A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid     

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     

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     

Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts

Two radioimmunoassays have been developed which allow the parallel quantitation of free as well as conjugated natural (+)-abscisic acid (ABA) directly and separately, in unpurified plant extracts. The differential specificity of antisera has been achieved by coupling ABA through C₁ (for total ABA determination) or C₄ (for free ABA determination), respectively, to proteins to obtain the immunogenic conjugates. Compounds structurally related to ABA, such as, dihydrophaseic acid or phaseic acid, do not interfere with either of the assays, even when present in more than ten-fold excess. Other related compounds, such as, violaxanthin or xanthoxin, do not cross react at all. Both antisera respond to (+)-ABA but show very low immunoreactivity with (-)-ABA. As little as 27 pg of ABA (serum for free ABA) or 47 pg (serum for total ABA) may be detected and the measuring ranges are from 0.2–8 and 0.2–30 pmol, respectively. Average recoveries are greater than 99%. Using these assays, more than 100 samples can be assayed for free and conjugated ABA per day. Levels of free ABA, as determined by radioimmunoassay (RIA), correlated well with those reported in the literature. Levels of conjugated ABA were found to be generally higher than previously reported for ABA after alkaline hydrolysis of the extracts. Conjugated ABA accumulates during aging of leaves and levels of conjugated ABA up to 17-fold higher than those of free ABA have been detected in senescent leaves of Hyoscyamus niger L. Evidence was obtained for the presence of ABA conjugates other than the glucose ester in some plants.Abbreviations ABA abscisic acid - BHT 2,6-di-t-4-methyl phenol - BSA bovine serum albumin - HSA human serum albumin - RIA radioimmunoassay - TLC thin-layer chromatography - EDC 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide · HCl Part 11 in the series: Use of Immunoassay in Plant Science     

An enzyme-immunoassay of abscisic acid in potato (Solanum commersonii) cultured cells

Estimation of abscisic acid (ABA) content in potato (Solanum commersonii) suspension-cultured cells with an enzyme-immunoassay (EIA) was investigated. In crude extracts of potato cultured cells or even after simple clean-up using C₁₈ cartridge, EIA based on commercial monoclonal antibodies (Idetek, Inc) failed to detect any ABA content. An interference could be removed by partitioning against ethyl acetate after the C₁₈ cartridge so that the EIA yielded an estimate of ABA similar to that determined by high pressure liquid chromatography and gas chromatography-mass spectrometry analysis. These results demonstrate the presence of metabolites in potato cultured cell extract that prevent the binding of ABA to its binding site but not the binding of tracer.Abbreviations ABA abscisic acid - EIA enzyme-immunoassay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.     

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)     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

Changes in the ratio of cis-trans to trans-trans abscisic acid during ripening of apple fruits

Immediately after harvest, abscisic acid (ABA) extracted from fruits of the apple cultivar Golden Delicious comprised solely the cis-trans isomer. During postharvest ripening, however, trans-trans ABA accumulated and finally exceeded the level of cis-trans ABA. The two geometrical isomers were separated and identified by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. After purification by HPLC the putative trans-trans isomer yielded considerable quantities of cis-trans ABA, when irradiated with UV light. This isomerization was more rapid than the reverse reaction. The physiological significance of the accumulation of trans-trans ABA is discussed, as well as the applications of these results in the use of trans-trans ABA as an internal standard during the extraction and quantification of ABA from plant tissues.Abbreviations ABA 2-cis-4-trans abscisic acid - t-ABA 2-trans-4-trans abscisic acid - ECD electron capture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PVP water insoluble polyvinylpyrroli-done - UV ultraviolet     

Determination of femtomol quantities of gibberellic acid by radioimmunoassay

A highly sensitive and specific radioimmunoassay which allows the detection of as little as 5 fmol (2 pg) of gibberellic acid (GA₃) in crude plant extracts is described. Antisera of high affinity and titer were obtained by immunizing rabbits with a conjugate of carboxyl-coupled GA₃ and bovine serum albumin. [¹²⁵I]Gibberellic acid-[N-(p-hydroxybenzyl) putrescine]amide of high specific activity, used as the immunotracer, is readily displaced by gibberellic acid methyl ester but not by free gibberellic acid. Thus, methylation of extracts prior to analysis is required. The assay is very specific; besides GA₃, only the closely related GA₇ is highly immunoreactive. Various gibberellins, related compounds, as well as other classes of plant hormones do not interfere with the assay. Levels of immunoreactive gibberellins (GA₃, GA₇) in actively growing tissues, among them cell suspension cultures of 33 different species, were determined.Abbreviations ABA abscisic acid - GC-MS gas chromatography-mass spectroscopy - TLC thin-layer chromatography - GA gibberellin Part 17 in the Series: Use of Immunoassay in Plant Science     

