Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings

Seven day old seedlings of Pisum sativum L., cv. Kleine Rheinländerin, were wilted for 3 days. After partially removing the roots, they were rewatered and at the same time radioactive abscisic acid([1-¹⁴C]ABA, spec. activity 1.7·10⁸d s^-1mmol^-1) was applied for 1 h via the xylem of the roots. After 24 h, 4 days, and 12 days the seedlings were extracted and the metabolites of ABA were analyzed by means of thin-layer and gas chromatography in combination with mass spectrometry, autoradiography, and scintillation counting. Phaseic acid (PA) and dihydrophaseic acid (DPA) were identified as metabolites of ABA. The presence of another ABA-metabolite was also demonstrated. From its mass spectrum it has been postulated that this metabolite is 4-desoxy-ABA. In addition to these substances, several other metabolites, which are more polar than ABA and its known degradation products, were present in the seedlings. The quantity and number of these unknown metabolites increased with time.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen bis(5-phenyloxazole)     

Gibberellin-like substances in Bryophyllum daigremontianum and the distribution and persistence of applied gibberellin A3

Summary Acidic extracts of the long-short-day plant Bryophyllum daigremontianum contain two gibberellin (GA)-like substances called fractions I and II. In plants under permanent short-day (SD) conditions the levels of both I and II are very low. In continuous long days (LD) the total GA content is approximately 20 times higher than in SD, mainly due to an increased level of II. Extracts of plants induced to flower by the shift LDSD show a further increase in the level of II. Application of GA₃ to plants in SD causes normal flower formation, but the level of fraction II remains as low as in vegetative plants in permanent SD.Approximately 10% of the GA₃ applied could still be recovered from leaves and inflorescences after 45 days, indicating that GA₃ is very stable in Bryophyllum, Most of the GA₃ recovered was still associated with the treated leaves, but small amounts could be detected in other leaves and in inflorescences. Results of grafting experiments indicate that these low levels of GA₃ are adequate to induce production of the floral stimulus.     

Immunoaffinity chromatography of abscisic acid combined with electrospray liquid chromatography-mass spectrometry

Polyclonal antibodies with high specificity for C1-immobilised (+)-cis,trans-abscisic acid (ABA) were raised, characterised by enzyme-linked immunosorbent assay (ELISA) and used for preparation of an immunoaffinity chromatography (IAC) gel. The detection limit of the ELISA was approximately 4.6x10(-10)mol/L. Sensitive electrospray liquid chromatography-mass spectrometry (LC-ESI-MS) methods were also developed with detection limits below 0.1x10(-12)mol. The IAC allowed quick, single-step processing of samples prior to the analyses. The LC-ESI-MS and LC-ELISA techniques were used for comparative estimation of endogenous ABA levels in immunoaffinity purified extracts of normal and water-stressed Nicotiana tabacum L. leaves. The analytical approaches were validated using deuterium- and tritium-labelled internal standards, respectively. The IAC method was found to be highly effective, sensitive and convenient for isolating the target analyte from plant material.     

Gibberellin A20 content of Bryophyllum daigremontianum under different photoperiodic conditions as determined by gas-liquid chromatography

Summary The gibberellin A₂₀ content of the long-short-day plant Bryophyllum daigremontianum (R. Hamet and Perr.) Berg. under different photoperiodic conditions was determined by gas-liquid chromatography. Purified extracts from ten plants were adequate for quantitative analysis by this method. The level of GA₂₀ increased following transfer from long-day (LD) to short-day (SD) conditions until after 38 SD it was three times higher on a dry weight basis than in comparable plants under continuous LD. No GA₂₀ could be detected in extracts of plants under permanent SD. These results are in agreement with earlier data obtained by assaying Bryophyllum extracts with the d-5 mutant of corn.     

Definitive identification of indole-3-acetic acid and abscisic acid in shoots of Coleus blumei by gas chromatography-mass spectrometry

Mass spectra provide definitive identification of indole-3-acetic acid and abscisic acid in shoots of Coleus blumei, a species used for studying the hormone control of plant development since the early 1930s.     

