Analysis of picogram quantities of indole-3-acetic acid by high performance liquid chromatography-fluorescence procedures

The use of spectrofluorimeter coupled to a reverse phase high performance liquid chromatography column permits selective detection of indole-3-acetic acid at the low picogram level. The value of the technique is demonstrated by the analysis of endogenous IAA in elongating shoots, xylem sap and callus of Douglas-fir. The data are also used to illustrate a procedure whereby the accuracy of chromatographic analyses can be verified within definable probability limits.Abbreviations GC-MS combined gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - SEC steric exclusion chromatography - SICM selected ion current monitoring Technical Paper No. 5379 from the Oregon State University Agricultural Experiment Station     

Apical dominance and the levels of indole acetic acid in Phaseolus lateral buds

Combined gas chromatography-mass spectrometry procedures have been used to establish that the indole acetic acid levels of lateral buds from Phaseolus seedlings rise following removal of the shoot apex.Abbreviations GC gas chromatograph - GC-MS combined gas liquid chromatography mass spectrometry - bis-TMS bis-trimethylsilyl - IAA indole acetic acid - MPM multiple-peak monitoring - MS mass spectrometer - GLC gas liquid chromatography - TLC thin-layer chromatography     

The current status of physiology and biochemistry of brassinosteroids

Brassinolide, first isolated from pollen of rape as a plant growth promoting substance, has been found to be widely distributed in the plant kingdom. Over thirty endogenous analogues, called collectively brassinosteroids, have been isolated and identified. As a new class of plant hormones, brassinosteroids show not only growth promoting activity but also other physiological effects on the growth and development of plants and draw attention as promising chemicals for practical application in agriculture. This review describes the current status of the studies on the natural occurrence, analysis, physiological actions, metabolism and biosynthesis of brassinosteroids.Abbreviations ABA abscisis acid - BR brassinosteroid - GA gibberellin - GC-MS combined gas chromatography-mass spectrometry - GC-SIM combined gas chromatography-selected ion monitoring - HPLC high performance liquid chromatography - IAA indole-3-acetic acid     

Gibberellins and the procera mutant of tomato

The procera mutant of tomato (Lycopersicon esculentum L.) has a phenotype which is remarkably similar to that of normal tomatoes treated with exogenous gibberellin (GA), indicating that it might be a GA over-producer. However, analysis of endogenous GAs by gas chromatography-mass spectrometry showed that Procera actually has lower levels of GA₂₀ and GA₁ than normal. The reason for these anomalously low GA levels is not clear, as there was no difference between procera and normal plants in their ability to metabolize [³H]GA₂₀. The procera mutant responded to exogenous gibberellic acid with increased extension growth, but the proportional response for a given dose of GA was the same in procera and normal plants. It therefore appears that the procera mutation does not directly affect either the GA status of the plant, or its ability to respond to GA.Abbreviations GA gibberellin - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - MeTMSi methyl trimethylsilyl - SIM selected ion monitoring     

Identification of abscisic acid from shoots of Salix pentandra

Abscisic acid isolated from vegetative shoots of Salix pentandra L. has been identified by high performance liquid chromatography and combined gas chromatography-mass spectrometry.     

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     

Indolyl-3-acetic acid from shoots of Salix pentandra

Indolyl-3-acetic acid from vegetative shoots of Salix pentandra L. has been isolated and identified by high performance liquid chromatography and combined gas chromatography-mass spectrometry.     

Comparison of gas chromatography-mass spectrometry,radioimmunoassay and bioassay for the quantification of gibberellin A9 in norway spruce (Picea abies (L.) Karst.)

Quantitative estimates of gibberellin A₉ in Norway spruce extracts obtained by gas chromatography-mass spectrometry, radioimmunoassay (RIA_ and bioassay were compared after successive purifications of the extracts. The extracts were assayed in several dilutions with and without the addition of standard gibberellin A₉, thus showing the effect of extract components on the response of the assays. Radioimmunoassay produced estimates comparable to gas chromatography-mass spectrometry after one purification step by high-performance liquid chromatography. Extracts purified by polyvinylpyrrolidone-column chromatography and solvent partitioning but not high-performance liquid chromatography resulted in inaccurate RIA estimates. The performance of the RIA could be monitored by logit-log transformations of the standard curve and extract dilution curve and by calculating the slope of the standard addition curve. It was, however, not possible to correct for the interference caused by extract components by the standard addition procedure. Quantifications by Tan-ginbozu dwarf-rice bioassay were accurate, but a large and unpredictable variation makes it unsuitable for quantitative determinations.Abbreviations FW fresh weight - GA₉ gibberellin A₉ - GA₉–Me methylated GA₉ - GC-MS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - MID multiple-ion detection - RIA radioimmunoassay     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay     

Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography     

Identification of gibberellin A1 in the embryo suspensor of Phaseolus coccineus

By combined gas chromatography-mass spectrometry the gibberellin present in suspensors of heart-shaped embryos of Phaseolus coccineus has been identified as Gibberellin A₁ (GA₁). The amount of GA₁ in 2000 suspensors (452 mg), as estimated by gas chromatography. was 4g. The presence of GA₁ in suspensors of P. coccineus is discussed in relation to our present knowledge of the occurrence of many gibberellins in developing seeds and immature fruits of the same species.Abbreviations FID flame ionization detector - GA gibberellin - GC gas chromatography - MS mass spectrometry - PGC preparative gas chromatography - Stage A heart-shaped embryo - Stage B cotytedonary embryo - TMS trimethylsilyl     

Occurrence and identification of jasmonic acid and its amino acid conjugates induced by osmotic stress in barley leaf tissue

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.Abbreviations JM methyl jasmonate - JA jasmonic acid - JIP(s) jasmonate-induced protein(s) - PEG polyethylene glycol - RIA radioimmunoassay - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry - R _t retention time - IAA indole-3-acetic acid     

The endogenous gibberellins of vegetative and reproductive tissue of G2 peas

The gibberellins (GAs) of both vegetative (leaves and stems) and reproductive (pods and seeds) tissue of the G2 strain of peas Pisum sativum L. were characterized in purified extracts by a combination of sequential silicic-acid partition column chromatography, and gas chromatography-mass spectrometry. Gibberellins A₁₉, A₂₀, A₂₉ and an A₂₉ catabolite were identified in both types of tissue. Gibberellins A₉, A₁₇ and A₄₄ were also found in pods and seeds.Abbreviations FID Ilame ionization detector - GA(s) gibberellin(s) - GC gas chromatograph(y) - HPLC high performance liquid chromatograph(y) - LD long day - MS mass spectrum(a) or mass spectrometer(ry) - SD short day     

Dihydrozeatin riboside,a minor cytokinin from the leaves of Phaseolus vulgaris L.

Dihydrozeatin riboside has been identified in the leaves of decapitated bean plants by Sephadex LH20 chromatography and combined gas chromatography-mass spectrometry. The relationship between the cytokinins isolated and identified from this system and those previously reported in Phaseolus is discussed.Abbreviations DHZ dihydrozeatin - DHZOG dihydrozeatin-O--D-glucoside - DHZR dihydrozeatin riboside - GCMS combined gas chromatography-mass spectrometry - GLC gas-liquid chromatography - TIC total ion current - TMS trimethylsilyl - Z zeatin - ZR zeatin riboside     

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     

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     

Identification of a diene conjugated component of human lipid as octadeca-9,11-dienoic acid

The predominant diene conjugated acyl residue in triacylglycerols, cholesteryl esters and phospholipids in human serum was identified by high performance liquid chromatography and capillary gas chromatography-mass spectrometry. It is an octadeca -9,11-dienoic acid.     

Cytokinin metabolism in Phaseolus vulgaris L.

The major cytokinins in stems of decapitated, disbudded bean plants have been identified by enzymic degradation, Sephadex LH20 and reversed phase high performance liquid chromatography, and by combined gas chromatography-mass spectrometry as 6-(4-hydroxy-3-methylbut-trans-2-enylamino)-9--D-ribofuranosylpurine (zeatin riboside), 6-(4-hydroxy-3-methylbutylamino)-9--D-ribofuranosylpurine (dihydrozeatin riboside), and the 5-phosphates of these compounds (zeatin ribotide and dihydrozeatin ribotide). Minor cytokinins in this tissue were tentatively identified as dihydrozeatin-O--D-glucoside and zeatin ribotide-O--D-glucoside. [8-¹⁴C-]Dihydrozeatin appeared to be rapidly metabolized to dihydrozeatin ribotide when supplied to segments of stems from decapitated plants. These results are discussed in relation to the metabolism and distribution of cytokinins in the whole plant.Abbreviations TEAB triethyl ammonium bicarbonate - UV ultra-violet - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - TMS trimethyl silyl     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography     

Growth regulators,Rhizobium and nodulation in peas

The cytokinin content of roots and nodules of pea and the culture supernatants from two strains of Rhizobium leguminosarum has been examined. Roots, nodules and wild-type Rhizobium culture medium contained very little cytokinin as indicated by bioassay. Chemical ionisation gas chromatography-mass spectrometric analysis of the isopentenyladenine content of the culture medium from the Rhizobium strains confirmed that the content of the wild-type was low (approx. 1 ng dm^-3) but that it was increased by the introduction of the Agrobacterium Ti plasmid into the Rhizobium strain.Abbreviations CI chemical ionisation - GCMS gas chromatography-mass spectrometry - HPLC high performance liquid chromatography - iPAde isopentenyladenine - MIM multiple ion monitoring