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)     

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)     

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     

Preparation and Validation of an Antiserum Specific for Non-Derivatized Gibberellins

An antiserum recognizing free gibberellins (GAs) was preparedby immunizing rabbits with a GA₄-BSA conjugate. A radioimmunoassay(RIA) and an enzyme-linked immunosorbent assay (ELISA) wereset up using this antiserum. This antiserum showed high cross-reactivityto the so-called active GAs, such as GA¹, GA₃, GA⁴ and GA₇.The range for measurements of these gibberellins extended from30 fmol to 3 pmol in both RIA and ELISA. Extracts from immature seeds of P. vulgaris were subjected todetermination of GA, by RIA and GC/SIM. The two assays providedsimilar results, indicating the high degree of reliability ofthe immunoassay. ²Present address: Department of Agricultural Chemistry, UtsunomiyaUniversity, Mine-machi 350, Utsunomiya, 321 Japan ( Accepted August 8, 1990)     

Quantitative Analysis of Endogenous Gibberellins in Normal and Dwarf Cultivars of Rice

Endogenous levels of gibberellins in shoots and ears of twodwarf rice (Oryza sativa L.) cultivars, Tan-ginbozu (dx mutant)and Waito-C (dy mutant), were analyzed and compared with thoseof normal rice cultivar, Nihonbare. The endogenous levels of13-hydroxylated gibberellins in Tan-ginbozu were much lowerthan those in Nihonbare. In Waito-C, the levels of GA₁₉ andGA₂₀ in the shoots were higher but that of GA₁ was lower thanthe levels of these gibberellins in Nihonbare. These resultssupport the hypothesis that the dy gene controls the 3ß-hydroxylationof GA₂₀ to GA₁ while the dx gene controls a much earlier stepin the gibberellin biosynthesis. Our results indicate that GA₁is the active gibberellin that regulates the vegetative growthof rice. The endogenous levels of GA₄ in the ears of the twodwarf cultivars of rice were higher than the level of GA₄ inthe ears of the normal cultivar, Nihonbare suggesting that thebiosynthesis of gibberellin is not blocked in the anthers ofthe dwarf rice. (Received April 27, 1989; Accepted July 12, 1989)     

Control by Light of Hypocotyl Elongation and Levels of Endogenous Gibberellins in Seedlings of Lactuca sativa L.

Four 13-hydroxygibberellins, gibberellin A₁ (GA₁), 3-epi-GA₁,GA₁₉ and GA₂₀ were identified by full-scan GC/MS in extractsof lettuce seedlings (Lactuca sativa L. cv. Grand Rapids). Theresults suggest that the early-13-hydroxylation biosyntheticpathway to GA₁ functions in the lettuce seedlings. It was alsofound that GA₁ is active per se in the control of hypocotylelongation in lettuce seedlings. To investigate the relationshipbetween control by light of hypocotyl elongation and levelsof endogenous GAs in lettuce, endogenous levels of GAs werequantified by radioimmunoassay in seedlings that had been grownfor 5 days in the dark (5D) and in those that had been grownfor 4 days in the dark and then under white light for 1 day(4D1L). The endogenous level of GA₁ in the upper and lower partsof hypocotyls in 5D seedlings was about four times higher thanthat in 4D1L seedlings. The response of explants (hypocotylsegments with cotyledons) from dark-grown seedlings to GA₁ isknown to be similar in the dark and under white light when theexplants are treated with inhibitors of the biosynthesis ofGA. Therefore, the above information suggests that the highlevel of GA₁ in hypocotyls of dark-grown seedlings is responsiblefor the rapid elongation of hypocotyl, while irradiation bywhite light decreases the endogenous level of GA₁ in the hypocotylswith a resultant decrease in the rate of hypocotyl elongation. (Received March 13, 1992; Accepted May 21, 1992)     

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)     

Effect of GA3, GA4$7, GA5 and GA9 on the sex expression of Luffa acutangula var. H-2

Gibberellins A₃, A_4$7, A₅, and A₉ increase the number of malebuds and flowers, but inhibit the opening of female flowersand shift their position to a higher node in Luffa acutangula.GA₃ is the most effective followed by GA_4$7, GA₅ and GA₉. (Received November 19, 1971; )     

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 of Gibberellins in Cell-Free Extracts of Anthers from Normal and Dwarf Rice

Cell-free extracts were prepared from anthers of normal anddwarf rice (Oryza sativa L.), and the metabolism of radioisotope-labeledgibberellins in the extracts was analyzed by HPLC and gas chromatography-massspectrometry (GC/MS). GA₁₂ was converted to GA₁₅ and GA₃₄ inthe extracts. GA₂₀ was converted to GA¹, GA₈ and GA₂₉, but GA₉was converted only to GA₃₄. The extracts of the dwarf cultivar,Waito-C (dy mutant), showed the same 3ß-hydroxylationactivity as did those of the normal cultivar, Nihonbare, indicatingthat the dy gene is not expressed in the anthers. These resultssuggest that the regulation of the biosynthesis of gibberellinsin rice is organ-specific. (Received November 9, 1989; Accepted January 10, 1990)     

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)     

Zinc Deficiency Affects the Levels of Endogenous Gibberellins in Zea mays L.

