Time of initiation of the barley endosperm response to gibberellin A3, gibberellin A14 and kaurene

Summary Gibberellin A₁₄ and kaurene, precursors of gibberellin A₃, are active in the barley endosperm reducing-sugar assay, but require longer incubation periods for activity to be observed than does GA₃. This finding shows that the incubation period must be considered when determining whether a compound is active in this assay. The longer incubation periods required by GA₁₄ and kaurene may reflect reduced rates of penetration, or reduced activity within the cell, or the time required for conversion to a physiologically active form.     

The presence of gibberellin A20 in Stevia rebaudiana and its significance for the biological activity of steviol

Gibberellin-like substances of stems and leaves from Steviarebaudiana were analyzed and gibberellin A₂₀ was identifiedby gas chromatography-mass spectrometry. The presence of GA₂₀in S. rebaudiana is significant for the interpretation of thegibberellin activity of steviol. It indicates that steviol,the C-13 hydroxykaurenoic acid, may function as a precursorfor C-13 hydroxy-gibberellins and not as a gibberellin analog. ¹ This work was supported by National Science Foundation GrantGB 17304 to M. R. and by a Research Grant-in-Aid from SigmaXi to L. M. A. The research described is from a dissertationsubmitted by L. M. A. in partial fulfillment of the requirementsfor the Ph.D. degree. ² Present address: Laboratory of Plant Morphogenesis, Departmentof Biology, Manhattan College, Bronx, N. Y. 10471, U. S. A. (Received June 12, 1978; )     

Studies of Sites of Action of a New Plant Growth Retardant (E)-1-(4-Chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol (S-3307) and Comparative Effects of Its Stereoisomers in a Cell-Free System from Cucurbita maxima

(E)-1-(4-Chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol(S-3307), a new plant growth retardant, was investigated todetermine the inhibitory sites in gibberellin biosynthesis andthe comparative effects of its stereoisomers in a cell-freesystem from Cucurbita maxima. After treatment with S-3307, theincorporation of [¹⁴C]mevalonic acid into GA₁₂-aldehyde andGA₁₂ clearly was inhibited, and kaurene accumulated. Feedingexperiments showed that S-3307 inhibited the oxidation of kaurene,kaurenol and kaurenal, but did not affect the oxidation of kaurenoicacid. Thus the reaction sites of S-3307 in gibberellin biosynthesiswere shown to be the three oxidation steps from kaurene to kaurenoicacid. Because S-3307 has an asymmetric center and a tri-substituteddouble bond, there are four stereoisomers; two optical isomersand two geometrical isomers. The activities of these isomersas a growth retardant and gibberellin biosynthesis inhibitorwere examined with a rice seedling assay and the cell-free system.The relative activity of these isomers was basically the samein the two assay methods. The (S)-(E) form was the most active,being 7-fold more active in the cell-free system and 70-foldmore active in the bioassay than the (R)-(E) form. The (RS)-(Z)form showed no activity in the cell-free system, but weak dwarfingeffects in the rice seedling assay. (Received January 19, 1985; Accepted April 13, 1985)     

Fluctuation of Endogenous Levels of Free and Conjugated Gibberellins throughout Seed Maturation in Leucaena leucocephala (Lmk) De Wit

Combined gas chromatography-selected ion monitoring (GC-SIM)analysis of a purified extract from seeds of Leucaena leucocephalashowed the presence of GA₁, GA₈, GA₁₇, GA₁₉, GA₂₀, GA₂₃, GA₂₉and GA₅₃. GA₁, GA₈ and GA₂₉ were also found both as glucosylester-like and glucosyl ether-like conjugates, and GA₂₀ as aglucosyl ester-like conjugate; these conjugates were analyzedas free GAs after enzyme hydrolysis. GA₂₃ was shown to be themain free gibberellin in immature seeds (268 ng/seed), thoughits level drastically decreased during their maturation. GA₁was the main free C₁₉-gibbercllin and GA₁ glucosyl ester-likeand glucosyl ether-like conjugates were found at the highestlevels in the seeds prior to maturation. Fluctuation of endogenouslevels of gibberellins is discussed in terms of seed development. (Received May 9, 1984; Accepted August 25, 1984)     

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; )     

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)     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings III. Gibberellin specificity in its enhancing effect on IAA-induced elongation

Pretreatment effects of different gibberellins, helminthosporicacid, cyclic AMP and Kinetin on subsequent IAA-induced elongationwere tested in cucumber hypocotyl sections. Gibberellin A₇ wasmore active than GA₃, while gibberellin A₃ was almost inactive.Both helminthosporic acid and cyclic AMP mimicked GA₃-action,though the degree of their activity was less. Kinetin pretreatmentresulted in marked inhibition of IAA-induced elongation. Thepretreatment effect of GA₃ was also reflected in a greater responceof the sections to synthetic auxins. (Received October 6, 1973; )     

Studies on the Relationship between Gibberellin Metabolism and Daylength in Normal and Non-flowering Red Clover (Trifolium pratenseL.)

