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.     

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)     

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)     

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)     

Gibberellins in Suspensors of Phaseolus coccineus L. Seeds

Analysis of extracts from 6300 (1.2 grams fresh weight) Phaseolus coccineus suspensors by combined gas chromatography-mass spectrometry has demonstrated the presence of five C₁₉-gibberellins, GA₁, GA₄, GA₅, GA₆, GA₈, and one C₂₀-GA, GA₄₄. The major GAs present were GA₁ and GA₈. Data are discussed in relation to previous results obtained in P. coccineus seed as well as in the embryo-suspensor system.     

Effects of Auxin and Gibberellin on Conidial Germination and Elongation of Young Hyphae in Gibberella fujikuroi and Penicillium notatum

In Gibberella fujikuroi and Penicillium notatum, IAA, 2,4-Dand GA₃ promoted conidial germination and the elongation ofyoung hyphae. The promotive effects of IAA and GA₃ were additive.In both fungi, the concentrations of endogenous auxin and gibberellinin the culture media were 10^–10 to 610^–12M. (Received April 27, 1985; Accepted August 12, 1985)     

Effects of a New Plant Growth Regulator Prohexadione Calcium (BX-112) on Shoot Elongation Caused by Exogenously Applied Gibberellins in Rice (Oryza sativa L.) Seedlings

The effects of a novel plant growth regulator (PGR) prohexadionecalcium (BX-112; calcium 3,5-dioxo-4-propionylcyclohexanecarboxylate)on shoot elongation caused by exogenously applied GA₁, GA₃,GA₄₎ GA₁₉ and GA₂₀ were investigated in rice (Oryza sativa L.cv. Nihonbare and cv. Tan-ginbozu) seedling test. Dependingon the dose, BX-112 reduced shoot elongation in both cultivarscaused by GA₁₉ and GA₂₀, but not by GA₁. When a high dose ofBX-112 (e.g. 250 ng/plant and over) was applied with GA₁, orGA₄, shoot elongation was even promoted. This promotive effect,however, was not observed with GA₃. These results suggest thatBX-112 inhibits gibberellin (GA) biosynthesis in the rice plantat the 3ß- and 2ß-hydroxylation of GAs,namely steps of activation and inactivation, respectively. (Received September 6, 1989; Accepted November 27, 1989)     

Activity of Gibberellins A1 to A0 in the Avena First-leaf Bioassay, and Location after Chromatography

Activity curves are determined for gibberellins A₁ to A₀ bythe Avena first-leaf bioassay method. Gibberellins A¹, A₄ andA₅ can be detected at 10^-11 or 10^-10 g/ml and give optimum activityof approximately 230 per cent elongation (water controls = 100per cent). Gibberellins A²A³, and A⁹ can be detected at 10^-3g/mland give optimum activity of approximately 200 per cent. GibberellinsA₆ and A₇ can be detected at 10^-5g/ml; GA₇ gives optimum activityof around 190 per cent. All the gibberellins except GA₈ canbe detected by this bioassay method after chromatography inn-butanol: 1.5 N ammonia (3: 1) and benzene: acetic acid: water(4: 2: 1) when applied to the paper at concentrations from O.Ito µg. The sensitivity of the method is compared withthat of other gibberellin bioassay methods.     

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)     

Identification of gibberellin A1 in the embryo suspensor of Phaseolus coccineus

By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A₁ (GA₁). The amount of GA₁ in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA₁ in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID flame ionization detector - GA gibberellin - GC gas chromatography - MS mass spectrometry - PGC preparative gas chromatography - Stage A heart-shaped embryo - Stage B cotytedonary embryo - TMS trimethylsilyl     

Interaction of a Brassinosteroid with IAA and GA3 in the Elongation of Cucumber Hypocotyl Sections

