Effects of a Plant-Growth Regulator, Prohexadione, on the Biosynthesis of Gibberellins in Cell-Free Systems Derived from Immature Seeds

Inhibition of the biosynthesis of gibberellins by prohexadione,3,5-dioxo-4-propionylcyclo-hexanecarboxylic acid, was studiedwith cell-free systems derived from immature seeds of Cucur-bitamaxima, Phaseolus vulgaris and Pisum sativum. Prohexadione,at a concentration of 10–⁴ M, inhibited C-7 oxidationof GA₁₂-aldehyde, C-20 oxidation of GA₁₅, conversion of C₂₀-gib-berellinsto C₁₉-gibberellins, 3ß-hydroxylation, 2,3-dehydrogenationof GA²⁰, 2,3-epoxidation of GA₅ and 2ß-hydroxylationof GA₉ and GA₂₀. The 3ß-hydroxylase activity appearedto be more sensitive to prohexadione than were the C-20 oxygenaseand the 2ß-hydroxylase activities. The conversionof mevalonic acid to GA₁₂-aldehyde and the 13-hydroxylationof GA₁₂ were not affected by prohexadione at a concentrationof 3 ? 10^–4 M. All of the steps inhibited by prohexadioneare oxidation steps catalyzed by soluble enzymes that require2-oxoglutarate, Fe²⁺ and oxygen, and all of them occur distalto the synthesis of GA₁₂-aldehyde in the biosynthesis of gibberellins. (Received April 4, 1990; Accepted September 14, 1990)     

Functional Significance of Acid Phosphatase Distribution During Embryo Development in Pisum sativum L

The distribution of P₁, ester P and acid-insoluble nucleic acidP has been studied in relation to acid phosphatase activity(EC 3. 1. 3. 2) in the component parts of developing pea seeds(Pisum sativum L.). Despite the favourable pH of the liquidcontents of the embryo sac (pH 5.5), only very low acid phosphataseactivity was detected in this fluid (c. 0.01 units per seed).Potential substrates for phosphatase action were in fact absentfrom the secretion, the only form of P present being P_i, inconcentrations up to 8 mM. The data support the hypothesis thatthe high acid phosphatase activities which develop in the seed-coatsare involved in regulating the supply of P as P₁ to the developingembryo. Pisum sativum L., pea, embryo development, acid phosphatase, phosphorus, seed-coats, seed development     

Gibberellic Acid Controls the Secretion of Acid Phosphatase in Aleurone Layers and Isolated Aleurone Protoplasts of Avena fatua

The role of gibberellic acid (GA₃) in controlling the secretion(across the plasma membrane) and release (through the cell wall)of acid phosphatase (E.C. 3.1.3.2 [EC] .) from Avena aleurone layershas been investigated. Evidence from this comparative studywith intact aleurone layers and isolated aleurone protoplastsreveals that the secretion of acid phosphatase is under GA₃control. The mechanism underlying secretion and release of theenzyme from aleurone cells is discussed. Key words: Avena fatua, Acid phosphatase, Aleurone protoplasts, Gibberellic acid, Secretion     

Metabolism of [14C]GA20 in a Cell-Free System from Developing Seeds of Phaseolus vulgaris L.

The metabolism of [¹⁴C]GA₂₀ during seed maturation of Phaseolusvulgaris was studied using cell-free systems from embryos rangingin age from 10 DAF (day after flowering) to 24 DAF. Enzyme preparationsfrom very immature seeds actively converted GA₂₀ to GA¹, GA₅and GA₆. The ratio of incubation products suggested the biosyntheticpathway of GA₂₀—GA₅—GA₆. Fluctuation in the levelsof endogenous C₁₉-GAs, namely GA₁, GA₄, GA₅, GA₆, GA₈, GA₉ andGA₂₀ was analyzed by GC-SIM using internal standards to compareenzyme activity with the levels of endogenous GAs. AlthoughGA₁, GA₄ and GA₆ showed maximum levels on 20 DAF, enzyme activitydecreased during seed maturation and showed weak activity on20 DAF. ¹Graduate student of the University of Tokyo, Department ofAgricultural Chemistry, Bunkyo-ku, Tokyo 113, Japan. ³Present address: Pesticides Research Laboratory, TakarazukaResearch Center, Sumitomo Chemical Co., Ltd., Takarazuka, Hyogo655, Japan. (Received December 17, 1987; Accepted March 30, 1988)     

