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.     

In vitro glucosylation of gibberellins

In maturing fruits ofPhaseolus coccineus a soluble glucosyltransferase activity occurs which converts gibberellins into their O-glucosides. The enzyme glucosylates GA₃ and structurally closely related gibberellins (GA₇ and GA₃₀) to their 3-O-glucosides by transfer of glucose preferentially from UDP-glucose. From cell suspension cultures ofLycopersicon peruvianum cytosolic glucosyltransferases were isolated which in the presence of UDP-glucose converted GA₇ and GA₉ to the corresponding glucosyl esters. In both cases numerous other gibberellins failed to serve as substrates. Thus, the enzymes are UDP-glucose: gibberellin glucosyltransferases of considerable substrate specificity.     

Gibberellin-like substances in root exudate of Vitis vinifera

Summary The levels of gibberellin (GA)-like activity in the root exudate of two seedless varieties of Vitis vinifera were examined by the barley endosperm assay, and compared with levels determined for other parts of the plant. That activity was due to GA-like substances was confirmed with dwarf-5 corn.When acidic, ethyl acetate soluble GA-like substances from sap and leaf extracts were chromatogrammed on thin layers of silica gel in chloroform/ethyl acetate/formic acid (50:50:1), activity moved to the same Rf as GA₃ and GA₁ (Rf 0.05–0.25). However, substances inhibitory to the barley endosperm assay were detected in both sap and leaf extracts. In the above solvent system the inhibitor(s) co-chromatogrammed with a GA₁/GA₇ mixture, and with abscisin II. The GA-like activity co-chromatogrammed with GA₃ on paper developed in isopropanol/ammonia/water (10:1:1).Calculations on the rate of gibberellin movement from the roots seemed to be compatible with levels of activity in the leaves, although these levels could also be a reflection of the general gibberellin level in the plant.The relevance of the findings is discussed.     

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A₃, A_{4 + 7} (GA₃, GA_{4 + 7}), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA₃ or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA₃, GA_{4 + 7} or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.     

Enhanced production of gibberellin A4 (GA4) by a mutant of Gibberella fujikuroi in wheat gluten medium

Mutants of Gibberella fujikuroi with different colony characteristics, morphology and pigmentation were generated by exposure to UV radiation. A mutant, Mor-189, was selected based on its short filament length, relatively high gibberellin A₄ (GA₄) and gibberellin A₃ (GA₃) production, as well as its lack of pigmentation. Production of GA₄ by Mor-189 was studied using different inorganic and organic nitrogen sources, carbon sources and by maintaining the pH of the fermentation medium using calcium carbonate. Analysis of GA₄ and GA₃ was done by reversed-phase high-performance liquid chromatography and LC-MS. The mutants of G. fujikuroi produced more GA₄ when the pH of the medium was maintained above 5. During shake flask studies, the mutant Mor-189 produced 210 mg l^?1 GA₄ in media containing wheat gluten as the nitrogen source and glucose as the carbon source. Fed-batch fermentation in a 14 l agitated fermenter was performed to evaluate the applicability of the mutant Mor-189 for the production of GA₄. In 7-day fed-batch fermentation, 600 mg l^?1 GA₄ were obtained in the culture filtrate. The concentration of GA₄ and GA₃ combined was 713 mg l^?1, of which GA₄ accounted for 84% of the total gibberellin. These values are substantially higher than those published previously. The present study indicated that, along with maintenance of pH and controlled glucose feeding, the use of wheat gluten as the sole nitrogen source considerably enhances GA₄ production by the mutant Mor-189.     

