear relationship was observed between internode growth and the logarithm of GA₈ levels. A similar relationship was found on comparing GA₂₀ metabolism in the three genotypes le ^d, le and Le. The former mutation results in a more severe dwarf phenotype than the le allele (which has previously been shown to reduce the 3-hydroxylation of GA₂₀ to GA₁). These results indicate that GA₂₀ has negligible intrinsic activity and support the contention that GA₁ is the only GA active per se in promoting stem growth in pea.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography     

Purification and partial characterization of a gibberellin 2β-hydroxylase fromPhaseolus vulgaris

A gibberellin 2β-hydroxylase has been purified from mature seeds ofPhaseolus vulgaris. The enzyme is of molecular weight 36,000 and has the characteristics of a dioxygenase; the cofactors areα-ketoglu-tarate, Fe²⁺ and ascorbate, and activity is stimulated by catalase. The V_max of the enzyme is 6.86 nmole h^?1 mg^?1, and the Km values for [1,2-³H₂]GA₁ andα-ketoglutarate are 0.085 μM and 21 μM, respectively. The purified enzyme preparation catalyzes hydroxylation of GA₁, GA₄, GA₉, and GA₂₀ but exhibits a marked preference for the 3-hydroxylated gibberellins as substrate.     

Ontogenetic variation in levels of gibberellin A1 in Pisum

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

Identification of a Novel Gibberellin (GA85) in Very Young Seedlings of Brassica campestris cv. Tobin

A new gibberellin (GA) was identified from extracts of cotyledons of 7 day-old canola seedlings (Brassica campestris cv. Tobin). This GA is 12α-hydroxy-GA₁ and has been assigned the trivial name of GA₈₅. Isolation was monitored by the Tan-ginbozu dwarf rice micro-drop assay after each high-performance liquid chromatography (HPLC) step. Identification was based on Kovats retention index (KRI) and the mass spectrum of the methyl ester, trimethylsilyl ether (MeTMSi) derivative after analysis by gas chromatography-mass spectrometry (GC-MS) in comparison with an authentic sample of 12α-hydroxy-GA₁. Based on quantitation by the dwarf rice micro-drop assay, GA₈₅ is one of the major biologically active GAs in cotyledons of young canola seedlings.     

Activity of Antheridiogen from the Fern Anemia phyllitidis in Three Flowering Plant Bioassays

Pure samples of the antheridiogen of Anemia phyllitidis (A_An) were tested for their ability to affect the growth of dwarf corn (d₅) and lettuce seedlings, and to influence α-amylase production by barley half-seeds. Stimulation of dwarf corn growth and barley amylase production was, on a molar basis, from 1/2 to 1/250 that given by GA_3. In lettuce, A_An had a synergistic effect with low levels of GA₃; alone, A_An was inhibitory or ineffective. Therefore, in addition to having a close chemical resemblance to gibberellin, A_An induces similar, but not identical physiological responses in flowering plants as well as ferns.     

The Structure of Gibberellin A23 and the Biological Properties of 3,13-Dihydroxy C20-Gibberellins

Two aldehydic C₂₀-gibberellins, L-2 and L-4, were isolated from the immature fruits of yellow lupine (Lupinus luteus L.). L-2 was shown to have the structure II and named gibberellin A₂₃. L-4 was identified as gibberellin A₁₉(VI). Two new C₂₀-gibberellins, tentatively called 3,13-dihydroxy GA₁₅(IV) and 13-hydroxy GA₁₅(VIII), were derived from gibberellins, A₂₃ and A₁₉, respectively. The biological activities of four 3,13-dihydroxy C₂₀-gibberellins-GA₁₈(I), GA₂₃(II), GA₂₈(III) and 3,13-dihydroxy GA₁₅(IV), which were isolated from the fruits except for 3,13-dihydroxy GA₁₅—were compared in six gibberellin bioassays.     

Isolation and Structure of a Novel C20 Gibberellin in Bamboo Shoots

A new gibberellin C₂₀H₂₆O₆, tentatively named “bamboo gibberellin”, was isolated from water extract of bamboo shoots (Phyllostachys edulis) through successive purification procedures: countercurrent distribution, charcoal column chromatography, and silicic acid adsorption and partition chromatography. Its structure was established as lβ-methyl-4aα-fromyl-7α-hydroxy-8-methylenegibbane-lα,10β-dicarboxylic acid (VI) from analysis on infrared, NMR and mass spectra of its methyl ester.     