Identification and Quantification of Cambial Region Hormones of Eucalyptus globulus

Gibberellins GA₁, GA₄, GA₈, GA₉, GA₁₉, GA₂₀, GA₂₉, GA₄₄, GA₈₁,indole-3-acetic acid (IAA) and abscisic acid (ABA) were identifiedin cambial region tissues of Eucalyptus globulus by comparingmass spectra and Kovats retention indices with those of authenticstandards. Using stable isotope labelled internal standardsGA₁₉, GA₂₀ and GA₄₄ were quantified at levels of 2–7 ng(g fr wt)^-1, other GAs were present at levels < 1 ng (g frwt)^-1. Levels of IAA and ABA ranged from 417–1, 140 ng(g fr wt)^-1 and 86–305 ng (g fr wt)^-1 respectively. Thepresence of brassinosteroid-like substances was also indicatedbased on activity in the rice seedling leaf inclination assay. (Received April 28, 1995; Accepted June 20, 1995)     

Poly(A) polymerase in Dormant Embryos of Triticum durum

Triticum durum‘Cappelli’ has a ‘relative’dormancy which can be broken by dry after-ripening at room temperature.The breakage of dormancy in the embryos of T. durum , is accompaniedby a decline in content and a different degree of synthesisof poly(A)⁺RNA. This work studies the activity of poly(A) polymerase(E.C. 2.7.7.19), the enzyme which permits polyadenylation. Anincrease in the activity of this enzyme in parallel with theenhanced rate of germination is revealed. Since poly(A) polymeraseactivity is the same in dormant and non-dormant dry embryos,it seems that the activity of the enzyme is not involved inthe breakage of dormancy. The use of cycloheximide and cordycepinshows the presence of enzymes with different origins: a storedenzyme and one bound to a long lived mRNA, present in dormantand non-dormant embryos, plus an enzyme bound to newly synthesizedmRNA which is mainly active in non-dormant embryos. Since dormancycould be the result of an interaction between hormones, thiswork analyses the effects of GA₃and ABA on poly(A) polymerase.GA₃enhanced poly(A) polymerase activity only in dormant embryoswhile ABA inhibited this activity only in non-dormant embryos.Cycloheximide applied to excised wheat embryos represses thestimulatory and inhibitory effects of GA₃and ABA, respectively.The hormone action on poly(A) polymerase activity is thus dependenton de novo protein synthesis. Results using cordycepin suggestthe presence of a stored mRNA for poly(A) polymerase, togetherwith hormonal regulation of enzyme activity at a translationallevel. Copyright 1999 Annals of Botany Company Triticum durum , wheat, dormancy breakage, poly(A) polymerase, GA₃, ABA, germination.     

Effects of Gibberellins on Seed Germination of Phytochrome-Deficient Mutants of Arabidopsis thaliana

Experiments were carried out to explore the involvement of gibberellins(GAs) in the light-induced germination of Arabidopsis thaliana(L.) Heynh, using wild type (WT) and phytochrome-deficient mutants(phyA, phyB and phyAphyB deficient in phytochrome A, B and Aplus B, respectively). Seed germination of WT and phytochrome-deficientmutants was inhibited by uniconazole (an inhibitor of an earlystep in biosynthesis of GA, the oxidation of ent-kaurene) andprohexadione (an inhibitor of late steps, namely, 2rß-and 3rß-hydroxylation). This inhibition was overcomeby simultaneous application of 10^-5 M GA₄. The relative activityof GAs for promoting germination of uniconazole-treated seedswas GA₄>GA₁=GA₉>GA₂₀. The wild type and the phyA and phyBmutants had an increased response to a red light pulse in thepresence of GA₁, GA₄, GA₉, GA₂₀ and GA₂₄ but there were no significantdifferences in activity of each GA between the mutants. Therefore,neither phytochrome A nor hytochrome B appears to regulate GAbiosynthesis from GA₁₂ to GA₄ during seed germination, sincethe conversion of GA₁₂ to GA₉ is regulated by one enzyme (GA20-oxidase). However, GA responsiveness appears to be regulatedby phytochromes other than phytochromes A and B, since the phyAphyBdouble mutant retains the photoreversible increased responseto GAs after a red light pulse. (Received February 13, 1995; Accepted July 11, 1995)     

