Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [¹⁴C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).Abbreviations ABA abscisic acid - RIA radioimmunoassay - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

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     

Abscisic acid and related compounds in phloem exudate of Yucca flaccida haw. and coconut (Cocos nucifera L.)

Phloem sap collected from Yucca and coconut inflorescence stalks was shown to contain abscisic acid (ABA) and trace amounts of 2-trans ABA. In coconut sap, two compounds probably derived from ABA with mass spectra consistent with their being dihydrophaseic acid and either hydroxyphaseic acid or oxo-dihydrophaseic acid were also found to be present.Abbreviations ABA abscisic acid - TMSi trimethylsilyl - GLC(EC) gas chromatography (electron capture) - GC-MS gas chromatography=mass spectrometry     

Conversion of [14C]gibberellin A12-aldehyde to C19- and C20-gibberellins in a cell-free system from immature seed of Phaseolus coccineus L.

A cell-free system prepared from developing seed of runner bean (Phaseolus coccineus L.) converted [¹⁴C]gibberellin A₁₂-aldehyde to several products. Thirteen of these were identified by capillary gas chromatography-mass spectrometry as gibberellin A₁ (GA₁), GA₄, GA₅, GA₆, GA₁₅, GA₁₇, GA₁₉, GA₂₀, GA₂₄, GA₃₇, GA₃₈, GA₄₄ and GA₅₃-aldehyde, all giving mass spectra with ¹⁴C-isotope peaks. GA₈ and GA₂₈ were also identified but contained no ¹⁴C. All the [¹⁴C]GA₁₂-aldehyde metabolites, except GA₁₅, GA₂₄ and GA₅₃-aldehyde, are known endogenous GAs of P. coccineus.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC highperformance liquid chromatography - MVA mevalonic acid - S-2 2000-g supernatant     

Partial characterization of an antheridiogen of Anemia mexicana: Comparison with the antheridiogen of A. phyllitidis

An antheridiogen of Anemia mexicana Klotzsch has been partially characterized by combined gas chromatography-mass spectrometry and gas chromatography-Fourier transform/infra-red spectrometry. It is a C₁₉-gibberellin(GA)-like compound with one carboxyl group, an exocyclic methylene group and a lactone ring. It also has one hydroxyl-group and one double-bond equivalent which has not been determined. On the basis of its mass spectrum, it is not identical to previously identified monohydroxy GAs with one ring double bond such as GA₅, GA₇, GA₃₁ and GA₆₂. By direct comparison of mass spectra, the antheridiogen of A. mexicana was also determined to be different from the antheridiogens of Anemia phyllitidis (L.) Swartz, Anemia hirsuta (L.) Swartz and Lygodium japonicum (Thunb.) Sw.Abbreviations GA(s) gibberellin(s) - GA_a gibberellin A_n - GC-FT/IR combined gas chromatography-Fourier transform/infra-red spectrometry - GC-MS combined gas chromatography-mass spectrometry - IR intra-red spectrometry - KRI Kovats Retention Index - m/z mass/charge - TLC thin-layer chromatography - TMSi trimethylsilyl     

Endogenous Gibberellins in the Developing Liquid Endosperm of Tea

Changes in the kind and level of endogenous gibberellins (GAs) in the developing liquid endosperm of tea (Camellia sinensis L.) were investigated. Gibberellin A₁ (GA₁), GA₈, GA₁₉, GA₂₀, and GA₄₄ were identified by GC-MS or GC-SIM. Besides these early C-13 hydroxylated GAs, GA₃, iso-GA₃, and GA₃₈ were also identified. Of these GAs, GA₁ and GA₃ were the major gibberellins. The levels of these GAs were at a maximum in the globular embryo stage and then decreased rapidly during embryo maturation.     