Feeding experiments on tillers of a non-flowering red cloversegregate indicated that the promotive effect of gibberellinupon stem extension and flowering occured only under inductivelong days (LD). Furthermore, floral initiation took place inLD only when free gibberellin exceeded an unspecified criticallevel. Various intermediates in fungal gibberellin synthesis were fedin a similar fashion but none exerted any effect upon reproductivegrowth. Studies using ³H-gibberellin A₃ (GA₃) showed that innormal (flowering) material GA₃ was more rapidly metabolizedin short days than in long days. Qualitative differences ingibberellin metabolism between normal and non-flowering genotypeswere revealed by radio-isotope studies. Non-flowering materialrapidly degraded GA₃ under LD conditions to a biologically inactivecompound chromatographically indentical with allo-gibberic acid,whereas normal plants metabolized GA₃ more slowly producinga compund similar to gibberellenic acid. The implications ofthese results are discussed in terms of the mechanism of stemelongation and floral initiation.     

Changes in Gibberellin Content during Seed Ripening in Grasses

Floret samples of Aberystwyth S. 321 perennial ryegrass andS. 352 timothy were analysed for gibberellin content at 2–5day intervals from inflorescence emergence to seed harvest.Both species had a high gibberellin content at emergence whichdiminished during anther formation. After anthesis there wasa sharp increase in gibberellin content with maxima of 256 and116 GA3 µ equivalents/Kg dry matter occurring in S. 321and S. 352 respectively. During the final stages of seed maturation,the level fell steadily to reach a stable content prior to harvest.Biological activity in ryegrass extracts was always identifiedchromatographically as gibberellin A₃, (GA₃), but in timothy,GA₃ was replaced after anthesis by a more mobile component,chromatographically similar to gibberellin A₃. The relationshipof these observations to seed formation and ripening is discussed.     

Effects of a Plant Growth Regulator, Prohexadione Calcium (BX-112), on the Elongation of Rice Shoots Caused by Exogenously Applied Gibberellins and Helminthosporol, Part II

Prohexadione calcium (BX-112) is a novel plant growth regulatorthat inhibits the late stages of the biosynthesis of gibberellinsin plants. Fourteen kinds of gjbberellin, helminthosporol and'helminthosporic acid were applied simultaneously with BX-112to rice seedlings (Oryza sativa L. ), and their growth-promotingactivities in terms of shoot elongation were examined. The growth-promotingactivities of GA₁, GA₄, GA₁₈, GA₂₂, GA₂₃, GA₃₈, helminthosporoland helminthosporic acid were not inhibited by BX-112, but thoseof GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, GA₃₁, GA₄₄ and GA₅₃ were inhibited.These results suggest that 3ß-hydroxylation is animportant and necessary step in the biosynthesis of gibberellinsthat promote shoot elongation in rice seedlings. The weak promotionof shoot elongation by GA₂₂ in the presence of BX-112 suggeststhat the effect of a hydroxyl group at C-18 of GA₂₂ might beable to mimic the effect of the 3ß-hydroxyl groupof GA₁. Helminthosporol and helminthosporic acid may promotethe shoot elongation of rice by mimicking physiologically activegibberellins and not by stimulating their biosynthesis. ¹Part I is the previous paper by Nakayama et al. (1990a) ³Present address: Frontier Research Program RIKEN, Wako-shi,Saitama, 351-01 Japan. (Received June 26, 1991; Accepted September 4, 1991)     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings II. Effect of GA3-pretreatment on IAA-induced elongation

IAA-induced growth of light-grown cucumber hypocotyl sectionsis markedly enhanced by GA₃-pretreatment of the sections; thereis a distinct synergism between IAA and GA₃. Water pretreatmentalso enhances IAA-induced growth. On the other hand, IAA-pretreatedsections showed practically no further growth in response topost treatment with GA₃. The enhancing effect of GA₃ is obtainedwith only 30 min pretreatment, the maximum effect occuring with2 hr pretreatment. Pretreatment longer than 8 hr is less effective.This enhancing effect of GA₃ can be observed soon after posttreatment with IAA. The response of GA₃-pretreated sectionsto IAA is greater in pretreatment with higher concentrationsof GA₃, and higher degrees of synergism between IAA and GA₃are obtained at IAA concentrations less than 10^-4 M. This synergisticinteraction between GA₃ and IAA is more marked in aged hypocotylsections than in young sections. From these results we concludedthat gibberellin sensitizes hypocotyl cells to the subsequenteffect of auxin on cell elongation. (Received October 6, 1973; )     