Specific binding of gibberellin A1 to aleurone grain fractions from wheat endosperm

A subcellular fraction enriched in aleurone grains isolatedin glycerol from aleurone layers of wheat endosperm specificallyand reversibly bound GA₁-(³H). Specific binding of GA₁ to otherfractions including spherosomes, nuclei, mitochondria, and plasmamembranes was negligible. The K_d of binding to aleurone grainswas 1.5 µM and the number of specific binding sites 0.45pmoles per mg protein. The presence of Ca⁺⁺ ions was absolutelyrequired for binding. Abscisic acid which inhibits giberellinaction in vivo prevented specific GA₁-binding in vitro. GA₁-bindingto aleurone grains is important to the primary action of thehormone which may involve mobilization of reserves from thealeurone grain-spherosome complex for utilization in membranebiogenesis. ¹ Present address: Section of Cytology, Yale University Schoolof Medicine, New Haven, CT 06510, U.S.A. ³ Present address: Laboratoire de Biologie V?g?tale, Ecole NormaleSup?rieure, 24 rue Lhomond, 75231 Paris, France. (Received March 28, 1977; )     

Abscisic acid and the accumulation, biological activity and metabolism of four derivatives of [3H]gibberellin A1 in barley aleurone layers

Tritiated GA₁ and four of its synthetic derivatives were studiedin relation to their biological activity, uptake and metabolismby barley aleurone layers. Incubation was done in the presenceand absence of ABA. Tentative identification of some of themetabolites was made by TLC and GLC radiocounting of the metaboliteand its acid hydrolyzed derivative. Only GA₁ promoted -amylase synthesis. Uptake ranged from 20to 42%, varying with the derivative. ABA enhanced uptake of[³H]GA₁ and [³H]pseudoGA₁ and inhibited uptake of [³H]ketoGA₁the Wagner-Meerwein rearrangement product of [³H]GA₁ Uptakeof [³H]GA₁ methyl ester ([³H]GA₁-Me) and [³H]dihydroGA₁ wasunaffected by ABA. [³H]GA₁ was converted to an amphoteric GA₁ derivative ([³H]amphoGA₁)and [³H]GA₁-glycosyl ester. GA₁-Me was metabolized to four products,all of them GA₁ derivatives, including an apparent amphotericGA₁ derivative. DihydroGA₁ was quite stable; only one metabolitewas produced in sufficient yield to analyze. This product didnot cochromatograph with either of the expected acid hydrolyzedepimers of [³H]dihydroGA₁. [³H]ketoGA₁ was readily metabolizedto one product, probably the glycoside. [³H]pseudoGA₁ remainedessentially unmetabolized. Metabolism of all compounds testedwas not dramatically affected by ABA. Surprisingly, no metabolitesfrom hydroxylation at the 2-position were found. ¹ Present address: Monsanto Agricultural Co., 800 N. LindberghBlvd., St. Louis, MO 63166, U.S.A. (Received January 31, 1977; )     

Inoculation with Azospirillum lipoferum Affects Growth and Gibberellin Status of Corn Seedling Roots