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [2¹⁴-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.Abbreviations ABA abscisic acid - 3,3-DGA 3,3-dimethyl-glutaric acid - DPA dihydrophaseic acid - PA phaseic acid - GCMS gas chromatography-mass spectrometry     

Gibberellins in immature seeds and dark-grown shoots of Pisum sativum

Gibberellins (GAs) A₁₇, A₁₉, A₂₀, A₂₉, A₄₄, 2OH-GA₄₄ (tentative) and GA₂₉-catabolite were identified in 21-day-old seeds of Pisum sativum cv. Alaska (tall). These GAs are qualitatively similar to those in the dwarf cultivar Progress No. 9 with the exception of GA₁₉ which does not accumulate in Progress seeds. There was no evidence for the presence of 3-hydroxylated GAs in 21 day-old Alaska seeds. Dark-grown shoots of the cultivar Alaska contein GA₁, GA₈, GA₂₀, GA₂₉, GA₈-catabolite and GA₂₉-catabolite. Dark-grown shoots of the cultivar Progress No.9 contain GA₈, GA₂₀, GA₂₉ and GA₂₉-catabolite, and the presence of GA₁ was strongly indicated. Quantitation using GAs labelled with stable isotope showed the level of GA₁ in dark-grown shoots of the two cultivars to be almost identical, whilst the levels of GA₂₀, GA₂₉ and GA₂₉-catabolite were significantly lower in Alaska than in Progress No. 9. The levels of these GAs in dark-grown shoots were 10²- to 10³-fold less than the levels in developing seeds. The 2-epimer of GA₂₉ is present in dark-grown-shoot extracts of both cultivars and is not thought to be an artefact.Abbreviations cv cultivar - GA_n gibberellin A_n - GC gas chromatography - GC-MS combined gas chromatographymass spectrometry - HPLC high-pressure liquid chromatography - KRI Kovats retention index - MeTMSi methyl ester trimethylsilyl ether     

Identification of Endogenous Gibberellins and Abscisic Acid from Dormant Bulbils of Dioscorea japonica (Japanese Yam)

Four gibberelJins (GAs), GA₁₉, GA₂₀, GA₂₄, and GA₅₃, were identified by gas chromatography-mass spectrometry (GC-MS) from the dormant bulbils of Dioscorea japonica Thunb. ex Murray (Japanese yam), suggesting that two biosynthetic pathways of GAs, early 13- and non-13-hydroxylation pathways, are operating in the bulbils. Abscisic acid was also identified by GC-MS.     

The effect of photoperiod on the levels of seven endogenous gibberellins in the long-day plant Agrostemma githago L.

The following seven gibberellins (GAs) have been identified by gas chromatography-mass spectrometry in shoots and leaves of the long-day plant Agrostemma githago: GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, and 3-epi-GA₁. The levels of these compounds were measured, using selected ion monitoring, during photoperiodic induction. The levels of GA₄₄, GA₁₉, GA₁₇, and GA₂₀ all increased to a peak at eight long days (LD), followed by a decline, while the levels of GA₁ and 3-epi-GA₁ did not reach a peak until 12 LD. The level of GA₅₃ remained steady over the first 10–12 LD. Later in the LD treatment the levels of GA₅₃, GA₄₄, GA₁₉, and GA₁₇ increased again. The rate of metabolism of all GAs except GA₅₃ was higher after 12–16 LD than under short days. These data thus provide indirect evidence for an effect of photoperiodic induction on GA turnover in A. githago.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - LD long day(s) - MeTMS trimethylsilylether of the methyl ester - SD short day(s) - SIM selected ion monitoring     

Accumulation of C19-gibberellins in the gibberellin-insensitive dwarf mutantgai ofArabidopsis thaliana (L.) Heynh