Zinc deficiency in Zea mays L. markedly reduced the level ofGA₁, but not GA₂₀, suggesting blockage of 3rß-hy-droxylation.The level of IAA was also decreased although not as markedly.Castasterone was affected less than IAA by zinc deficiency. (Received February 24, 1997; Accepted June 24, 1997)     

Biological Activities of the Methyl Ester of Gibberellin A73, a Novel and Principal Antheridiogen in Lygodium japonicum

The synthetic methyl ester of GA₇₃ (GA₇₃-Me) and the naturalantheridiogen of Lygodium japonicum showed almost the same activityto induce the formation of antheridia in dark-grown protonemataof L. japonicum at concentrations of 10^-14 M and higher. Thus,it appears that the principal antheridiogen in L. japonicumis GA₇₃-Me. GA₇₃-Me inhibited formation of ar-chegonia in light-grownprothallia of L. japonicum at concentrations of 10^-11 M andhigher and induced germination of spores in the dark in thisspecies at the same range of concentrations. GA₇₃(free acidform) promoted growth of seedlings of dwarf rice and hypocotylsof cucumber seedlings at dosages of and above 1 and 100 ng/plant,respectively. Eight compounds related to GA₇₃-Me, includingantheridiogens of Anemia phyllitidis and Anemia mexicana, wereactive in inducing an-theridial formation in L. japonicum, althoughtheir activities were considerably lower than that of GA₇₃-Me. (Received August 24, 1988; Accepted November 28, 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)     

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)     

Metabolism of gibberellins in a cell-free system from immature seeds of Phaseolus vulgaris L.

The biosynthetic steps from gibberellin A₁₂-aldehyde (GA₁₂-aldehyde) to C₁₉-GAs were studied by means of a cell-free system from the embryos of immature Phaseolus vulgaris seeds. Stable-isotope-labeled GAs were used as substrates and the products were identified by gas chromatography-mass spectrometry. Gibberellin A₁₂-aldehyde was converted to GA₄ via non-hydroxylated intermediates and to GA₁ via 13-hydroxylated intermediates. 13-Hydroxylation took place at the beginning of the pathway by the conversion of GA₁₂-aldehyde to GA₅₃-aldehyde. The conversion of GA₂₀ to GA₅ and GA₆ was also shown but no 2-hydroxylating activity was found. Endogenous GAs from embryos and testas of 17-dold seeds were re-examined by gas chromatography-selected ion monitoring using stable-isotopelabeled GAs as internal standards. Gibberellins A₉, A₁₂, A₁₅, A₁₉, A₂₃, A₂₄, and A₅₃ were identified for the first time in P. vulgaris, in addition to GA₁, GA₄, GA₅, GA₆, GA₈, GA₁₇, GA₂₀, GA₂₉, GA₃₇, GA₃₈ and GA₄₄, which were previously known to occur in this species. The levels of all GAs, except the 2-hydroxylated ones, were greater in the embryos than in the testas. Conversely, the contents of GA₈ and GA₂₉, both 2-hydroxylated, were much higher in the testas than in the embryos.Abbreviations GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - GC-SIM gas chromatography-selected ion monitoring - HPLC high-performance liquid chromatography - TLC thin-layer chromatography - m/z ion of mass     

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)     

Comparison of the endogenous gibberellins in the shoots and roots of vernalized and non-vernalized chinese spring wheat seedlings

Endogenous gibberellins (GAs) in Chinese Spring wheat seedlings were isolated by high performance liquid chromatography (HPLC) and identified by combined capillary gas chromatography-selected ion monitoring (GC-SIM). Gibberellins A₁, A₃, A₁₉, A₂₀, A₄₄, and A₅₃ were identified in the shoots, A₁₉ and A₂₀ in the roots. The identification of these 13-hydroxylated GAs demonstrates the presence of the early-13-hydroxylation pathway in wheat seedlings. Based on peak area of total ion response of five characteristic ions by GC-SIM, the approximate levels of GAs in the shoots is GA₄₄ > GA₁₉ > GA₁ = GA₃ > GA₂₀ for the non-vernalized wheat seedlings, and GA₄₄ > GA₁₉ > GA₅₃ = GA₃ > GA₁ = GA₂₀ for the vernalized wheat seedlings. The C₂₀ GAs, GA₅₃, GA₄₄ and GA₁₉, are present in shoots of the vernalized (flowering) wheat seedlings at somewhat higher levels than that in the non-vernalized (rapidly growing) wheat seedlings. Approximate levels of the C₁₉ GAs, GA₂₀, GA₁ and GA₃ were lower in the shoots of the vernalized wheat seedlings than in the non-vernalized wheat seedlings. The conversion of GA₁₉ to GA₂₀ (C₂₀ to C₁₉ GAs) may be a rate-limiting step in the vernalized wheat seedlings.     

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)