Maize (Zea mays L., hybrid Cargill 147) seedlings, grown inaseptic conditions, were inoculated with three strains of Azospirillumlipoferum (Al op 33, Al iaa 320, and ATCC 29708) or culturedin different concentrations of indol-3-acetic acid (IAA) orgibberellin A₃ (GA₃). After 48 h, root length, root surfacearea, root dry weight, and shoot dry weight were measured inall treatments. Gibberellin content was evaluated in selectedroots of control and Azospirillum inoculated seedlings by gaschromatography-mass spectrometry-selected ion monitoring withthe use of deuterated gibberellins as internal standards. Thethree strains of A. lipoferum, IAA (2 ng ml^–1), and GA₃(40 to 400 pg ml^–1) significantly enhanced root growth.Improvement of root hair growth and density was obtained mainlywith A. lipoferum strain Al op 33 and GA₃ 40 pg ml^–1.GA₃ was identified by gas chromatography-mass spectrometry (byboth, full scan and selected ion monitoring) in a free acidfraction from roots of the seedlings inoculated with A. lipoferum.In the roots of the non inoculated seedlings GA₃ was found afterhydrolysis of a fraction expected to contain glucosyl conjugates. (Received April 26, 1993; Accepted September 27, 1993)     

Biological activities of rishitin, an antifungal compound isolated from diseased potato tubers, and its derivatives

Rishitin, a norsesquiterpene alcohol, found in infected, resistantpotato-tuber tissue completely inhibited zoospore germinationand germtube elongation of Phytophthora infestans (MONT.) DEBARY at 10^–3M. There was little difference in sensitivityto rishitin among races of Phytophthora infestans. IAA-inducedelongation of Avcna coleoptile sections and GA₃-induced elongationof wheat leaf sections were also inhibited by rishitin. Theinhibition of IAA-induced elongation of Avena coleoptiles wasrelieved to some extent by increasing IAA concentration. However,little relief of the inhibition of GA₃-induced elongation ofwheat leaf sections was obtained by increasing GA₃ concentration.No plant injury was observed at this concentration of rishitin(10^–3M). Examination of a series of rishitin derivatives indicated thatthe hydroxyl group at C-3 is indispensable for antifungal activity.This activity was intensified by saturating the double bondbetween the rings of rishitin and/or that of the isopropenylgroup at C-7, though activity decreased when oxygenated functionalgroups were introduced into the side chain. Aromatization of the A ring did not lower biological activities.The antifungal activities of most rishitin derivatives almostparalleled their activities as plant growth retardants. However,some compounds without antifungal activity were active as growthretardants. ¹Studies on the phytoalexins (5). (Received August 14, 1968; )     

Total and Polar Lipid Biosynthesis during Crotalaria juncea Linn. Pollen Tube Growth: Effect of Gibberellic Acid, Indoleacetic Acid and (2-Chloroethyl)-phosphonic Acid

Gibberellic acid (GA₃) at 58 µM, indoleacetic acid (IAA)at 29 µM, and (2-chloroethyl) phosphonic acid (Ethephon)at 70 µM promoted pollen tube growth in Crotalaria junceapollen suspension cultures both in water and basal medium. GA₃stimulated [ l-¹⁴C]acetate incorporation into total lipids inboth media, whereas IAA enhanced incorporation in water culturesonly. On the contrary, Ethephon reduced the label in total lipidswhen supplemented in basal medium. Based on [l-¹⁴C lacetateincorporation into different phospho- and glycolipids, it isproposed that these growth regulators have a definite role inthe biosynthesis of lipid components of the membranes.     

Effects of Gibberellic Acid and Naphthaleneacetic Acid on Petiole Senescence and Subtended Peduncle Growth of Cyclamen persicum Mill

Removal of the blade from the leaf subtending the first flowerbud on Cyclamen persicum ‘Swan Lake’ plants causedthe petiole of that leaf to senesce, but had no effect on thegrowth of the flower peduncle in the debladed petiole's axil.A 10 mg NAA l^–1 application generally had no effect onpetiole senescence, peduncle elongation or flowering date whenapplied to the cut end of the petiole after blade removal. A25 mg GA₃ l^–1 application or a combination of 25 mg GA₃l^–1 application or a combination of 25 mg GA₃ l^–1plus 10 mg NAA l^–1 delayed petiole senescence and enhancedpeduncle elongation and subsequent flowering. No treatment significantlyaltered peduncle length at the time of flowering. Cyclamen persicum Mill, ‘Swan Lake’, tissue receptivity, flowering, GA₃, NAA     