A synthetic brassinosteroid, 22,23(S,S)-homobrassinolide (hBR),was examined for its interaction with IAA and GA₃ in the elongationof hypocotyl sections of light-grown cucumber (Cucumis salivusL. cv. Aonagajibai) seedlings. hBR alone was less active thanIAA. Its optimal concentration was around 10 µM and thelowest effective concentration between 10 and 100 µM,which is more than 100 times higher than that of brassinolide.hBR was more active in sections from younger seedlings. Itsgrowth-promoting effect was negated or greatly reduced by inhibitorsof auxin-induced elongation such as p-chlorophenoxyisobutyricacid and kinetin. hBR acted synergistically with IAA and 2,4-Dbut not with GA₃ showing only an additive effect. Sequentialtreatment of sections with hBR and then with IAA also resultedin synergistic enhancement of auxininduced elongation, but whenthe order of treatment was reversed, hBR was inactive. The synergisticeffect was obtained with 1 h pretreatment with hBR and couldbe reduced by subsequent washing with water. There was no sequentialinteraction between hBR and GA₃. The synergistic pretreatmenteffects of hBR and GA₃ were simply additive to each other. Amembrane-bound ATPase inhibitor, dicyclohexylcarbodiimide, inhibitedthe hBR-induced elongation, but did not affect GA₃-induced elongation.The findings led to the conclusion that brassinosteroids enhanceauxin action and possess growth-promoting activity which isindependent of that of gibberellin. (Received November 9, 1984; Accepted February 18, 1985)     

Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

Uptake of Gibberellin Methyl Esters by Spores and Induction of Dark Germination in Lygodium japonicum

In the fern Lygodium japonicum, the effect of the exogenousapplication of two gibberellin methyl esters, gibberellin A₄methyl ester (GA₄Me) and gibberellin A₂₀ methyl ester (GA₂₀Me)on spore germination in the dark and uptake of GA₄Me and GA₂₀Meby spores was investigated. Tritiated GA₄Me and GA₂₀Me wereprepared and used as radioactive tracers. The activity of GA₄Mewas more than 100-fold that of GA₂₀Me for the induction of sporegermination. When treated for 24 h, the activity for inducingspore germination remained after removal of the gibberellinmethyl esters from the medium. The amount of GA₄Me taken upby spores was more than three times that of GA₂₀Me throughoutthe 24 h time course of treatment. The uptake of both gibberellinmethyl esters was proportional to the external concentrationfor the range of concentrations between 10^–9 M and 10^–6M. When treated with the tritiated gibberellin methyl estersat 10^–6 M and 10^–7 M for 24 h, most of the gibberellinmethyl esters taken up by the spores were not metabolized. Althoughthe uptake of the two gibberellin methyl esters differed by3- to 5- fold, their abilities to induce spore germination differedby more than 100-fold. Therefore, the difference in the activityof the two gibberellin methyl esters regarding the inductionof spore germination could not be explained solely by the differencein their uptake. (Received January 11, 1988; Accepted May 26, 1988)     

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)     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

THE RATIO OF CALCAREOUS AND ORGANIC SHELL COMPONENTS OF FRESHWATER SPHAERIID CLAMS IN RELATION TO WATER HARDNESS AND TROPHIC CONDITIONS

Shells from 14 populations of sphaeriid clams including Sphaeriumstriatinum, S. simile, Pisidium walkeri, Musculim partumeiumand M. iransversum were analyzed for organic carbon (µgCmg^–1 shell), nitrogen (µg,N mg^–1 shell) andCaCO_s (%CaCO₃ of total clam dry weight). Habitat waters wereassessed for total hardness (expressed as ppm CaCo₃), ppm Ca,ppm Mg, conductivity (µmho) and suspended organic Carbon(µgCl^–1). For all populations, shell organic C andN are positively correlated and there is an inverse relationshipbetween the amounts of shell CaCO₃ and shell organic carbon.Trophic considerations give the best correlation with shelltype at the generic level of consideration since species ofMusculium are found at the opposite end of the trophic scale(eutrophic) from all other populations studied. For S. striatinum,the most extensively studied species, the amount of shell CaCO₃is inversely related to water hardness. The selection of shellsin the Sphaeriidae is discussed in relation to structural-functionalneeds and habitat conditions ¹ Present Address: Department of Biology, Syracuse University,Syracuse, New York 13210, U.S.A. ² Present Address: Department of Zoology, Miami University,Oxford, Ohio 45056, U.S.A. (Received 5 December 1978;     

Effects of Gibberellins and Prohexadione on the Activities of Oryzain and {alpha}-Amylase in Rice Seeds

