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)     

Gibberellin Production in Cultured Cells of Nicotiana tabacum

Endogenous gibberellins (GAs) in several kinds of crown gallcells and cultured cells derived from normal tissue of Nicotianatabacum were systematically analyzed by gas chromatography-selectedion current monitoring (GC-SICM) after chromatographic purifications,and GA₁, GA₉, GA₁₉ and GA₂₀ were identified. Agrobacterium tumefaciens,a pathogen of crown gall, was confirmed not to produce GAs inits culture. We also investigated endogenous GAs of mother plant,tobacco, and found the same kinds of GAs as in cultured cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received May 19, 1982; Accepted July 22, 1983)     

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)     

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     

Metabolism and Translocation of Gibberellins in Seedlings of Pharbitis nil. (I) Effect of Photoperiod on Stem Elongation and Endogenous Gibberellins in Cotyledons and Their Phloem Exudates

Gibberellin A₁, (GA₁), GA₁₉, and GA₂₀ in phloem exudates andcotyledons of seedlings of Pharbitis nil cv. Violet, grown underdifferent photoperiodic conditions, were qualitatively and semi-quantitativelyanalyzed by a combination of high performance-liquid chromatography(HPLC) and radioimmunoassays (RIA). The levels of GA₁₉ and GA₂₀were higher in cotyledons from plants grown under dark treatment(DT) conditons of 16 h-light/8 h-dark for 6 days followed by8 h-light/16 h-dark for 3 days than in those grown under continuouslight (CL) for 9 days. This relationship was also observed forthe GAs in phloem exudates, although the levels were much lowerthan in the cotyledons. When GAs were applied to the cotyledons,elongation of the epicotyl was promoted more by GA₂₀ than byGA₁ or GA₁₉, especially under the CL treatment. The relativeeffect of GA₁ and GA₂₀ on the epicotyl elongation was reversedwhen these GAs were applied to epicotyls pre-treated with prohexadione,an inhibitor of 2-oxoglutarate-dependent dioxygenases. ³Present address: Frontier Research Program, The Institute ofPhysical and Chemical Research (RIKEN), 2-1 Hirosawa, Wakoshi,Saitama, 351-01 Japan ⁴Present address: Laboratory of Horticulture, Faculty of Agriculture,Nagoya University, Nagoya, 464-01 Japan     

Metabolism and Translocation of Gibberellins in the Seedlings of Pharbitis nil (II). Photoperiodic Effects on Metabolism and Translocation of Gibberellins Applied to Cotyledons

We investigated the metabolism and translocation of two gibberellins(GAs), [³H]GA₂₀ and [³H]GA₁, which were applied at low concentrationto the cotyledons of Pharbitis nil (cv. Violet). Seedlings weregrown under three different photoperiodic conditions: continuouslight (CL-CL), continous light followed by short day conditions(CL-DT) and long day conditions followed by short day conditions(DT-DT). Translocation of the applied [³H]GAs from cotyledonsto hypocotyls was promoted by DT for all GAs examined. Whilethe conversion of the translocated [³H]GA₁ to [³H]GA₈ and itsconjugates was rapid in hypocotyl, the conversion of translocated[³H]GA₂₀ to [³H]GA₂₉ was slow. Radioactivity in epicotyls wasdetected much more rapidly on application of [³H]GA₂₀ than of[³H]GA₁, [³H]GA₈ and [³H]GA₂₉ and their conjugates. The conversionof [³H]GA₂₀ to [³H]GA₁ in the epicotyl was more rapid underCL-CL conditions. This result in consistent with the higherlevel of endogenous GA₁ existing in epicotyls under CL-DT thanDT-DT conditions. However, when [³H]GA₁ was applied to the cotyledon,only small amounts of [³H]GA₈ and its conjugates were detectedin the epicotyl regardless of the photoperiodic conditions.This result may suggest that the translocation and metabolismof [³H]GA₂₀ from cotyledons to epicotyl was faster under CL-CLthan DT-DT conditions and may correlate with the increased epicotylelongation of GA₂₀ treated plants under CL-DT than DT-DT conditions. (Received June 28, 1995; Accepted November 2, 1995)     

