Purification and measurement of abscisic Acid and indoleacetic Acid by high performance liquid chromatography

A procedure was selected for the simultaneous extraction and purification of abscisic acid (ABA) and indoleacetic acid (IAA). Unnecessary steps were eliminated and an accumulation of aqueous phase was avoided. The superior performance of diethyl ether (compared to ethyl acetate) for bulk purification and the superior resolution provided by 250 millimeter columns packed with 5-micrometer spherical particles of strong anion exchanger and octadecylsilane (C18) greatly facilitated the purification of samples. A fixed-wavelength (254 nanometer) ultraviolet detector and a fluorescence detector connected in series on a high performance liquid chromatograph permitted nondestructive monitoring and measurement of ABA and IAA. Derivatization was not necessary for chromatography or for detection. Isocratic elution with simple mobile phases gave sharp peaks. A few simple precautions minimized losses. Recoveries through the entire procedure averaged about 75% for ABA and about 50% for IAA. Purified ABA and IAA fractions were usually free of interfering contaminants. Identities were confirmed by gas chromatography-mass spectrometry.     

Determination of IAA and ABA in the same plant sample by a widely applicable method using GC-MS with selected ion monitoring

A method for the purification and subsequent quantification of indole-3-acetic acid (IAA) and abscisic acid (ABA) from the same sample of highly pigmented green tissue has been developed and tested in several species. Solvent partitioning and high-performance liquid chromatography (HPLC) were used for purification. Separate fractions from HPLC-containing IAA and ABA were analyzed by gas chromatography-mass spectrometry (GC-MS) using selected-ion monitoring (SIM). Isotope dilution was used to correct for incomplete recovery. Results are presented for tissue samples from 11 different species and five different plant organs. The method can be completed, for both IAA and ABA, for two samples in 8 h by an experienced technician. IAA and ABA were the dominant peaks in the gas chromatograms from HPLC-purified samples, and amounts of about 1 ng can be detected. The extract was partitioned into an aqueous solution of pH 9.5, a step suspected of ester hydrolysis. By analyzing samples known to contain esters of IAA and ABA and comparing the results with methods which excluded this step, we have shown that this partitioning does not result in erroneously high values due to ester hydrolysis. A direct comparison of the method with one in which HPLC was not employed indicates that our method measures IAA and ABA in samples in which these compounds are not detectable when HPLC is omitted. Thus, HPLC is an essential purification step for samples where contaminating compounds co-purify with IAA and ABA through the solvent-partitioning steps.     

Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole-3-acetic acid in plants

Introduction  – Jasmonic acid (JA), abscisic acid (ABA) and indole‐3‐acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection. Objective  – To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species. Methodology  – Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C₁₈ cartridges. The final extracts were derivatised with diazomethane and then measured by GC‐MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure. Results  – Sequential elution of the assimilates from the C₁₈ cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones. Conclusion  – A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue. Copyright © 2008 John Wiley & Sons, Ltd.     

Study of Parameters Involved in the Determination of IAA and ABA in Plant Materials

A procedure for the combined purification of both indolyl-3-aceticacid (IAA) and abscisic acid (ABA) is presented. The procedureis designed to obtain accurate quantifications with minimallosses and within a relatively short time. The prepurification procedure makes optimal use of C18 prepackedcartridges. A purification step by high pressure liquid chromatographyis included and discussed. The quantification of IAA is performed with the 2-methylindolepyroneassay (2-MIP-assay). An alternative way of correcting for aspecificfluorescence is proposed and tested. ABA is analysed by GLC-ECD. The accuracy of the overall method is tested using differentapproaches for both ABA and IAA. Using this method, extractionby homogenization is compared to extraction by diffusion withouthomogenization. Key words: IAA, ABA, Purification     

Abscisic Acid Alters the Metabolism of Indole-3-Acetic Acid in Senescing Flowers of Cucumis melo L