Effects Produced on Tomato Plants, Lycopersicum esculentum, by Seed or Root Treatment with Gibberellic Acid and Indol-3yl-Acetic Acid

Growth in lengths of tomato stems and leaves was acceleratedby 5.0 µg gibberellic acid (GA₂) applied to the seed,or by 5.0, 0.5, and 0.05 µg given to the roots. Treatmentwith 5.0 µg also decreased bud number and lengthened thetime between bud appearance and fruit formation on the firsttruss by 1–8 days. Smaller amounts applied to roots shortenedthis time by 1–4 days. Indol-3yl-acetic acid at 0.5 µghad no effect, nor was simultaneous application of GA₃ and IAAto the roots more effective than GA₃, alone. Single applicationsof very small amounts of GA₃ to seeds or seedling roots thusproved capable of changing growth-rates of stems, leaves, andtrusses.The effects of treating tomatoes with GA₂ and with culturesof Azotobacter chroococcum, which contain small amounts of GA₃,and IAA, are compared.     

Effect of Hormones on Sucrose Uptake and on ATPase Activity of Citrus sinensis L. Osbeck Leaves

Carbohydrate accumulation in young, fully expanded leaves ofCitrus sinensis L. Osbeck is affected by the presence of thefruitlet on the shoot. Previous work gave evidence that gibberellinsmay be involved in this 'fruit effect'. In the present workwe have studied the effect of gibberellic acid (GA₃) on ¹⁴C-sucroseuptake by leaf discs and whether its action could be due toa modulation of the plasma membrane ATPase, which maintainsthe H⁺ gradient that drives H⁺/sucrose co-transport. The effect of GA₃ on ¹⁴C-sucrose uptake depended on the osmolarityof the assay medium. At 300 mOsm a reduction in the uptake ratewas observed. The inhibitory effect of the hormone disappearedafter preincubating the leaf discs with para-chloromercuri-phenylsulphonicacid (PCMPS), a sulphydril binding inhibitor. ATPase activityof isolated plasma membrane vesicles was inhibited by IAA treatments,while GA₃ or ABA did not affect this enzyme, even after a 3h preincubation period. However, in the absence of a surfactantin the assay medium, GA₃, together with turgor pressure, modulatedplasma membrane ATPase activity, possibly through modificationsof membrane permeability. The hormone effect on ¹⁴ C-sucroseuptake may involve action on the sucrose carrier.Copyright 1994,1999 Academic Press Abscisic acid, Citrus sinensis, gibberellic acid, indoleacetic acid, orange, osmotic pressure, plasma membrane ATPase, ¹⁴C-sucrose uptake     

Partial Purification and Characterization of Gibberellin 3{beta}-hydroxylase from Immature Seeds of Phaseolus vulgaris L.

Gibberellin 3/ß-hydroxylase,a 2-oxoglutarate-dependentdioxygenase that catalyzes the hydroxylation of GA₂₀ to GA₁,was purified 313-fold from immature seeds of Phaseolus vulgarisL. The mol wt of the enzyme was estimated to be 42,000 by gelfiltration HPLC and SDS-polyacrylamide gel electrophoresis.The enzyme exhibited maximum activity at pH 7.7. The Km valuesfor [2,3-³H]GA20 and [2,3-³H]GA, were 0.29µu and 0.33µm, respectively. The enzyme requires 2-oxoglutarate asa cosubstrate; the K_m value for 2-oxoglutarate was 250µMusing [³H]- GA₂₀ as a substrate. Fe²⁺ and ascorbate significantlyactivated the enzyme at all purification steps, while catalaseand BSA activated the purified enzyme only. The enzyme was inhibitedby divalent cations Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺ and Hg²⁺.3ß-Hydroxylation of [³H]- GA₂₀ was also inhibitedby non-radioactive GA₅, GA₉,GA₁₅, GA₂₀ and GA₄₄. The possiblesite of 3ß-hydroxylation in gibberellin biosynthesisis discussed in terms of the substrate specificity of partiallypurified gibberellin 3ß-hydroxylase. (Received February 29, 1988; Accepted June 3, 1988)     