Identification of benzophenanthridine alkaloids from Bocconia arborea by gas chromatography-mass spectrometry

A methanol extract of the bark of Bocconia arborea was fractionated on silica gel and the fractions analysed using gas chromatography coupled with mass spectrometry (GC-MS). Several benzophenanthridine alkaloids were identified including dihydrosanguinarine, oxysanguinarine, 11-acetonyldihydrochelerythrine, dihydrochelerythrine, chelerythrine, chelerythridimerine and angoline as the principal constituents. The results show that the direct GC-MS analysis of these alkaloids is possible with a clear distinction between the compounds. The technique is shown to be a valuable tool and an alternative technique to classical phytochemical procedures permitting the fast analysis of alkaloids mixtures.     

Identification of gibberellins A17, A25, A45, abscisic acid,phaseic acid,and dihydrophaseic acid in seeds of Pyrus communis

Gibberellin A₁₇, abscisic acid, and 4′-dihydrophaseic acid were identified by GC-MS of derivatized extracts from both immature and mature seeds of pear. Immature seeds also contained phaseic acid, gibberellins A₂₅ and A₄₅, and two presumed mono-hydroxylated derivatives of GA₄₅, one of which was tentatively identified as 3β-hydroxy-GA₄₅. Several presumed metabolites of abscisic acid were detected in both mature and immature seeds.     

Identification of gibberellins A20 and A29 in seed of Pisum sativum cv. Progress No. 9 by combined gas chromatography-mass spectrometry

Summary The gibberellin A₁ (GA₁)-like and GA₅-like fractions from immature seeds of Pisum sativum cv. Progress No. 9 were identified by combined gas chromatography-mass spectrometry as GA₂₉ and GA₂₀ respectively.     

Determination of urinary trans,trans-muconic acid by gas chromatography-mass spectrometry

A sensitive and specific method for the determination of trans,trans-muconic acid (t,t-MA) in urine is described. After clean-up on an anion-exchange cartridge, t,t-MA was derivatized with BF₃-methanol to the dimethyl ester and analyzed by gas chromatography-mass spectrometry (GC-MS), with 2-bromohexanoic acid as an internal standard. The limit of detection was 0.01 mg/l, the coefficient of variation for duplicate analysis in a series of urine samples (n = 50) was 2.6% and the recovery rate ranged from 93.3 to 106.3%. The between-day and within-day precision for the analysis were 7.4 and 14.6%, respectively. The method was applied to the determination of t,t-MA in urine samples from smokers and non-smokers. The mean concentration of t,t-MA in urine of 10 smokers was 0.09 ± 0.04 mg/g creatinine and was significantly (p = 0.012) higher than that found in urine of 10 non-smokers (0.05 ± 0.02 mg/g creatinine). In contrast to the results obtained with the commonly used high-performance liquid chromatographic ultraviolet detection (HPLC-UV) methods, no interference between t,t-MA and other urinary compounds was found. This GC-MS method is both specific and sensitive for biomonitoring of low environmental benzene exposure.     

Identification and measurement of indoleacetic and abscisic acids in the cambial region of Picea sitchensis (Bong.) Carr. by combined gas chromatography-mass spectrometry

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified by combined gas chromatography-mass spectrometry (GCMS) in fractions obtained by diffusion and extraction from bark peelings of Sitka spruce. A procedure is described for the quantitative analysis of IAA and ABA levels in the same extract using the GCMS technique of single-ion current monitoring. This procedure was used to measure the diffusible, free, and bound fractions of IAA and ABA in the cambial region of Sitka spruce throughout one year; the range in concentration for these fractions was 0.06–0.30, 0.46–3.85, and 0.04–0.20 g/g oven-dry weight, respectively, for IAA, and 0–0.08, 0.03–2.21, and 0.13–0.66 g/g oven-dry weight, respectively, for ABA. Movement in the cambial region was found to be polar for endogenous IAA and nonpolar for endogenous ABA. Recoveries of [¹⁴C]IAA internal standards showed that 73–99.5% of the IAA was lost during purification, and that there could be up to 5-fold differences in recovery between purifications, indicating that IAA loss shold be measured in quantitative analyses.Abbreviations ABA aoscisic acid - GCMS combined gas chromatography-mass spectrometry - IAA indole-3-acetic acid - PVP polyvinylpyrrolidone - SICM single ion current monitoring - TMS trimethylsilyl     