Oryzains, cysteine proteinases of rice seeds, are induced byGA₃ in germinating rice seeds [Abe et al. (1987) Agric. Biol.Chem. 51: 1509]. The effects of GA₁, GA₃, GA₄, GA₉, and GA₂₀on the production of oryzain and -amylase were investigatedin embryoless half- and whole-seeds of rice (cv. Nipponbare).When gibberellins (GAs) were incubated with embryoless half-seeds,GA₁, GA₃ and GA₄ induced oryzain and -amylase, but GA₉, andGA₂₀ did not. GA₉ and GAM induced oryzain and -amylase productionin whole seeds, but this production was inhibited by the simultaneousapplication of prohexadione, an inhibitor of 2ß- and3ß-hydroxylation of GAs. Prohexadione did not inhibitthe activities of oryzain and -amylase induced by GA₁. Theseresults suggest that GAs possessing the 3ß-hydroxylgroup induce activities of oryzain and -amylase in rice seedsand that GA₉ and GA₂₀ have activity only after they are convertedmetabolically to active GAs, probably GA₄ and GA₁, respectively.GA₁, was more active than GA₄ in both half seeds and wholeseeds incubation. Oryzain and -amylase activities induced byGA₄ were significantly inhibited in the presence of 10^–4M prohexadione. This suggests that the conversion of GA₁, toGA₄ (13-hydroxylation) might be inhibited at a high dose ofprohexadione in whole seeds. ⁴Present address: Institute of Food Development, Kyung Hee University,Suwon 449-701, Korea     

Inhibition of flowering and growth in Pharbitis nil by the growth retardant Ancymidol

Flower formation and growth of the short day plant Pharbitisnil, strain "Violet", were inhibited when the growth retardantAncymidol was applied prior to an inductive dark period viacotyledons or roots. Inhibition of flower formation by Ancymidolcould be completely reversed by an application of gibberellinA₃ (GA₃) to the plumule before the inductive dark period. Adose of 0.01 µg GA₃/plant was almost sufficient to restoreflowering, but about a hundred times more GA₃ was required torestore the internode length to that of control. Ancimidol greatlyreduced the endogenous gibberellin content. (Received July 18, 1980; )     

Inhibition of flowering and growth in Pharbitis nil by the growth retardant Ancymidol

Flower formation and growth of the short day plant Pharbitisnil, strain "Violet", were inhibited when the growth retardantAncymidol was applied prior to an inductive dark period viacotyledons or roots. Inhibition of flower formation by Ancymidolcould be completely reversed by an application of gibberellinA₃ (GA₃) to the plumule before the inductive dark period. Adose of 0.01 µg GA₃/plant was almost sufficient to restoreflowering, but about a hundred times more GA₃ was required torestore the internode length to that of control. Ancimidol greatlyreduced the endogenous gibberellin content. (Received July 18, 1980; )     

Identification and Semi-Quantification of Gibberellins from the Pollen and Anthers of Zea mays by Immunoassay and GC/MS

Radioimmunoassays and enzyme-linked immunosorbent assays formethyl esters of gibberellins A₁, A₃, A₄, and A₇ were establishedusing an antiserum specific for GA₁-Me. The antiserum was characterizedby high titer and specificity for such C₁₉-GAs with 3ß-hydroxylgroup as GA₁, GA₃, GA₄ and GA₇. Combination of this antiserumand HPLC enabled us to identify and quantify GA, and GA₄ fromthe pollen of Zea mays with a high degree of reliability. Similarly,identification and quantification of GA₉ and GA₂₀ were alsomade possible by use of an antiserum specific for GA₂₀-Me. Combineduse of immunoassays and GC/MS enabled us to identify nine GAsfrom the pollen and four from the anthers of Zea mays. The identificationof non-13-hydroxylated GAs, such as GA₄ and GA₉, in additionto 13-hydroxylated GAs from the pollen and the anthers suggeststhat the early-non-hydroxylation pathway, as well as the early-13-hydrox-ylationpathway, operates in the male reproductive organs of Zea mays,and that the organ-specific biosynthesis and/or localizationof GAs in Zea mays is similar to that in Oryza saliva. (Received May 7, 1990; Accepted August 20, 1990)