Effect of nitrogen stress and abscisic acid on nitrate absorption and transport in barley and tomato

The potential of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) roots for net NO ₃ ^- absorption increased two-to five fold within 2 d of being deprived of NO ₃ ^- supply. Nitrogen-starved barley roots continued to maintain a high potential for NO ₃ ^- absorption, whereas NO ₃ ^- absorption by tomato roots declined below control levels after 10 d of N starvation. When placed in a 0.2 mM NO ₃ ^- solution, roots of both species transported more NO ₃ ^- and total solutes to the xylem after 2 d of N starvation than did N-sufficient controls. However, replenishment of root NO ₃ ^- stores took precedence over NO ₃ ^- transport to the xylem. Consequently, as N stress became more severe, transport of NO ₃ ^- and total solutes to the xylem declined, relative to controls. Nitrogen stress caused an increase in hydraulic conductance (L _p) and exudate volume (J _v) in barley but decrased these parameters in tomato. Nitrogen stress had no significant effect upon abscisic acid (ABA) levels in roots of barley or flacca (a low-ABA mutant) tomato, but prevented an agerelated decline in ABA in wild-type tomato roots. Applied ABA had the same effect upon barley and upon the wild type and flacca tomatoes: L _p and J _v were increased, but NO ₃ ^- absorption and NO ₃ ^- flux to the xylem were either unaffected or sometimes inhibited. We conclude that ABA is not directly involved in the normal changes in NO ₃ ^- absorption and transport that occur with N stress in barley and tomato, because (1) the root ABA level was either unaffected by N stress (barley and flacca tomato) or changed, after the greatest changes in NO ₃ ^- absorption and transport and L _p had been observed (wild-type tomato); (2) changes in NO ₃ ^- absorption/transport characteristics either did not respond to applied ABA, or, if they did, they changed in the direction opposite to that predicted from changes in root ABA with N stress; and (3) the flacca tomato (which produces very little ABA in response to N stress) responded to N stress with very similar changes in NO ₃ ^- transport to those observed in the wild type.Abbreviation and symbols ABA abscisic acid - J_v exudate volume - L_p root hydraulic conductance     

The permeability of the epidermal cell plasma membrane of barley leaves to abscisic acid

Uptake of ³H-labelled (±)-abscisic acid (ABA) into isolated barley (Hordeum vulgare L.) epidermal cell protoplasts (ECP) was followed over a range of pH values and ABA concentrations. The present results show that ABA uptake is not always linearly correlated with the external concentration of undissociated ABA (ABAH). At pH 7.25, ABA uptake exhibited saturation kinetics with an apparent K _m value of 75 mmol·m^–3 to tal ABA. This saturable transport component was inhibited by pretreating the protoplasts with 1 mol·m^–3 p-chloromercuribenzenesulfonic acid at pH 8.0, conditions that minimized the uptake of this acid sulfhydryl reagent. Moreover, the rate of (±)-[^3]HABA uptake was reduced by addition of 0.1 mol·m^–3 (±)-ABA to 41%, whereas the same concentration of (±)-ABA was approximately half as effective (46% of the inhibitory effect). Thus, it was concluded that only (±)-ABA competes for an ABA carrier that is located in the epidermal cell plasma membrane. The permeability of the epidermal cell plasma membrane was studied by performing a Collander analysis. At pH 6 the overall plasma-membrane permeability of epidermal cells was similar to that of guard cells but was about two times higher than that of mesophyll cells.Abbreviations ABA abscisic acid - ABA^– anion of ABA - ABAH undissociated ABA - 2,4-D 2,4-dichlorophenoxyacetic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - ECP deepidermal cell protoplast - K_r partition coefficient - M_r relative molecular mass - NEM N-ethylmaleimide - PCMBS p-chloromercuriben zenesulfonic acid - P_s permeability coefficient We are grateful to Barbara Dierich for expert technical assistance, to Prof. H. Gimmler (Lehrstuhl für Botanik I, Universität Würzburg, FRG) for helpful discussions and to the Deutsche Forschungsgemeinschaft (SFB 251, TP 3) for financial support.     

Kinetin-promoted transport of assimilates in stems of Phaseolus vulgaris L.