Abscisic acid,phaseic acid and gibberellin contents associated with dormancy and germination in barley

Analyses of abscisic acid (ABA), ent -kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley ( Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent -kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent -kaurenoids and GAs, including GA_1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent -kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent -kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.     

Identification of dihydroetorphine in biological fluids by gas chromatography-mass spectrometry

A method for the monitoring of dihydroetorphine hydrochloride, a powerful anaesthetic and analgesic drug, in biological fluids was developed, involving GC-MS with multiple selected-ion monitoring. Dihydroetorphine was extracted from human blood and urine with dichloromethane and then derivatized with N-heptafluorobutyrylimidazole after having been concentrated to dryness. A dihydroetorphine monoheptafluorobutyl derivative was formed, which showed good behaviour in GC-MS with electron impact ionization. Its molecular ion, m/z 609, and its main fragments, m/z 576, 534, 522 and 508, were selected as the ions for identification owing to their relative peak intensities and characteristics. The target drug was identified based on its retention time, its selected multiple ions and their relative intensities. This method was successfully used for the detection of dihydroetorphine in blood and urine from a dihydroetorphine addict and a poisoned patient, respectively.     

Quantification of urinary 5-aminolevulinic acid by gas chromatography-mass spectrometry

In this report, we describe a method for the specific quantification of urinary 5-aminolevulinic acid. It is based on gas chromatographic mass spectrometric measurement of the trimethylsilyl ester of the ethylacetoacetate pyrrole derivative of 5-aminolevulinic acid. Selected ion monitoring (SIM) was used for quantification using 3-hydroxymyristic acid as an internal standard. The detection limit of this method was 0.1 nmol/ml. This method was applied to the determination of urinary 5-aminolevulinic acid from a tyrosinemia type I patient and normal subjects, and 21.4 mmol/mol creatinine and 0.54+/- 0.49 mmol/mol creatinine (n = 7), respectively, were detected. Less than 0.2 ml urine was sufficient for the determination of 5-aminolevulinic acid in healthy subjects.     

Genetic analyses of interactions among gibberellin, abscisic acid, and brassinosteroids in the control of flowering time in Arabidopsis thaliana

Background

Genetic interactions between phytohormones in the control of flowering time in Arabidopsis thaliana have not been extensively studied. Three phytohormones have been individually connected to the floral-timing program. The inductive function of gibberellins (GAs) is the most documented. Abscisic acid (ABA) has been demonstrated to delay flowering. Finally, the promotive role of brassinosteroids (BRs) has been established. It has been reported that for many physiological processes, hormone pathways interact to ensure an appropriate biological response.

Methodology

We tested possible genetic interactions between GA-, ABA-, and BR-dependent pathways in the control of the transition to flowering. For this, single and double mutants deficient in the biosynthesis of GAs, ABA, and BRs were used to assess the effect of hormone deficiency on the timing of floral transition. Also, plants that over-express genes encoding rate-limiting enzymes in each biosynthetic pathway were generated and the flowering time of these lines was investigated.

Conclusions

Loss-of-function studies revealed a complex relationship between GAs and ABA, and between ABA and BRs, and suggested a cross-regulatory relation between GAs to BRs. Gain-of-function studies revealed that GAs were clearly limiting in their sufficiency of action, whereas increases in BRs and ABA led to a more modest phenotypic effect on floral timing. We conclude from our genetic tests that the effects of GA, ABA, and BR on timing of floral induction are only in partially coordinated action.     