Experiments were conducted to investigate indole-3-acetic acid (IAA) and abscisic acid (ABA) metabolism associated with postanthesis senescence of ovaries from nonpollinated muskmelon (Cucumis melo L.) flowers. Flowers attached to the vine were allowed to senesce for 4 days after anthesis or were harvested at full anthesis and aged for the same time interval with or without access to water. The IAA ester, amide-linked forms of IAA, free ABA, and ABA esters increased in senescent ovaries from flowers left attached to the vine. Detaching flowers from the vine resulted in an accumulation of free and amide-linked IAA in the senescing ovary but suppressed accumulation of ester IAA. Free ABA failed to increase in ovaries detached from the vine. Subjecting detached flowers to water stress had no effect on the endogenous level of free ABA but resulted in the accumulation of ABA ester and suppression of any increase in free IAA. However, detached flowers treated with 0.1 millimolar ABA accumulated 75% less free IAA and initiated the synthesis of ester IAA. Detached flowers treated with ABA also accumulated high levels of ester ABA. These results suggest that the metabolism of free IAA in muskmelon ovary tissue is regulated in situ and not the consequence of external synthesis and importation. ABA appears to be transported into the senescing ovary from an external source and alters the IAA metabolism in such a manner as to suppress the level of free IAA while stimulating accumulation of the ester IAA.     

Involvement of Abscisic Acid and Indole-3-acetic Acid in the Flowering of Pharbitis nil

The involvement of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the regulation of flowering of Pharbitis nil was investigated through exogenous applications and analyses of endogenous levels. Both hormones inhibited the flowering of P. nil when they were applied before or after a single 15-h dark treatment. The inhibitory effect of ABA and IAA was significant when they were applied before the dark treatment, and the application to plumules was more effective than that to cotyledons. In all applications, the inhibitory effect of IAA was stronger than that of ABA. Endogenous levels of ABA and IAA in the plumules were compared between flower-inductive (15-h dark treatment) and noninductive (continuous light) light conditions. There was no significant difference in the ABA level between light and dark conditions, whereas the level of IAA was decreased by the dark treatment. These results suggest that biosynthesis and/or catabolism of IAA is affected by the light treatment and therefore may be involved in the regulation of early flowering processes in the apex. The inhibitory effects of ABA and IAA were reversed by an application of gibberellin A₃, indicating that gibberellin A₃ counteracts the flowering processes affected by ABA and IAA. Application of aminoethoxyvinylglycine restored the flowering response inhibited by IAA, which suggests the possibility that the inhibitory effect of IAA is the result of enhanced ethylene biosynthesis. Received November 22, 1996; accepted February 17, 1997     

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     

Rapid separation and quantification of abscisic Acid from plant tissues using high performance liquid chromatography

Abscisic acid (ABA) was purified from soybean (Glycine max [L.]) seed extract using a preparative high performance liquid chromatography (HPLC) procedure. The preparative procedure was rapid (70 minutes per sample), required no prior partitioning for purification and was quantitative as demonstrated with an internal standard of [2-¹⁴C]ABA, of which 98.9% was recovered.     

Concentrations of Abscisic Acid and Indole-3-Acetic Acid in Soybean Seeds during Development

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in seed parts were determined during reproductive development of soybean plants (Glycine max [L.] Merr. cv `Chippewa 64'). The concentration of ABA and IAA changed independently in individual seed parts with time. Measurement of the level of ABA and IAA in whole seeds masked the changes which occurred in individual seed tissues. The concentration of ABA was generally highest and that of IAA was generally lowest in the embryonic axis of soybean seeds. In the testa, the IAA concentration was generally highest while the ABA concentration was generally the lowest compared to other parts of the seed.     

Simultaneous quantitation of indole 3-acetic Acid and abscisic Acid in small samples of plant tissue by gas chromatography/mass spectrometry/selected ion monitoring

Indole 3-acetic acid (IAA) was analyzed in apple, orange, and prune tissue by GC-MS by monitoring the protonated molecular ion of its methyl ester at mass to charge ratio (m/z) 190 together with the major fragment ion at m/z 130 and the corresponding ions from the methyl esters of either [²H₄]IAA (m/z 194, 134) or [²H₅]IAA (m/z 195, 135). Abscisic acid (ABA) was analyzed by monitoring the major fragment ions of its methyl ester at m/z 261 and m/z 247 and the corresponding ions from the methyl ester of [²H₃]ABA (m/z 264, 250). Detection limits for IAA and ABA were 1 and 10 picograms, respectively using standards and 1 nanogram per gram dry weight for both phytohormones in plant tissue.     