Similarities between gibberellins and related compounds in inducing Acid phosphatase and reducing sugar release from barley endosperm

Barley endosperm halves release acid phosphatase in response to several gibberellins and gibberellin precursors. Seed halves incubated with 10⁻⁷m GA₃ at 29° begin to release phosphatase after 11 hr and release it for another 26 hr in response to GA₃. After 37 hr, the rate of release slows to that of seed halves incubated without GA₃. GA₃ is active at 10⁻¹⁰m and maximally active at 10⁻⁷m. Comparative activity of 12 gibberellins and gibberellin precursors is GA₁ = GA₃ > GA₂ > GA₄ = GA₇ > GA₅ = GA₁₃ > GA₁₄ > GA₈ = GA₉ > (−)kaurenoic acid > (−)-kaurene. These compounds show the same order of activity and approximately the same relative activity in inducing reducing sugar release as in inducing phosphatase activity. The activity of each compound increases with its presumed position in a biosynthetic pathway leading from kaurene to GA₃. This correlation suggests that activity may be a reflection of the efficiency of conversion to an active form within the seed half.     

Contents and recovery of gibberellins in monoecious and gynoecious cucumber plants

Diffusates from seedlings and root exudates from 6-week-old plants of a monoecious line of cucumber, Cucumis sativus L., contained considerably higher levels of gibberellin-(GA-) like substances than did those from plants of an isogenic gynoecious line. Most of the GA-like activity was found in a chromatogram region typical of GA₁ and GA₃; some activity, particularly in root exudates, appeared also at an R_F similar to that of GA₄ and GA₇.

When seedlings were treated with ³H-labeled GA₁, more radioactivity was found in the diffusates from monoecious seedlings than from gynoecious ones. The same was true of biological activity in root diffusates from older plants which had been treated with gibberellin A₄₊₇.

In conjunction with evidence present in literature, these results support the idea that endogenous GAs play a part in the regulation of sex expression in cucumber, relatively high levels favoring the formation of staminate flowers.

     

Dormancy break of celery (Apium graveolens L.) seeds by plant derived smoke extract

Seed dormancy of a highly-dormant cultivar of celery (Apium graveolens L.) was broken by combinations of plant-derived smoke extract or N⁶-benzyladenine (BA) and gibberellins A_4/7 (GA_4/7) in the dark at temperatures between 18 and 26°C. A less dormant cultivar which responded to GA_4/7 alone showed no additional response to smoke extract or BA. Neither smoke extract nor BA affected either cultivar in the dark in the absence of GA_4/7. The partial dormancy-breaking effect of short exposures to red-light was also enhanced by smoke extracts in this highly-dormant cultivar. The results suggest that smoke extracts act in a similar way to cytokinins, by enhancing gibberellin activity in the celery seed system.Abbreviations BA N⁶-benzyladenine - GA_4/7 A₄ and A₇ gibberellin mixture     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

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.     

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.     

Localization of Stem Elongation Control in Cucumis sativus L

Reciprocal grafts, and applications of gibberellin (GA) and indoleacetic acid (IAA) were used to localize the site of control for stem elongation in cucumber (Cucumis sativus L.). Dwarf and tall plants were reciprocally grafted to determine influence of stems and roots on stem elongation. At 21 days there were no significant differences in length between stems grafted to their own roots and those grafted to roots of the other type. GA₃, GA₄₊₇, and IAA were applied to seedlings with and without live apical buds. Seedlings with live apical buds responded to level of added GA, but not to added IAA. GA₄₊₇ was more effective than GA₃. Hypocotyls of tall plants responded more to both GA treatments than did those of the dwarves when both types had live apical buds. When either GA₄₊₇ or IAA was applied to seedlings with dead apical buds, elongation of the hypocotyl responded to level of the growth regulator, but there was no difference in response between the dwarf and tall plants.     

