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.
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acetic acid-alkaline phosphatase
1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride
gas chromatographymass spectrometry
goat anti-rabbit IgG
peroxidase/rabbit antiperoxidase complex
Browning, G. (1980) Endogenous cis, trans-abscisic acid and pea seed development: evidence for a role in seed growth from changes induced by temperature. J. Exp. Bot. 31, 185–197
Clark, M.F. (1981) Immunosorbent assays in plant virology. Annu. Rev. Phytopath 19, 83–106
Clark, M.F., Bar-Joseph, M. (1984) Enzyme immunosorbent assays in plant virology. In: Methods in virology vol. 7, pp. 51–85, Maramorosch, K., Kaprowski, H., eds. Academic Press, New York
Cornish, K., Zeevaart, J.A.D. (1985) Movement of abscisic acid into the apoplast in response to water stress in Xanthium strumarium L. Plant Physiol. 78, 623–626
Gronemeyer, H. (1985) Photoafinity labelling of steroid hormone binding sites. Trends Biochem. Sci. 10, 264–267
Harvey, D.M.R. (1982) Freeze substitution. J. Microsc. 127, 209–221
Hornberg, C., Weiler, E.W. (1984) High affinity binding sites for abscisic acid on the plasmalemma of Vicia faba guard cells. Nature 310, 321–323
Knox, R.B. (1982) Methods of locating and identifying antigens in plant tissues. In: Techniques in immunocytochemistry, vol. 1, pp. 205–238, Bullock, G.R., Petrusz, P., eds. Academic Press, New York
Metens, R., Deus-Neumann, B., Weiler, E.W. (1983) Monoclonal antibodies for the detection and quantitation of the endogenous plant growth regulator, abscisic acid. FEBS Lett. 160, 269–272
Perbal, G., Leroux, Y., Driss-Ecole, D. (1982) Mise en évidence de l'AIA-5-3H par autoradiographie dans le coleoptile de Blé. Physiol. Plant. 54, 167–173
Quarrie, S.A., Lister, P.G. (1984) Evidence of plastid control of abscisic acid accumulation in barley (Hordeum vulgare L.). Z. Pflanzenphysiol. 114, 295–308
Rosher, P.H., Jones, H.G., Hedden, P. (1985) 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. Planta 165, 91–99
Sossountzov, L., Sotta, B., Maldiney, R., Sabbagh, I., Miginiac, E. (1986) Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chenopodium polyspermum L. Planta 168, 471–481
Sotta, B., Sossountzov, L., Maldiney, R., Sabbagh, I., Tachon, P., Miginiac, E. (1985) Abscisic acid localisation by light microscopic immunohistochemistry in Chenopodium polyspernum L. J. Histochem. Cytochem. 33, 201–208
Sternberger, L.A. (1979) Immunocytochemistry (2nd edn.), Wiley, New York
Sternberger, L.A., Hardy, P.H., Cuculis, J.J., Meyer, H.G. (1970) The unlabelled antibody enzyme method of immunohistochemistry. Preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in dientification of spirochetes. J. Histochem. Cytochem. 18, 313–333
Weiler, E.W. (1980) Radioimmunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts. Planta 148, 262–272
Weiler, E.W. (1984) Immunoassay of plant growth regulators. Annu. Rev. Plant Physiol. 35, 85–95
Yalow, R.S. (1978) Radioimmunoassay: a probe for the fine structure of biological systems. Science 200, 1236–1245
Zavala, M.E., Brandon, O.L. (1983) Localisation of a phytohormone using immunochemistry. J. Cell. Biol. 97, 1235–1239
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Skene, D.S., Browning, G. & Jones, H.G. Model systems for the immunolocalisation of cis, trans abscisic acid in plant tissues. Planta 172, 192–199 (1987). https://doi.org/10.1007/BF00394587
- Abscisic acid (fixation immunolocalisation)
- Daucus (abscisic acid)
- Immunolocalisation (model systems)
- Pisum (abscisic acid)