Levels of Indol-3yl-acetic Acid and Acid Inhibitors in Green and Etiolated Bean Seedlings (Phaseolus vulgaris)

Indol-3yl-acetic acid (IAA) was identified in Phaseolus vulgaris L. Shoot tissue of seedlings, exposed to light for 5 days, had a higher level of IAA than etiolated seedlings of the same age. The content of IAA increased in green seedlings during light treatment for 5–12 days. No increase could be measured in dark-grown seedlings. Inhibitory substances appeared at different R_f-values. The main part was identical to the inhibitor-β complex and occurred in a higher amount in light-grown seedlings than in etiolated taller ones. One part of the inhibitor-complex appeared to be abscisic acid (ABA). It is suggested that both IAA and acid inhibitors may play an important role in the control of stem growth and differentiation, although light effects on other hormones and regulatory systems cannot be ignored.     

Effects of pod removal on the transport and accumulation of abscisic Acid and indole-3-acetic Acid in soybean leaves

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the second most recently expanded trifoliolate leaf were determined during reproductive development of soybean (Glycine max [L.] Merr cv `Chippewa 64'). The concentration of ABA in leaves was constant during most of the seed filling period until the seeds began to dry. The concentration of IAA in the leaves decreased throughout development. Removal of pods 36 hours prior to sampling resulted in increased concentrations of ABA in leaves during the period of rapid pod filling but had little effect on the concentration of IAA in leaves. ABA appears to accumulate in leaves after fruit removal only when fruits represent the major sink for photosynthate.

ABA and IAA moving acropetally and basipetally in petioles of soybean were estimated using a phloem exudation technique. ABA was found to move mostly in the basipetal direction in petioles (away from laminae). IAA, primarily in the form of ester conjugate(s), was found to be moving acropetally (toward laminae) in petioles. The highest amount of IAA ester(s) was found in petiole exudate during the mid and late stages of seed filling. Removal of fruits 36 hours prior to exudation reduced the amount of IAA ester recovered in exudate, suggesting that fruits were a source of the IAA conjugate in petiole exudate.

     

Abscisic Acid, Auxin, and Ethylene in Explant Abscission

Experiments with explants of Phaseolus vulgaris L., cv. CanadianWonder, show that abscission and the associated rise in oarboxymethyl-cellulaseactivity in the separation zone are initiated by a peak in ethyleneproduction during senescence of pulvinar tissue distal to thezone. Distal applications of abscisic acid (ABA) induce an earlierpeak in ethylene production, increase cellulase activity, andpromote abscission. ABA is more effective in these ways if treatmentis delayed from 0 to 24 h after excision. With increasing concentrations of ABA the maximum rate of ethylene production is achievedsooner. Indol-3yl-acetic acid (IAA) and ABA are antagonisticin this system and have opposing effects. IAA retards the timeof peak ethylene-production and delays abscission. Explantsmay be retained for long periods without abscinding if incubatedin an ethylene-free atmosphere: the addition of ethylene forany one 24-h period (except the first 24 h after excision) willinduce abscission. The initial period of insensitivity to ethyleneis extended by distal applications of IAA. Ethylene-inducedabscission can be inhibited by IAA applied up to 72 h afterexcision provided the ethylene is not applied first. It is proposedthat abscission in the explant is controlled at two levels:(1) an auxin-dependent stage determining the duration of insensitivityto ethylene; (2) the timing of a rise in ethylene productionin senescing tissue distal to the separation zone. An auxin-ethylenebalance-mechanism at the separation zone is discussed.     

NO and IAA Key Regulators in the Shoot Growth Promoting Action of Humic Acid in Cucumis sativus L.

Previous studies have reported that a purified sedimentary humic acid (PHA) was able to increase the concentration of nitric oxide (NO), indole-acetic acid (IAA) and ethylene in cucumber roots. Here, we investigated if these effects are functionally related to the ability of PHA to improve shoot growth. The effect of specific inhibitors of NO, IAA and ethylene functionality and signaling on PHA-induced shoot growth was studied. Likewise, the effect of these inhibitors on the synthesis and activity of the phytoregulators concerned by PHA action in cucumber roots was also explored. The results show that shoot growth promoted by PHA is due to an increase of IAA concentration in the root through both a NO-dependent and a NO-independent pathway. In addition, the increased ethylene production in the root is regulated by an IAA-dependent pathway. Finally, results also showed that the increase of ABA concentration in the root is regulated through both IAA- and ethylene-dependent pathways. In summary, the shoot growth promoting action of PHA involves a complex hormonal network. On one hand, the PHA action is functionally linked to increments in NO and IAA concentration in roots. And on the other hand, PHA action also increases ethylene and ABA root concentration mediated by NO-IAA dependent pathways.     