Gibberellins and Heterosis in Maize : II. Response to Gibberellic Acid and Metabolism of [H]Gibberellin A(20)

Two maize inbreds, CM7 and CM49, and CM7 × CM49, their F₁ hybrid (which displayed significant heterosis), were examined with regard to response to exogenous gibberellin A₃ (GA₃), and in their ability to metabolize GA₂₀, a native GA of maize. The leaf sheath elongation response to GA₃ was far greater for the imbreds than for their hybrid. The inbreds also displayed significant elongation of the leaf blades in response to GA₃, whereas the hybrid was unaffected. Promotion of cell division in the leaf sheath of CM7 and the hybrid was effected by GA₃, but no promotion of cell elongation was observed in CM49, even though significant leaf sheath elongation occurred. Shoot dry weight of both inbreds was significantly increased by GA₃, but response by the hybrid in this parameter was slight and variable. Root dry weight of CM7 was significantly increased by GA₃, but was unchanged in CM49 and the hybrid. Thus, inbred shoot dry weight increases effected by GA₃ were not at the expense of the root system. Rapid metabolism of [2,3-³H]GA₂₀ occurred in all genotypes, although genotypic differences were observed. The hybrid had the highest rates of metabolism to GA glucosyl conjugate-like substances. Oxidative metabolism was also fastest in the hybrid, followed by CM7, and slowest in CM49, the slowest-growing inbred. Thus, rate of GA₂₀ metabolism is under genetic control in normal (i.e. not dwarfed) maize genotypes. These results, taken together with previous reports that the hybrid has significantly enhanced levels of endogenous GA-like substances, suggest that GA play a role in the expression of heterosis in maize.     

Interaction of Gibberellic Acid and Indole-3-acetic Acid in the Growth of Excised Cuscuta Shoot Tips in Vitro

Gibberellic acid (GA₃) induced a marked elongation of 2.5-centimeter shoot tips of Cuscuta chinensis Lamk. cultured in vitro. In terms of the absolute amount of elongation, this growth may be the largest reported for an isolated plant system. The response to hormone was dependent on an exogenous carbohydrate supply. The hormone-stimulated growth was due to both cell division and cell elongation. The growth response progressively decreased if GA₃ was given at increasingly later times after culturing, but the decreased growth response could be restored by the application of indole-3-acetic acid (IAA) to the apex. Explants deprived of GA₃ gradually lost their ability to transport IAA basipetally, but this ability was also restored by auxin application. The observations are explained on the basis that: (a) the growth of Cuscuta shoot tip in vitro requires, at least, both an auxin and a gibberellin; and (b) in the absence of gibberellin the cultured shoot tip explants lose the ability to produce and/or transport auxin.     

Competition for in vitro [h]gibberellin a(4) binding in cucumber by substituted phthalimides: comparison with in vivo gibberellin-like activity

Certain N-substituted phthalimides (NSPs) have gibberellin (GA)-like activity in a number of GA bioassays. The interaction between representative NSPs and a protein fraction from cucumber (Cucumis sativus L.) hypocotyls that has GA-binding characteristics consistent with those expected of GA receptors was studied. Analysis of in vitro equilibrium saturation data indicated the presence of only one class of high affinity [³H]GA₄ binding sites (K_d ~ 30 nanomolar, n = 0.25 picomole per milligram of protein). In the presence of 6 or 60 micromolar 1-[3-chlorophthalimido]-cyclohexanecarboximide (AC-94,377), the K_d for [³H]GA₄ increased, whereas the maximum number of saturable [³H]GA₄ binding sites did not change significantly. The dissociation of [³H]GA₄ from its binding sites was complex and was best described by a bi-exponential equation. AC-94,377 did not affect the rates of [³H]GA₄ dissociation from its binding sites. These results implied that AC-94,377 and [³H]GA₄ compete for binding to the same sites. A correlation was observed between the activity of over 20 NSPs in the cucumber hypocotyl bioassay and their in vitro affinity for the GA binding sites. Our observations lend further support to the notion that certain GA binding proteins in cucumber cytosol are GA receptors and also provide a molecular explanation for the GA-like in vivo activity of some NSPs.     

Radioactive gibberellin a(5) and its metabolism in dwarf peas

Radioactive gibberellin A₅ (³H-GA₅) was synthesized from gibberellic acid. When it was applied to dwarf peas grown in the dark, an average of 3% was converted to another acid gibberellin within 48 hours. The biological activity of the metabolite did not account for the response to applied GA₅. GA₅ is therefore assumed to be biologically active per se.³H-GA₅ did not appear to form a stable complex with a macromolecule in pea shoots. When injected into dwarf pea pods, ³H-GA₅ was readily metabolized by maturing seed to more water-soluble substances and to two other acidic compounds. This metabolism continued even throughout germination of the seed without reconversion of the metabolites to GA₅. It is concluded that “bound” GA₅ plays no part in the germination of dwarf pea seeds.     