The endogenous gibberellins (GAs) from shoots of the GA-insensitive mutant,gai, ofArabidopsis thaliana were analyzed and compared with the GAs from the Landsberg erecta (Ler) line. Twenty GAs were identified in Ler plants by full-scan gas chromatography-mass spectrometry (GC-MS) and Kovats retention indices (KRI's). These GAs are members of the early-13-hydroxylation pathway (GA₅₃, GA₄₄, GA₁₉, GA₁₇, GA₂₀, GA₁, GA₂₉, and GA₈), the non-3,13-hydroxylation pathway (GA₁₂, GA₁₅, GA₂₄, GA₂₅, GA₉, and GA₅₁), and the early-3-hydroxylation pathway (GA₃₇, GA₂₇, GA₃₆, GA₁₃, GA₄, and GA₃₄). The same GAs, except GA₅₃, GA₄₄, GA₃₇, and GA₂₉ were detected in thegai mutant by the same methods. In addition, extracts fromgai plants contained GA₄₁ and GA₇₁. Both lines also contained several unknown GAs. In Ler plants these were mainly hydroxy-GA₁₂ derivatives, whereas in thegai mutant hydroxy-GA₂₄, hydroxy-GA₂₅, and hydroxy-GA₉ compounds were detected. Quantification of seven GAs by GC-selected ion monitoring (SIM), using internal standards, and comparisons of the ion intensities in the SIM chromatograms of the other thirteen GAs, demonstrated that thegai mutant had reduced levels of all C₂₀-dicarboxylic acids (GA₅₃, GA₄₄, GA₁₉, GA₁₂, GA₁₅, GA₂₄, GA₃₇, GA₂₇, and GA₃₆). In contrast,gai plants had increased levels of C₂₀-tricarboxylic acid GAs (GA₁₇, GA₂₅, and GA₄₁) and of all C₁₉-GAs (GA₂₀, GA₁, GA₈, GA₉, GA₅₁, GA₄, GA₃₄, and GA₇₁) except GA₂₉. The 3β-hydroxylated GAs, GA₁ and GA₄, and their respective 2β-hydroxylated derivatives, GA₈ and GA₃₄, were the most abundant GAs found in shoots of thegai mutant. Thus, thegai mutation inArabidopsis results in a phenotype that resembles GA-deficient mutants, is insensitive to both applied and endogenous GAs, and contains low levels of C₂₀-dicarboxylic acid GAs and high levels of C₁₉-GAs. This indicates that theGAI gene controls a step beyond the synthesis of an active GA. Thegai mutant is presumably a GA-receptor mutant or a mutant with a block in the transduction pathway between the receptor and stem elongation. We thank Dr. L.N. Mander, Australian National University, Canberra, for providing [²H]gibberellins, Dr. B.O. Phinney, University of California, Los Angeles, USA for [¹³C]GA₈, and Dr. D.A. Gage, MSU-NIH Mass Spectrometry Facility (grant No. DRR00480), for advice with mass spectrometry. This work was supported by a fellowship from the Spanish Ministry of Agriculture (I.N.I.A.) to M.T., by the U.S. Department of Energy under Contract DE-ACO2-76ERO-1338, and by U.S. Department of Agriculture grant No. 88-37261-3434 to J.A.D.Z.     

Identification and localization of gibberellins in maturing seeds of the cucurbit Sechium edule,and a comparison between this cucurbit and the legume Phaseolus coccineus

Twenty known gibberellins (GAs) have been identified by combined capillary gas chromatography-mass spectrometry in extracts from less than 10 g fresh weight of maturing seeds of the cucurbit Sechium edule Sw. The GAs are predominantly 3- and-or 13-hydroxylated. This is the first reported identification of non-conjugated 13-hydroxylated GAs in a cucurbit. Gibberellin A₈ and gibberellin A₈-catabolite are the major GAs in terms of quantity and are largely accumulated in the testa. The catabolites of 2-hydroxylated GAs are ,-unsaturated ketones which no longer possess of a -lactone. They were hitherto known only in legumes. The presence of GA₈-catabolite as a major component of Sechium seeds indicates that the distribution of these GA-catabolites may be more widespread than previously envisaged. The localization of known GAs in maturing seeds of the legume Phaseolus coccineus L. was found to resemble closely that in Sechium. Gibberellin A₈, a putative conjugate of GA₈ and GA₈-catabolite are accumulated in the testa. The localization in the testa of end-products of the GA-biosynthetic pathway, which was first observed in maturing seeds of Pisum sativum, and is now described in Phaseolus and Sechium, may be a general feature of seed development.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry     