A Simplified Method for the Quantitative Determination of Indoleacetic Acid by High Performance Liquid Chromatography with a Fluorometric Detector

Quantitative analysis of endogenous IAA contents of radish plantextracts by high performance liquid chromatography with a fluorometricdetector was refined by using a C-18 SEP-PAK cartridge for thepurification procedure and indolepropionic acid as the internalstandard. The C-18 SEP-PAK cartridge efficiently purified theIAA from the acidic ether-soluble fraction of the plant extractswhen the pH of the fraction injected into the cartridge waskept under 3.5. Both of the recovery rates for IAA and indolepropionicacid were over 90% for the extraction and purification procedure.The endogenous IAA content in green radish seedlings, correctedby internal standard methods with indolepropionic acid, was20.2 ng/g fr wt. This was in good agreement with the value correctedwith radiolabeled ¹⁴C-IAA, 20.8 ng/g fr wt. (Received May 18, 1983; Accepted August 31, 1983)     

The Effect of Auxin and Abscisic Acid on the Catabolism of RNA

Abscisic acid (ABA) reduced growth in a root test (lentil),but the inhibition observed was less noticeable than that producedby using indol-3yl-acetic acid (IAA) alone. When both ABA andIAA were employed together, ABA acted as a growth-antagonistof IAA. ABA produced a strong inhibition of the total RNA accumulationand accelerated the RNase activity, while IAA strongly stimulatedthe RNA accumulation and greatly inhibited RNase activity. WhenABA and IAA were tested together, ABA also acted as an antagonistof TAA.     

Simultaneous Determination of Indole-3-Acetic Acid and Abscisic Acid as the Pentafluorobenzyl Derivative with Electron-Capture Gas Chromatography

Indole-3-acetic acid (IAA) and abscisic acid (ABA) were converted in to pentafluorobenzyl esters by α-bromo-2, 3, 4, 5, 6-pentafluorotoluene at 55℃. The derivatization took about 90 minutes. The two esters generated were able to be simultaneously determined with electron-capture gas chromatography. The method is simple and sensitive. The minimal test does: 10-14g for IAA and 10-13g for ABA.     

Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers

A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA.     

Control of the Co2 Responses of Stomata by Indol-3ylacetic Acid and Abscisic Acid

Previous studies of stomatal behaviour on detached epidermisof Commelina communis L. have suggested that abscisic acid (ABA)and C02 act independently to cause stomatal closure. Evidenceis presented here that if indol-3ylacetic acid (IAA) is addedto the medium used for incubating the epidermis, an interactionbetween ABA and Co₂ becomes apparent. Increasing concentrationsof IAA reduce the ability of the stomata to respond to CO₂,and ABA appears to antagonize this effect. Recognition of therole of IAA enables us to reconcile earlier conflicting reportsconcerning the interdependence of effects of ABA and Co₂on stomata.     

THE REGULATION OF ABSCISSION AND IAA BY SENESCENCE FACTOR AND ABSCISIC ACID

The effects of the endogenous “senescence factor” (SF) and synthetic (±)-abscisic acid (ABA) on basipetal transport of indoleacetic acid (IAA) in Coleus blumei Benth. were studied. When used to pretreat explant petioles, both SF and ABA accelerated abscission and decreased basipetal movement of IAA through petiolar sections but had no effect on acropetal movement, which was negligible in all experiments. SF decreased the intensity but not the velocity of the basipetal movement of IAA. Pretreatment with either SF or ABA decreased the free IAA and increased the IAA-aspartate extractable from the sections. The results support the hypothesis that SF and ABA hasten abscission by lowering the amounts of IAA transport in the tissue. Known properties of SF are similar to those of ABA. Efforts are under way to elucidate the chemical identity of SF.