Enzyme formation and release by isolated barley aleurone layers

Aleurone layers, with testa attached, were prepared from degermed, decorticated barley with the aid of a fungal enzyme preparation. The preparations appeared intact under the scanning electron microscope. By using antibiotics only in an early stage preparations were obtained uncontaminated by micro-organisms and which, when incubated under optimal conditions with gibberellic acid, GA₃, produced near-maximal amounts of α-amylase. The enzyme accumulated in the tissue before it was released into the incubation medium. Daily replacement of the incubation medium, containing GA₃, depressed the quantity of α-amylase produced. α-Amylase was also produced in response to gibberellins GA₁, GA₄ and GA₇ and, to a much lesser extent, helminthosporol and helminthosporic acid. A range of other substances, reported elsewhere to induce α-amylase formation, failed to do so in these trials. At some concentrations, glutamine marginally enhanced the quantity of enzyme formed during prolonged incubations. It is confirmed that α-glucosidase occurs in the aleurone layer and embryo of ungerminated barley, and increases in amount during germination. GA₃ is shown to enhance this increase. When embryos arc burnt, to prevent gibberellin formation, no rise in α-glucosidase levels occurs unless GA₃ is supplied to the grains. As the activity of α-glucosidase and other enzymes have been determined as ‘α-amylase’ by some assay methods, their alterations in activity in response to GA₃ necessitates a re-evaluation of the evidence for de novo) synthesis of α-amylase in aleurone tissue.     

Effect of nine different gibberellins on stem elongation and flower formation in cold-requiring and photoperiodic plants grown under non-inductive conditions

Summary The effect of gibberellins A₁ through A₉ on stem elongation and flower formation in five plants was tested. The plants wereMyosotis alpestris and a biennial strain ofCentaurium minus (cold-requiring plants),Silene armeria andCrepis parviflora (long-day plants), andBryophyllum crenatum (a long-short-day plant). The two former plants were maintained on non-inductive temperatures and long days, the three latter on short days, InMyosotis, flower formation was only obtained with GA₇ and GA₁, the latter being relatively less active. InCentaurium GA₃ was the most effective, followed by GA₁, GA₄ and GA₇ and perhaps GA₅ and GA₉. InSilene, flower formations was induced only by GA₇. InCrepis, the most effective gibberellins were GA₄ and GA₇, inBryophyllum, GA₃, GA₄ and GA₇. Thus, the different gibberellins exhibited considerable differences in their activity with respect to flower induction, and different plants exhibited in this respect certain specific differences in their sensitivity to the various gibberellins. Except inCrepis, flower initiation as a result of gibberellin treatment was always preceded by substantial stem or internode elongation; however, the correlation between the effect of the different gibberellins on stem elongation and flower induction was not in all cases complete. No correlation of the flower-inducing and elongation-promoting activity with the chemical structure of the different gibberellins could be recognized.With 2 Figures in the TextWork in part supported by the National Science Foundation, grants G-16408 and G-17483.     

Gibberellin retards chlorophyll degradation during senescence of Paris polyphylla

Chlorophyll (Chl) degradation was found to be related to the endogenous gibberellin (GA) content in shoots during senescence in the perennial plant Paris polyphylla var. yunnanensis (Franch.). Treatment with gibberellic acid (GA₃) significantly increased the content of endogenous GAs (GA₄ + GA₇), retarded the senescence of shoots, and the degradation of proteins and Chl. Chlorophyllase, Mg-dechelation and peroxidase activities increased more in control plants than in those treated with GA₃. GA₃ treatment also protected lipoxygenase activity, which decreased significantly in control plants.