Chromatography of acid phytohormones on columns of Sephadex LH‐20 and insoluble poly‐N‐vinylpyrrolidone, and application to the analysis of conifer extracts

Sandberg, Göran; Dunberg, Arne; Odén, Per Christer

doi:10.1111/j.1399-3054.1981.tb04490.x

Cited by 17 publications

(3 citation statements)

References 15 publications

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“…Thus an unequivocal quantitative determination with a detection limit of about 0.1 ng was achieved without further purification by High-Performance Liquid Chromatography (HPLC). GC-ECD was the most frequently employed method for quantification of ABA in coniferous needles (Little et al 1978;Sandberg et al 1981;Johnson and Ferrell 1982;Oden and Dunberg 1984;Frenzel et al 1987;Puttonen 1987;Squire et al 1988;Naumann et al 1988). A chromatogram of the purified extract using optimai separation conditions is shown in Fig.…”

Section: Methodical Resultsmentioning

confidence: 97%

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

Kraus

Ziegler

1993

Trees

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Summary. The amount of abscisic acid (ABA) in needles of silver fir from a natural location was investigated with regard to position in the crown, damage, seasonal variation, and needle age. Because of problems of quantification of ABA in coniferous needles, which contain numerous secondary plant products, a method for reliable determination of both isomers cis-trans-ABA (c-ABA) and transtrans-ABA (t-ABA) was developed. By means of gas chromatography (GC) using an electron capture detector (ECD) and a programmed temperature vaporizer (PTV) injector complete separation of both compounds was achieved. Two different pairs of fir were investigated -in each case a damaged and a healthy tree. Needles from both trees from the first and the second pair collected in September contained 500-1100 ng c-ABA/g fresh weight (FW), and the concentrations of t-ABA varied from 400 to 700 ng/g FW. Investigations from the second pair show highest amounts of 2900 ng/g Fw c-ABA and 1800 ng/g FW of t-ABA in May and June. For the first pair a higher c-ABA content was found in needles from the top of the crown than in those from the middle and the base. This difference could not be confirmed in the analysis of the second pair. Because of the strong natural deviation no statistically significant difference between the healthy and the damaged tree was found. The first pair of firs examined showed a higher t-ABA concentration than the second one. In this case the highest amount was found in the top of the crown. Methodical mistakes during the clean-up procedure and in quantification by gas chromatography could be excluded. The presence of c-and t-ABA in the purified extract was corroborated by mass spectrometry. With regard to the seasonal variation both isomers of ABA show an unequivocal trend. The maximum concentration is achieved in May to June, whereas the content is minimal inCorrespondence to: H. Ziegler August/September. In any case the level of t-ABA is lower than that of c-ABA. No correlation between the amount of ABA and the needle age could be established.

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Section: Methodical Resultsmentioning

confidence: 97%

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

Kraus

Ziegler

1993

Trees

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“…Cette différence dans l'intensité de la rhizogenèse serait due à une dégradation partielle des dérivés indoliques rhizogènes lors des opérations d'extraction et de fractionnement, plutôt qu'à un partage du pouvoir rhizogène dans les trois groupes de fractions A, B et C. Cette explication semble plus plausible dans la mesure où les Les dérivés indoliques d'origine animale ou végétale sont des composés très instables. Ils s'oxydent facilement en pré-sence d'oxygène et de peroxydes (Hinman et Lang 1965;Sandberg et al 1981aSandberg et al , 1981b) et sont à la fois thermolabiles et photolabiles (Atsumi et al 1976;Sweetser et Swartzfager 1978;Hemberg et Tillberg 1980). L'emploi d'antioxydants (Lino et Carr 1982;Sandberg et al 1984Sandberg et al , 1985 n'est pas toujours couronné de succès.…”

Section: Discussionunclassified

Production de composés indoliques rhizogènes par le ver de terre <i>Lumbricus terrestris</i>

Harti¹,

Saghi²,

Molina³

et al. 2001

Can. J. Zool.

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L'application in vitro de l'extrait brut total de vers (Lumbricus terrestris) à différentes dilutions entraîne une stimulation de la rhizogenèse des plantules de haricots (Phaseolus vulgaris). L'effet rhizogène observé est similaire à celui enregistré en présence de l'acide indole acétique, substance de croissance connue chez les végétaux et utilisée ici comme témoin à différentes concentrations. Le fractionnement de l'extrait brut de vers par chromatographie sur colonne a permis d'isoler trois groupes de fractions. Seul le groupe de fractions polaires présente un effet rhizogène notable; cet effet est cependant inférieur à celui observé respectivement en présence de l'extrait brut total de vers et de l'acide indole acétique. Les analyses de l'extrait brut de vers par chromatographie sur couches minces, utilisant des réactifs et des systèmes chromatographiques appropriés, ont révélé l'absence de l'acide indole acétique et la présence probable d'autres dérivés indoliques à comportement chromotographique neutre à basique. Les composés indoliques présumés identifiés sont le méthyl-tryptophane, la sérotonine et l'hydroxyle acide indole acétique. Les analyses par spectrométrie de masse couplées à la chromatographie en phase gazeuse, effectuées après fractionnement et purification du groupe de fractions rhizogènes, ont révélé la présence de l'hydroxyle acide indole carboxylique; celui-ci serait pré-sent sous forme de plusieurs isomères de position. Abstract: In vitro application of total gross extract of earthworms (Lumbricus terrestris) in diverse dilutions stimulates rhizogenesis in young bean plants (Phaseolus vulgaris).The observed effect is similar to that of indol acetic acid, a well-known growth enhancer in plants, used here as a control in various concentrations. Fragmentation of worm extract by column chromatography results in three groups of fractions. Only the polar group of fractions has a significant rhizogenous effect, which is, however, inferior to that observed in the presence of total gross extract of worms or of indol acetic acid. Gross extract analyses using thin layer chromatography, with appropriate chromatography systems and reagents, revealed that indol acetic acid is not present, but is probably replaced by other indol-derived substances that have a neutral to basic chromatographic behaviour. These presumed indol-derived substances are identified as methyltryptophane, serotonin, and hydroxy-indol acetic acid. Analyses using mass spectrometry combined with gas chromatography, following fragmentation and purification of the group of rhyzogenous fractions, have revealed the presence of hydroxy-indol carboxylic acid, which seems to take the form of several isomeres.