Germination inhibitors in the pine seed coat

Inhibitors from (Pinus pinea L.) seed coats were separated using paper chromatography, thin layer chromatography, and a Sephadex G-10 column. The inhibitory activity was resolved into several fractions. One of these behaved similarly to abscisic acid. It has exhibed the same properties as ABA in thin-layer chromatography, paper chromatography, and Sephadex G-10 chromatography and in UV absorption and fluorescence spectra. These germination inhibitors, present in the seed coats, are involved in the regulation of P. pinea seed germination.Abbreviations ABA abscisic acid - TLC thin-layer chromatography     

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     

Abscisic acid,xanthoxin and violaxanthin in the caps of gravistimulated maize roots

The occurrence and distribution of abscisic acid (ABA), xanthoxin (Xa) and the carotenoid violaxanthin (Va) were investigated in root tips of maize (Zea mays L. cv. Merit). In roots grown in the dark, Va and ABA were present in relatively high amounts in the root cap and in low amounts in the adjacent terminal 1.5 mm of the root. Xanthoxin was present in equal concentrations in both regions. In roots exposed to light, the ABA distribution was reversed, with relatively low levels in the root cap and high levels in the adjacent 1.5-mm segment. Light also caused a decrease in Va in both regions of the root and an increase in Xa, especially in the cap. In the maize cultivar used for this work, light is necessary for gravitropic curving. This response occurs within the same time frame as the light-induced ABA redistribution as well as the changes in the levels of Va and Xa. These data are consistent with a role for ABA in root gravitropism and support the proposal that Xa may arise from the turnover of Va.Abbreviations ABA abscisic acid - GC gas chromatography - HPLC high-performance liquid chromatography - GC-MS gas chromatography-mass spectroscopy - V_a violaxanthin - Xa xanthoxin     

Comparative quantitation of abscisic acid in plant extracts by gas-liquid chromatography and an enzyme-linked immunosorbent assay using the avidin-biotin system

In this report we describe an enzyme-linked immunosorbent assay (ELISA) for the quantitation of abscisic acid (ABA) in plant extracts. A microtitration plate is coated with an ABA-protein complex. The ABA, standard or sample, is then added to each well with a limiting quantity of rabbit anti-ABA antibodies. During the following incubation period, antibodies bind either to free or to bound ABA on the plates. After washing, bound antibodies are indirectly labelled in two steps by the means of biotinylated goat antirabbit immunoglobulin-G antibodies which act as a link between rabbit anti-ABA antibodies and an avidin-alkaline phosphatase complex. The relative enzyme activity bound is measured spectrophotometrically. The detection limit of this method is 5 pg ABA and the measuring range extends to 10 ng. Gas-liquid-chromatography controls, with an electron capture detector, show a good correlation with ELISA results obtained using extracts of Lycopersicon esculentum, Nicotiana tabacum and Pseudotsuga menziesii samples purified by high-performance liquid chromatography. This provides a good argument for the accuracy of the immunoenzymatic method. The indirect labelling of antibodies, with the avidin-biotin amplifying system, should make this technique suitable for the quantitation of other plant growth substances against which specific antibodies are available.Abbreviations ABA abscisic acid - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - GLC gas liquid chromatography - HPLC high-performance liquid chromatography - IgG Immunoglobulin G - PBS phosphate-buffered saline     

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     

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     

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     

Sensitivity of Commelina stomata to abscisic acid

Stomata of Commelina leaves pre-opened by incubation in moist air were found to close within 30 min when supplied with abscisic acid (ABA) via the transpiration stream. Radioactive ABA had similar effects, but allowed the distribution of the compound within the leaf to be measured and correlated with stomatal movements to give estimates of the sensitivity of Commelina stomata. On a whole-leaf basis, less than 163 fmol ABA per mm² leaf area were present at the time of complete stomatal closure. This was close to other published estimates. By taking epidermal ¹⁴C measurements, however, it was possible to increase the accuracy of the estimate on the assumption that only ABA present in the epidermis was physiologically active. Thus, less than 235 amol ABA for stomatal complex were present at complete closure, and statistically significant narrowing of the stomatal aperture had occurred when between 12.6 and 45.4 amol per complex were present. The distribution of ABA within the epidermal tissue after transpiration-stream application was studied using microautoradiography, and the compound appeared to have accumulated within the stomatal complex.Abbreviations ABA abscisic acid - TLC thin-layer chromatography