Identification of gibberellin A20, abscisic acid,and phaseic acid from flowering Bryophyllum daigremontianum by combined gas chromatography-mass spectrometry

Summary The presence of abscisic and phaseic acid in a purified acidic extract from flowering plants of the long-short-day plant Bryophyllum daigremontianum [(R. Hamet and Perr.) Berg.] was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of their methyl esters. Gibberellin A₂₀ (GA₂₀) was identified by GC-MS of the methyl ester and the trimethylsilyl ether of the methyl ester. The following levels of the 3 compounds per kg fresh weight were estimated: Abscisic acid, 5.5 g; phaseic acid, 9.4g; gibberellin A₂₀, 0.8 g. When GA₂₀ and four other GAs were applied to Bryophyllum under shortday conditions, the order of effectiveness for induction of flower formation was: GA₂>GA₁>GA₅=GA₇>GA₂₀. The low biological activity of the native GA₂₀ is discussed.     

Identification of gibberellins in the rice plant and quantitative changes of gibberellin A19 throughout its life cycle

The major endogenous gibberellin (GA) in shoots, roots and ears of the rice plant, Oryza sativa L. japonica cv. Nihonbare, was identified as GA₁₉ by combined gas liquid chromatography-mass spectrometry (GC-MS) and GC-selected ion current monitoring (GC-SICM). Another GA present in these tissues in small quantity was tentatively identified as GA₁ by GC-SICM, and GA₄ may be present in the seeds (kernels) of 3rd-leaf-stage seedlings. Using GC-SICM, the GA₁₉ content was quantified throughout the life cycle of rice plants. It was found to reach high levels (ca. 10–15 g/kg fresh weight) in 3rd-leaf seedlings, at panicle initiation (shoots), and during heading and anthesis (ears). The levels of GA₁₉ in Oryza sativa indica cv. T-136 underwent changes closely similar to those found in Nihonbare. The growth-promoting activity in rice of exogenous GA₁₉ is generally considerably less than that of GA₁. It therefore seems possible that GA₁₉ functions as a pool GA. The level of active GAs such as GA₁ may be regulated by the rate of biosynthesis of GA₁₉ or its metabolic conversions.Abbreviations GA(s) gibberellin(s) - GA_n gibberellin A_n - GA_n-MeTMS trimethylsilyl ether of GA_n methyl ester - GC-MS combined gas liquid chromatography-mass spectrometry - GC-SICM combined gas liquid chromatography-selected ion current monitoring - TLC thin-layer chromatography     

Water stress and the catabolism of (±)-abscisic acid by excised leaves of Hordeum vulgare L. cv. Dyan

When excised, light-grown leaves of Hordeum vulgare were fed with (±)-[2-¹⁴C]-abscisic acid and stressed until they had lost 12% of their original fresh weight, marked changes in the distribution of radioactivity between abscisic acid and its catabolites were observed. Wilted leaves were less able than their turgid counterparts to transform (±)-[2-¹⁴C]-abscisic acid into 2-hydroxymethyl abscisic acid, dihydrophaseic acid and water-soluble conjugates of abscisic acid. Water stress had little effect on the production of phaseic acid although refeeding studies with [¹⁴C]-phaseic acid showed that the step from phaseic acid to dihydrophaseic acid was inhibited in wilted leaves. Evidence was obtained which suggested that these changes did not result from dilution of applied, radiolabelled substrate by endogenous abscisic acid. The catabolites of (±)-abscisic acid were identified by capillary gas chromatography-mass spectrometry.     