Semi-Quantification of GAX and GA4 in Male-Sterile Anthers of Rice by Radioimmunoassay

The levels of endogenous GA, and GA₄ in the anthers of a standardcultivar and the corresponding male-sterile, single gene mutantof rice, Oryza saliva L. (Japonica), were measured by radioimmunoassayusing GA₄-specific antiserum that showed highly specific immunoreactivitywith GA, and GA₄. The levels of these GAs in the anthers ofthe male-sterile mutant were about one-fifth to one-sixth ofthose in the normal cultivar, suggesting a correlation betweenthe endogenous levels of these GAs and the male sterility. ²Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Mine-machi 350, Utsunomiya, 321 Japan (Received August 8, 1990; Accepted March 7, 1991)     

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)     

A Modified Micro-Drop Bioassay Using Dwarf Rice for Detection of Femtomol Quantities of Gibberellins

The sensitivity of the micro-drop assay with dwarf rice (Oryzasativa L., cv. Tan-ginbozu and cv. Waito-Q to gibberellins (GAs)was increased conspicuously by the use of assay plants thathas been treated with uniconazole (S-3307), an inhibitor ofthe biosynthesis of GAs. The Tan-ginbozu plants treated withS-3307 responded to 10 fmol/plant of GA₃ (ca. 3.5 pg/plant)and to 30 fmol/plant of gibberellins A₁, A₄, A₇, A₁₉ and A₂₀.Waito-C plants treated with S-3307 responded to 10 fmol of GA₃and to 30 fmol/plant of gibberellins A₁, A₄ and A₇. GibberellinsA₉, A₁₉ and A₂₀ had much less of an effect on the treated Waito-Cplants than did gibberellins A₁, A₃, A₄ and A₇. Furthermore,treatment with S-3307 counteracted the inhibition of growthof both cultivars by abscisic acid. Thus, the modified micro-dropassay should prove very useful for the detection of minute amountsof GAs in plant extracts. (Received October 3, 1988; Accepted March 29, 1989)     

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)     

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)     

Changes in Endogenous Gibberellin Contents during Growth of Cultured Tobacco Cells

Changes in the contents of endogenous gibberellins (GAs) wereexamined in three kinds of cultured tobacco cells; a crown gallcell and two cultured cells derived from normal tissue of Nicotianatabacum. The relative amounts of the GAs were analyzed by systematicchromatographic purifications followed by GC-SIM. In all thecell lines examined, the content of GAj was the highest duringthe logarithmic phase of growth, indicating that GA₁ has a physiologicalrole in the growth of dedifferentiated cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received April 11, 1984; Accepted July 10, 1984)     

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.     

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)     

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; )     

Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

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)     

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)     

Effects of Gibberellins and Their Methyl Esters on Dark Germination and Antheridium Formation in Lygodium japonicum and Anemia phyllitidis

The effects of GA₃, GA₄ and GA₉ and their methyl esters on darkspore germination and antheridium formation of the ferns Lygodiumjaponicum and Anemia phyllitidis were investigated. Althoughall induced both germination and antheridium formation in Lygodium,only the gibberellins induced these effects inAnemia. (Received August 28, 1986; Revision received November 14, 1986. )     

A Crucial Role for Gibberellins in Stress Protection of Plants

The hypothesis that there is an intimate relationship betweengibberellin levels and plant stress protection has been testedusing near-isogenic lines of a normal and dwarf barley (Hordeumvulgare L.). Application of paclobutrazol (a triazole), inducedstress protection in the normal line and application of GA₃to the dwarf reversed the inherent stress tolerance. Reversalof the dwarf phenotype by specific gibberellins (GAs) suggeststhat the conversion of GA₂₀ to GA₁ and GA₉ to GA₄ has been compromised.These observations indicate that modulation of specific GAsplays a key role in stress protection. The preferential useof non-chemical technologies including enzyme regulation, phytochromeA overexpression, and breeding for induction of stress tolerancein plants are discussed. (Received November 26, 1998; Accepted March 11, 1999)