Use of an Acylcyclohexanedione Growth Retardant, LAB 198 999, to Determine Whether Gibberellin A(20) Has Biological Activity per se in Dark-Grown Dwarf (le) Seedlings of Pisum sativum

Dwarf (le⁵⁸³⁹) seedlings of Pisum sativum respond to gibberellin A₂₀ (GA₂₀) in the dark, although the same dosage of GA₂₀ applied to light-grown le⁵⁸³⁹ seedlings elicits no growth response. The acylcyclohexanedione growth retardant, LAB 198 999, which is known to inhibit gibberellin oxidation and in particular 3β-hydroxylation such as the conversion of GA₂₀ to GA₁, also inhibits the growth response of dark-grown dwarf (le⁵⁸³⁹) seedlings to GA₂₀. Thus, the biological activity of GA₂₀ in the dark appears to be a consequence of its conversion to GA₁, even though it is known from studies with light-grown seedlings that the le mutation reduces the conversion of GA₂₀ to GA₁.     

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     

Biological activity of some conjugated gibberellins

Summary The biological activity of several gibberellin (GA) conjugates was studied and compared with that of the corresponding free GAs. The following conjugates were included: O(3)--d-glucopyranosides of GA₁, GA₃ and GA₄; O(13)--d-glucopyranosides of GA₁, GA₃ and GA₅; O(13)--d-glucopyranosyl-GA₅--d-glucopyranosyl ester; GA₃--d-glucopyranosyl ester and GA₃--d-glucopyranosyl ester; N-GA₃-oyl-glycine, its methyl ester, N-GA₃-oyl-glycylglycine, and N-GA₃-oyl-proline. All compounds were synthesized chemically but some of them are known to occur as endogenous plant products, or to be formed in plants upon application of a free GA. Activity was determined in the dwarf pea, dwarf corn, dwarf rice, and lettuce hypocotyl bioassays. The GA conjugates were found to posses different relative activities depending on the chemical structure, the bioassay system, and the site of application (shoot or roots). It is concluded that the activity of GA conjugates as measured in different bioassays is based upon the ability of plant enzymes and possibly of certain microorganisms to hydrolyze glucosidic, glucosyl ester, and amide-like linkages.Gibberellins-XLVIII. Part XLVII=Adam et al. (1976b)     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Comparison of growth and gibberellin concentrations in shoots from orchard-grown standard and thermosensitive dwarf apple trees

Apple (Malus domestica Borkh.) trees were propagated by budding from selected fully grown hybrids that ranged in height from 1.5 to 8 m. The growth and development of the selected budded trees after 7 years in the orchard was similar to that of the parent trees. Additional grafting studies showed that the dwarfism was not associated with the roots. Differences in photosynthetic activity and associated processes were not related to the size difference between tissue culture-propagated orchard-grown standard cv. Golden Delicious and dwarf hybrid trees. Applications of GA₃ did not stimulate elongation of shoots of dwarf trees. Shoots of both standard and dwarf trees started to develop in mid-April when they contained nearly the same amounts of GA₁, GA₃ and GA₈, but standard shoots contained higher concentrations of GA₁₉, GA₂₀ and GA₂₉. On 2 June standard shoots were almost three times the length of dwarf shoots, but the number of leaves and area per leaf were nearly the same. The relative amounts of GAs on 12 May and 2 June for both plant types were similar to those on 20 April, except that GA₁₉, GA₂₀, GA₁ and GA₂₉ levels had declined. Gibberellin levels in standard shoots declined further between 2 and 22 June, after which there was no further shoot elongation or production of new leaves. Between 2 June and the end of the growing season, when summer temperatures were high, dwarf shoots continued to elongate slowly and to develop new leaves, which expanded little. During this time, the GA₁₉ content of dwarf shoots nearly doubled, whereas the amounts of GA₂₀, GA₁, GA₂₉ and GA₈ declined. By the end of the season, standard shoots were 40 cm in length with 20 leaves and dwarf shoots were 28 cm in length, but with 36 leaves. High summer temperatures appear to induce loss of GA-responsiveness in orchard-grown dwarf trees and to cause a reduced rate of conversion of GA₁₉ to GA₂₀ in these genotypes.     