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“…The aqueous phase was then adjusted to pH 2.5 with 6 N HCl and extracted three times with EtOAc. The acidic EtOAc fraction was taken to dryness in vacuo at 35°C, dissolved in 2 ml MeOH, and further purified on a combined Sephadex LH 20/PVP column (25). The fraction containing free GAs (from 35-159 ml) was collected, adjusted to pH 2.5, and partitioned three times against EtOAc.…”

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confidence: 99%

Endogenous Gibberellins in Elongating Shoots of Clones of Salix dasyclados and Salix viminalis

Junttila

Abe²,

Pharis³

1988

Plant Physiol.

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Elongating shoots of rapidly growing clones of Salix viminalis L. (clone and Salix dasyclados Wimm. (clone 908) harvested in early August were analyzed for endogenous gibberellins (GA). Distribution of GA-like activity, determined by Tan-ginbozu dwarf rice microdrop bioassay after reverse phase C,I high performance chromatography, was similar for both species. For S. dasyclados, combined gas chromatography-selected ion monotoring (GC-SIM) yielded identifications of GA,, GAS, GA,9, GA20, and GA29. Identifications of GA4 and GA, were also made using coinjections of known amounts of [17,. By bioassay, the main activity was GA,9-like in both species. Gibbereilin Al, GA,,, and GA2.concentrations were approximated by GC-SIM using co-injections of known amounts of [17,17-2H2]GAs. Both bioassay and GC-SIM results indicated very high concentrations of GA,, and GA20 (about 6000 nanograms per kilogram fresh weight shoot tissue using GC-SIM: 800 ng using bioassay), compared to the concentration of GA, (about 130 nanograms per kilogram fresh weight using either GC-SIM or bioassay).Various species of Salix and Populus have been extensively studied in recent years in connection with short rotation forestry or energy forestry (26). Studies with Salix pentandra have shown that cessation of apical elongation growth can be prevented by exogenous GAs4 (10), and further studies have indicated that endogenous GAs may have a significant function in regulation of shoot elongation in Salix (4,11,12). Gibberellins Al, A19, A20, and A29 have recently been identified as endogenous components in S. pentandra (4). Salix viminalis and Salix dasyclados are rapidly growing willow species with an obvious potential for biomass production. In willow, the biomass production is closely related to shoot elongation and in this connection knowledge of GA metabolism is especially interesting. Identification of endogenous GAs is a prerequisite for further studies, and in this paper we report identification of several endogenous GAs from two selected clones of S. viminalis and S. dasyclados. [8]), filtered, and partitioned three times against onethird volumes of toluene to remove Chl and other nonpolar compounds. The aqueous phase was then adjusted to pH 2.5 with 6 N HCl and extracted three times with EtOAc. The acidic EtOAc fraction was taken to dryness in vacuo at 35°C, dissolved in 2 ml MeOH, and further purified on a combined Sephadex LH 20/PVP column (25). The fraction containing free GAs (from 35-159 ml) was collected, adjusted to pH 2.5, and partitioned three times against EtOAc. The EtOAc fraction was purified further by DEAE Sephadex A25 anion exchange chromatography (2). The fraction containing free GAs (from 90-140 ml) was collected and taken to the aqueous phase in vacuo at 35°C, adjusted to pH 2.5, and extracted with EtOAc. The EtOAc fraction was taken to dryness, dissolved in MeOH, adsorbed onto Celite 545, and applied to a silicic acid partition column (5 g silicic acid) saturated with 0.5 M formic acid (5, 22), and GAlike substa...

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Chromatography of acid phytohormones on columns of Sephadex LH‐20 and insoluble poly‐N‐vinylpyrrolidone, and application to the analysis of conifer extracts

Cited by 17 publications

References 15 publications

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

Quantitative analysis of abscisic acid in needles of Abies alba Mill. by electron capture gas chromatography

Production de composés indoliques rhizogènes par le ver de terre <i>Lumbricus terrestris</i>

Endogenous Gibberellins in Elongating Shoots of Clones of Salix dasyclados and Salix viminalis

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