Effect of Gibberellic Acid, (2-Chloroethyl) Phosphonic Acid, Actinomycin-D and Cycloheximide on the Activity and Leaching of Some Hydrolases in Pollen Suspension Cultures of Crotalaria juncea

Gibberellic acid (GA₃) 20 mg 1^?1 and (2-chloroethyl) phosphonic acid (Ethephon) 10 mg 1^?1 enhanced pollen tube length in Crotalaria juncea L. GA₃ markedly increased the activity of amylase, acid phosphatase and β-glucosidase and enhanced leaching of amylase and acid phosphatase enzymes. The rise in the activity level and leaching of amylase and acid phosphatase after Ethephon treatment was comparatively less than that of GA₃ and the response to Ethephon was restricted to the 45 and 90 min period of culturing. Ethephon did not affect the activity/leaching of β-glucosidase significantly. Actinomycin-D (Act.D) 25 mg l^?1 and Cycloheximide (CH) 10 mg 1^?1 reduced the tube growth as well as activity of these enzymes suggesting their de novo synthesis during pollen tube growth.     

The Effect of Gibberellic Acid on Starch Breakdown in Sprouting Tubers of Solanum tuberosum L.

The treatment of potato tubers with 150 µmol dm^–3gibberellic acid (GA₃) stimulated starch breakdown and hexoseaccumulation in tuber tissues and the transfer of dry matterto stems. These effects could not be accounted for by enhancedactivities of starch phosphorylase, amylase and acid invertase.Indeed enzyme activities either declined or remained relativelyconstant as starch degradation and hexose accumulation proceeded.Changes in the rate of starch depletion were related to changesin sink strength and sink type, the onset of tuber initiationin controls causing the rate of starch degradation to exceedthat in GA₃-treated tissues, in which tuberization was inhibited. Solanum tuberosum L., gibberellic acid, starch breakdown     

Internode Length in Pisum. A New Allele at the le Locus

In Pisum sativum L. a third, more severe, allele at the internodelength locus le is identified and named le^d. Plants homozygousfor le^d possess shorter internodes and appear relatively lessresponsive to GA₂₀ than comparable le (dwarf) plants. Gene le^dmay act by reducing the 3ß-hydroxylation of GA₂₀ tothe highly active GA₁ more effectively than does gene le. Theresults indicate that le is a leaky mutant and therefore thatendogenous GA₁ influences internode elongation in dwarf (le)plants. Pisum sativum, peas, internode length, genetics, gibberellin, dwarf elongation     

Gibberellin A12 Is Converted to Gibberellin A53 in Cultured Cells of Nicotiana tabacum and Catharanthus roseus

The metabolism of GA₁₂ and its precursors was investigated incultured cells of seven cell lines of Nicotiana tabacum andthree cell lines of Catharanthus roseus using ^l4C-labeled substrates.The presence of a metabolic pathway from ent-7-hydroxykaurenoicacid to GA₅₃ via GA₁₂-aldehyde and GA₁₂ was demonstrated inthe cultured cells. GA₁₂ was effectively converted to GA₅₃ incells of BY-2, 2b-4, 2b-13 and CG from N. tabacum. By contrast,GA₅₃ was not converted to any other GAs in all of the linesof cells examined. The metabolism of C₁₉-GAs was also examinedusing ³H-labeled substrates. The conversion of GA₂₀ to GA₂₉and GA, and of GA₄ to GA₃₄ occurred more efficiently in cellsfrom C. roseus than in cells from N. tabacum. However, 13-hydroxylationof GA₄ and GA₉ was not observed in any of the cell culturesexamined. Among the various metabolites, GA₅₃, GA₂₉ and GA₃₄were identified by full-scan GC/MS. (Received December 20, 1990; Accepted May 27, 1991)