Gibberellin metabolism in excised lettuce hypocotyls: Response to GA9 and the conversion of [3H]GA9

Elongation growth and gibberellin (GA₉) metabolism in excised hypocotyls of lettuce (Lactuca sativa L. cv. Arctic) were investigated. Exogenously supplied GA₉ stimulates elongation of hypocotyl sections and this response is intermediate between that elicited by GA₁ or GA₂₀ and GA_4/7 mixture. Although uptake of radioactivity from [³H]GA₉ increases with time, this gibberellin does not accumulate in the tissue but is rapidly converted to a compound with HPLC properties resembling those of [³H]GA₂₀. After 2 h incubation in [³H]GA₉, the presumptive GA₂₀ represents 90% of the acidic ethyl acetate-soluble radioactivity in the tissue. Radioactivity is also associated with an acidic butanol-soluble fraction containing two components resolvable by HVE. The major component is similar in electrophoretic properties to a GA-glucosyl ether while the other compares to a GA-glucosyl ester. Conversion of [³H]GA₉ to its [³H]GA₂₀-like metabolite is reduced by addition of carrier GA₉ or GA_4/7 at concentrations as low as 1 M, while GA₁, GA₃ and L-proline are without effect. Formation of the GA₂₀-like compound can be blocked by the addition of 2,2-dipyridyl, and this inhibitory effect of dipyridyl can be reversed by addition of Fe²⁺. At 200 M dipyridyl, elongation growth as well as [³H]GA₉ metabolism are reduced by 80%. The relationship of the metabolism of GA₉ to the growth response is discussed.Abbreviations AB butanol-soluble - AE ethyl-acetate-soluble - GA gibberellin - GA₁, GA₄ gibberellin A₁, gibberellin A₄, etc. - TLC thin layer chromatography - HPLC high performance liquid chromatography - HVE high voltage electrophoresis     

Gibberellin biosynthesis in cell-free extracts from developing Cucurbita maxima embryos and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthetic pathways from GA₁₂-aldehyde, GA₁₂ and GA₅₃ were investigated in cell-free systems from developing embryos of Cucurbita maxima L. Gibberellin A₁₂-aldehyde and GA₁₂ were converted to GA₂₅, putative 12α-hydroxyGA₂₅, GA₁₃ and GA₃₉ as main products. Minor products were GA₄, GA₃₄ and, when GA₁₂ was the substrate, putative 12α-hydroxyGA₁₂. The intermediates GA₁₅ and GA₂₄ accumulated at low protein concentrations. The influence of various factors on GA₁₂ metabolism was examined. At low 2-oxoglutarate and ascorbate concentrations, or at acid pH, 3β-hydroxylated products predominated, whereas with increasing 2-oxoglutarate and ascorbate concentrations, or at neutral pH, the yield of 12α-hydroxylated GAs increased. Gibberellin A₅₃ was metabolised mainly to the C₂₀-GAs GA₄₄, GA₁₉, GA₁₇, GA₂₃ and GA₂₈, with the C₁₉-GAs GA₂₀, GA₁ and GA₈ as minor products. Only C₁₉-GAs were 2β-hydroxylated, which is a main characteristic of the embryo systems. In addition to GA₁₃, GA₂₅, GA₃₉, GA₄₃, GA₄₉, GA₅₈, GA₇₄, 12α-hydroxyGA₂₅ and GA₃₉ 3-isovalerate, which were known previously from embryos of C. maxima, GA₁, GA₄, GA₁₇, GA₂₈, GA₃₇, GA₃₈, GA₄₈, GA₈₅, 12α-hydroxyGA₃₇ and putative 12α-hydroxyGA₄₃ were identified as endogenous components by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Evidence for putative 2β-hydroxyGA₂₈ and GA₂₃ was also obtained but it was less conclusive because of contamination.     

Thermomorphogenic and Photomorphogenic Control of Stem Elongation in Fuchsia Is Not Mediated by Changes in Responsiveness to Gibberellins

Stem elongation in Fuchsia × hybrida was influenced by cultivation at different day and night temperatures or in different light qualities. Internode elongation of plants grown at a day (25°C) to night (15°C) temperature difference (DIF+10) in white light was almost twofold that of plants grown at the opposite temperature regime (DIF−10). Orange light resulted in a threefold stimulation of internode elongation compared with white light DIF−10. Surprisingly, internode elongation in orange light was similar for plants grown at DIF−10 and DIF+10. Flower development was accelerated at DIF−10 compared with DIF+10 in both white and orange light. To examine whether the effects of DIF and light quality on shoot elongation were related to changes in gibberellin metabolism or plant sensitivity to gibberellins (GAs), the stem elongation responses of paclobutrazol-treated plants to applied gibberellins were determined. In the absence of applied gibberellins paclobutrazol (>0.32 μmol plant⁻¹) strongly retarded shoot elongation. This inhibition was nullified by the application of about 10–32 nmol of GA₁, GA₄, GA₉, GA₁₅, GA₁₉, GA₂₀, GA₂₄, or GA₄₄. The results are discussed in relation to possible effects of DIF and light quality on endogenous gibberellin levels and gibberellin sensitivity of fuchsia and their effects on stem elongation. Received October 4, 1997; accepted December 17, 1997