Change of gibberellin and abscisic acid content during fruit development of Prunus persica

The change of endogenous gibberellin and ABA content was measuredduring the fruit development of Prunus persica. GA₅, GA₃₂ andtwo unidentified gibberellins, were found throughout the developmentalstages. GA₅, GA₃₂ and ABA showed maximum peaks in their contentsixty days after full bloom, which suggests that they play importantroles in fruit development. ¹ To whom correspondence should be addressed. (Received December 12, 1975; )     

Stimulation of lateral bud growth of pea by (--)-kauren-19-oic acid

(—)-Kauren-19-oic acid, a precursor of gibberellin A₃,stimulated outgrowth of the lateral bud attached to a stem segmentexcised from an etiolated Alaska pea seedling. There was nosignificant difference in the degree of KA-induced responseat effective concentrations of 0.1, 1 and 10 mg/liter. Thiscompound required a longer period for its marked activity tobe observed than did gibberellin A₃. (Received May 30, 1970; )     

Gibberellins and Growth Inhibitors in Spring Bleeding Sap, Roots and Branches of Juglans regia L.

The main gibberellin found in the spring bleeding sap of thewalnut tree was identified as GA₁₉ and quantified by gas chromatography-selectedion monitoring (817 pg/ml). The second minor gibberellin (12pg GA₃-equiv./ml) was chromatographically similar to GA₅₃. Bothgibberellins occur in their free forms and as neutral ester-likeconjugates. In the roots, these free gibberellins and an additionalgibberellin glucoside-like component were found in late winter. In the bleeding sap, branches and roots, abscisic acid was identifiedby gas chromatography and mass spectrometry. In the sap, itsamount was determined to be 34 ng/ml. ABA was not found in aconjugated form, but the branches contained a neutral ester-likeconjugate of 4'-dihydrophaseic acid, an ABA metabolite, whichafter hydrolysis was identified by combined gas chromatography-massspectrometry. (Received March 28, 1981; Accepted November 16, 1981)     

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)     

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.     

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)     

Phase Change in Hedera helix L.: III. THE EFFECTS OF GIBBERELLINS, ABSCISIC ACID AND GROWTH RETARDANTS ON JUVENILE AND ADULT IVY

Gibberellic acid, applied to delaminated petioles of rootedcuttings of juvenile and adult ivy initially induced internodeelongation and abnormal leaf development, and suppressed apicaldominance. Juvenile cuttings were affected only transientlyand soon reverted to normal growth. Adult cuttings, insteadof resuming normal growth after this initial response to GA₃,gradually developed many juvenile characteristics. Approximately16 weeks after treatment at 25 ?C nearly all shoots of adultcuttings had undergone complete rejuvenation. Lower temperaturereduced the speed of response to GA₃. A mixture of gibberellinsA₄ and A₇ had effects similar to those of GA₃ on the growthof juvenile and adult cuttings. Treatment of both phases ofivy with abscisic acid (ABA) induced no visible effects andwhen ABA was applied with GA₃ it did not reduce the responseof either phase to the gibberellin. CCC had a marked dwarfingeffect on juvenile ivy but did not induce pre-maturation. However,extraction of gibberellin-like substances from severely dwarfedplants suggested that CCC was not exerting its growth retardingeffect through an inhibition of gibberellin biosynthesis. AMO1618 did not retard growth of juvenile ivy cuttings.     

Mechanical Stress and Gibberellin: Regulation of Hollowing Induction in the Stem of a Bean Plant, Phaseolus vulgaris L.

In pole bean plants, mechanical stress (MS) inhibited stem elongationand induced radial thickening of the stem. Application of uniconazole,an inhibitor of gibberellin biosynthesis, also reduced stemgrowth but had no effect on stem diameter. Both MS and uniconazolesignificantly reduced hollowing of the first internodes, butonly the former increased ethylene evolution from the firstinternode. Application of GA₃ increased the length of the firstinternode and decreased its diameter in bush bean plants; thiswas accompanied by a significant promotion of stem hollowing.Aminooxyacetic acid (AOA) decreased ethylene evolution fromthe GA₃-treated internodes, though it did not reduce the GA₃-inducedhollowing of the first internodes. Application of GA₃ affectedneither ethylene evolution nor cellulase activity in the firstinternodes of bush bean plants. Application of GA₃ stimulatedmuch greater cell elongation in the center of pith tissue thanin the outer surrounding tissues, suggesting a possible physicalbreakage of the inner cells, which leads the hollowing of beanstems. These results suggest that gibberellin is a factor responsiblefor stem hollowing in bean plants. Because MS is known to reducegibberellin content in bean plants [Suge (1978) Plant Cell Physiol.21: 303] MS may inhibit stem hollowing by reducing the amountof endogenous gibberellin. (Received July 1, 1994; Accepted November 8, 1994)