Determination of muramic acid in organic dust by gas chromatography-mass spectrometry

A method is described for the quantitation of muramic acid, a marker of bacterial peptidoglycan, in organic dust. House dust samples were hydrolysed in hydrochloric acid and then extracted with hexane to remove hydrophobic compounds. The aqueous phase was evaporated, heated in a silylation reagent to form trimethylsilyl derivatives, and analysed by gas chromatography-mass spectrometry. The muramic acid derivative gave two peaks upon injection into the gas chromatograph-mass spectrometer. Injection of 10 pg of the derivative gave a signal-to-noise ratio of 17 for the dominating peak when using selected ion monitoring in the electron impact mode, and a linear calibration curve was achieved upon analysis of samples containing 5–1500 ng of muramic acid. In a house dust sample, 40 ng of muramic acid was found per mg of dust; the coefficient of variation was 8.2% (n = 6, 1.2 mg of dust analysed). The described method is rapid and simple to apply, and should therefore become widely used for measuring peptidoglycan in many types of environmental samples, including organic dust.     

Characterization of gibberellins from dark-grown Phaseolus coccineus seedlings by gas-liquid chromatography and combined gas chromatography-mass spectrometry

Summary An extract from 6000 dark-grown Phaseolus coccineus seedlings was purified by countercurrent distribution and G-10 Sephadex followed by gradient elution from a silicic acid partition column with increasing amounts of ethyl actetate in n-hexane. 25 fractions were collected and tested with the barley-aleurone, Tan-ginbozu dwarf-rice, lettuce, cucumber, dwarf-pea, d-1, d-2, d-3 and d-5 maize, oat first-internode, and sugarcane-spindle bioassays. Major gibberellin (GA)-like activity was detected in fractions 4 (500g GA₃-equivalents) and 12–13 (270 g GA₃-equivalents) with smaller amounts in fractions 6, 8–9, 15–16, 18, 20, 23 and 25. The extracts were also applied to AMO-1618=dwarfed Ph.-coccineus seedlings. Fractions 4, 8 and 12 promoted the growth of both light- and dark-grown seedlings. GA₁, GA₃, GA₄ and GA₈ were active in the Phaseolus bioassay but GA₈-glucoside was inactive.The biological and chromatographic properties of fractions 4, 8–9 and 12–13 correspond with those of GA₄, GA₁₉ and GA₁. The identity of GA₄ in fraction 4 was conclusively established by combined gas chromatography-mass spectrometry (GC-MS) of the methyl ester and the trimethylsilyl ether of the methyl ester. Gasliquid-chromatography peaks corresponding to these derivatives of GA₁₉ and GA₁ were detected on QF-1 and SE-33 columns but their intensities were too weak to permit conclusive identification by GC-MS.Supported by an S.R.C. StudentshipSupported by a NATO Grant.Supported by NRC Grant A-5727.     

Abscisic acid,abscisic acid-esters and phaseic acid in vegetative terminal buds of Acer pseudoplatanus during emergence from winter dormancy

Levels of free-abscisic acid and boundabscisic acid (alkaline hydrolyzable abscisic acidesters) in replicated samples of terminal vegetative buds of sycamore trees were measured during natural emergence from winter dormancy by gas chromatographic methods together with isotope dilution estimation of recovery rates. Not until after the buds had been released from true dormancy in January by winter chilling did any clear change occur in either abscisic acid (ABA) fraction, or in total ABA, on any basis of comparison. The percentage of total ABA present as the free acid declined at the end of true dormancy to approximately two-thirds of its value in the earlier winter months. It is concluded that glucosylation of ABA is unlikely to play a major part in the mechanism of release from dormancy in vegetative sycamore buds. At the end of true dormancy there was a large transient increase in what appeared to be phaseic acid, but this was not accompanied by any marked decrease in either free- or bound-ABA.Abbreviations ABA abscisic acid - TLC thin layer chromatography - GLC gas chromatography