Gibberellins in suspensor,embryo and integument from very young seeds of Phaseolus coccineus L.

Endogenous gibberellins (GAs) were extracted from suspensor, embryo and integument of very young seeds of Phaseolus coccineus L. and detected by combined gas chromatography-mass spectrometry (GC-MS). Results show the presence of one C₂₀-GA, GA₄₄ and five C₁₉-GAs in the suspensor: GA₁, GA₄, GA₅, GA₆ and GA₈, and four C₁₉-GAs in the integument: GA₁, GA₅, GA₆ and GA₈. Only traces of GA₁ and GA₅ were identified in the embryo. A compound structurally related to GAs was identified as tetrahydroxy-Kauranoic acid in suspensor, integument and, only in trace amounts, in the embryo.     

Gibberellin biosynthesis from gibberellin A12-aldehyde in a cell-free system from germinating barley (Hordeum vulgare L., cv. Himalaya) embryos

Gibberellin (GA) metabolism from GA₁₂-aldehyde was studied in cell-free systems from 2-d-old germinating embryos of barley. [¹⁴C]- or [17-²H₂]Gibberellins were used as substrates and all products were identified by combined gas chromatography-mass spectrometry. Stepwise analysis demonstrated the conversion of GA₁₂-aldehyde via the 13-deoxy pathway to GA₅₁ and via the 13-hydroxylation pathway to GA₂₉, GA₁ and GA₈. In addition, GA₃ was formed from GA₂₀ via GA₅. We conclude that the embryo is capable of producing gibberellins that can induce -amylase production in the aleurone layer. There was no evidence for 12- or 18-hydroxylation and GA₄ was neither synthesised nor metabolised by the system. All metabolically obtained GAs, with the exception of GA₃, were also found as endogenous components of the cell-free system in spite of ammonium-sulfate precipitation and desalting steps.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography We thank Mrs. G. Bodtke and Mrs. B. Schattenberg for preparing the barley embryos and the Deutsche Forschungsgemeinschaft for supporting this work.     

Identification of gibberellin A9 methyl ester as a natural substance regulating formation of reproductive organs in Lygodium japonicum

Prothallia of Lygodium japonicum (Thunb.) Sw. were aseptically cultured under white light in a mineral solution. Solvent fractionation of the resultant culture medium and subsequent preparative thinlayer chromatography yielded a fraction that induced antheridium formation and inhibited archegonium formation. Combined gas chromatography-selected ion monitoring analysis of this fraction confirmed the presence of gibberellin A₉ methyl ester (GA₉-me) as an antheridiogen and an inhibitor of archegonium formation. Exogenously applied [³H]GA₉ was rapidly converted to [³H]GA₉-me in the prothallial tissue. Authentic GA₉-me was active to 10^-10M in antheridium formation and to 10^-9M in the inhibition of archegonium formation.Abbreviations GAs gibberellins - GA_n gibberellin A_n - GA_n me, gibberellin A_n methyl ester - TLC thin-layer chromatography - GCSIM Combined gas chromatography-selected ion monitoring     

Hormones of young tassels of Zea mays

The ethyl acetate-soluble acids from an aqueous methanolic extract of young tassels from Zea mays plants were fractionated by treatment with PVP, then by chromatography on a column of celite-charcoal. Methylated and trimethylsilylated fractions were analysed by GC/MS and the following compounds were identified by comparison with reference spectra: GA₁₇, GA₁₉, GA₂₀, GA₄₄, GA₅₃, ABA, phaseic acid and dihydrophaseic acid. Evidence is also presented for the presence of metabolise C of ABA and of a 16,17-dihydro-17-hydroxy-derivative of GA₅₃. In addition, the presence of small amounts of GA₁, GA₈ and GA₂₉, was indicated from a derivatized fraction analysed by capillary GC/SICM.