Kinetin, applied as a dispersion in aqueous lanolin to the stumps of decapitated stems of P. vulgaris plants with their roots removed, was found to promote the transport of ¹⁴C- and ³²P-labelled assimilates to the site of hormone application. Measurement of photosynthetic rate of, and assimilate export rate from the source leaves, indicated that kinetin was not acting to promote assimilate transport by stimulating these processes. Moreover, it was found that the time between kinetin application and detection of an enhanced transport flux was independent of the distance over which kinetin would need to move to be present throughout the length of the transport pathway. These observations, together with the finding that lateral applications of kinetin to the stems resulted in an enhanced localized accumulation of assimilates, provided evidence that kinetin acted locally at its point of application to stimulate assimilate transfer.Abbreviations GA₃ gibberellic acid - IAA indol-3yl-acetic acid     

Modulation of germination of embryos isolated from dormant and nondormant barley grains by manipulation of endogenous abscisic acid

Dormant and non-dormant barley (Hordeum distichum L.) grains with identical genetic backgrounds were obtained by maturing grains under different climate conditions. When isolated embryos from dormant grains were incubated in a well containing a fixed volume of water (300 l), the germination rate and percentage were dependent on the embryo number per well. A higher embryo number per well was correlated with a lower germination rate and percentage. However, this was not the case for the embryos isolated from nondormant grains. During germination, the endogenous cis-abscisic acid (ABA) in isolated embryos from both dormant and nondormant grains was analyzed. The inhibitory effect on germination of a higher number per well of isolated dormant embryos was due to diffusion of endogenous ABA out of the embryos and accumulation of ABA in the incubation medium. Moreover, there was de-novo synthesis of ABA in embryos isolated from dormant grains during incubation but not in embryos isolated from nondormant grains. The inhibitory effect of ABA on germination of embryos isolated from dormant grains could be mimicked by addition of ABA or the medium in which dormant embryos had been placed. Embryos isolated from nondormant grains were insensitive to addition of ABA and medium from dormant embryos. Our results demonstrate that diffusion of endogenous ABA, de-novo ABA synthesis and ABA sensitivity play a role in the control of germination. It is proposed that dormancy-breaking treatments act via changes to these processes.Abbreviations ABA cis-abscisic acid - E/W embryo(s) per well Prof. K.R. Libbenga (Institute of Molecular Plant Sciences, Leiden University) is thanked for fruitful discussions. B.V.D. was partly supported by E.E.C. BIOTECH program PL 920175.     

The monoclonal antibody JIM19 modulates abscisic acid action in barley aleurone protoplasts

A panel of hybridoma products generated against pea (Pisum sativum L.) guard-cell protoplasts has been assayed for anti-abscisic acid (ABA) biological activity in barley (Hordeum vulgare L.) aleurone protoplasts. The effects of the antibodies on ABA-induced accumulation of mRNA transcribed from RAB-16, a gene responsive to ABA, were determined. Most of the antibodies, and culture medium, had no effect, but five monoclonal antibodies (MAbs) were found to inhibit ABA-induced RAB-16 gene expression and one MAb enhanced it. The effects of one inhibitory MAb, JIM19, were studied in some detail. These effects were specific to ABA-induced events, as incubation with JIM19 had no effect on the expression of a constitutively-expressed gene, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, and only a slight effect on the production of -amylase induced by gibberellic acid. Increasing concentrations of ABA in the incubation medium partly overcame the inhibitory effect of JIM19. Immunolabelling and biological activity remained together during immuno-purification of JIM19 from hybridoma culture supernatant. Immunoblotting of JIM19 to membrane preparations from barley aleurone protoplasts revealed that JIM19 recognised a number of proteins.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase - GCP guard-cell protoplast - MAb monoclonal antibody - RAB (gene) responsive to ABA We thank the Agricultural and Food Research Council and The Nuffield Foundation for financial support, Professor Keith Roberts (John Innes Institute, Norwich, UK) for advice and generous use of his laboratory and Jan Peart (John Innes Institute) for animal cell culture. S.J.N. is grateful to Professor Colin Hawkes (University of the West of England, Bristol) for his continued support of this project.     

Effects of abscisic acid on sequestration and exchange of Na+ by barley roots

Excised Na⁺-starved barley roots were suspended in solutions of Na⁺ in combination with NO ₃ ^- , Cl^-, and SO ₄ ^2- , and effects of the added phytohormone, abscisic acid (ABA), to the medium were determined. Abscisic acid increased the rate of Na⁺ (²²Na⁺) accumulation and the amount of Na⁺ deposited in the vacuoles. These stimulating effects of ABA were modified by anions following the sequence NO ₃ ^- >Cl^->SO ₄ ^2- . Testing whether the magnitude of the pH gradient across the plasmalemma of the cells of the root cortex affects rates of Na⁺ accumulation and their dependence upon ABA, we observed that, in the pH range from 4 to 8, the ABA-induced stimulation was strongest at pH 5.8, and least at pH 4. Changes in pH during the experiment caused changes in the rates of Na⁺ accumulation in agreement with experiments performed at constant pH values. Simultaneously with ABA-enhanced accumulation, loss of Na⁺ occurred. Loss of Na⁺ was strongest at pH 4 and was affected by anions, being greatest with SO ₄ ^2- and following the sequence SO ₄ ^2- >Cl^->NO ₃ ^- . On the basis of the finding that initial acceleration of uptake as well as loss of Na⁺ depended on the pH of the medium we suggest that, in barley roots, ABA stimulates an exchange of Na⁺ for H⁺ at the plasmalemma of the cortical cells. The results indicate that ABA-stimulated expulsion of Na⁺, in combination with ABA-stimulated sequestration in the vacuoles, constitutes one of the mechanisms which enable barley plants to tolerate higher than normal levels of Na⁺.Abbreviations ABA abscisic acid - FW fresh weight     

Determination of endogenous abscisic acid levels in immature cereal embryos during in vitro culture

Levels of endogenous abscisic acid (ABA) in immature wheat (Triticum aestivum cv. Timmo) and barley (Hordeum vulgare cv. Golden Promise) embryos have been determined by enzyme-linked immunosorbent assay. Embryos of both cereal species showed an increase in ABA content during development on the parent plant. Immature embryos were excised and cultured in vitro on nutrient media that led to precocious germination or on media containing 9% (w/v) mannitol that maintained their developmental arrest. Barley and wheat embryos responded to these culture conditions in an identical manner with respect to changes in morphology, fresh weight, protein and lectin content. However, in complete contrast, the ABA content of barley embryos increased by an order of magnitude during culture on mannitol, whereas that of wheat embryos showed no significant change. The results are discussed within the context of the role of ABA in the regulation of embryo development.Abbreviations ABA abscisic acid - BGA barley-germ agglutinin - dpa days post anthesis - ELISA enzyme-linked immunosorbent assay - GC-MS gas chromatography-mass spectrometry - WGA wheat-germ agglutinin     

The effect of abscisic acid on sugar levels in seedlings of Phaseolus vulgaris L. cv. redland pioneer

Abscisic acid (ABA) caused an increase in total-sugar and a 3-fold increase in reducing-sugar content in the roots of intact bean seedlings. The level of reducing sugars was also increased in the stem but total sugar levels remained unaffected by ABA. ABA also increased reducing-sugar content of the root in seedlings with cotyledons removed but in this case the reducing- and total-sugar contents of the leaf were reduced. However, ABA did not affect reducing- and total-sugar levels in excised bean root systems. The observed increase in sugar content of the root of intact bean seedlings appears to be the consequence of an ABA-induced stimulation of sugar transport from the shoot to the root. It is proposed that a relationship may exist between the effect of ABA on sugar transport and its effect on ion transport in excised root systems and in intact seedlings.     

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     

Production and diurnal utilization of assimilates in leaves of spinach (Spinacia oleracea L.) and barley (Hordeum vulgare L.)

Rates of CO₂ fixation during the light period and the rates of CO₂ release during the night period were measured using mature leaves from 39- to 49-d-old spinach (Spinacia oleracea L., US Hybrid 424; grown in 9 h light, 15 h darkness, daily) and mature leaves from 21-d-old barley (Hordeum vulgare L., cv. Apex; grown in 14 h light, 10 h darkness, daily). At certain times during the light and dark periods leaves were harvested for assay of their contents of soluble carbohydrates, starch, malate and the various amino acids. Evaluation of the results of these measurements shows that in spinach and barley leaves 46% and 26%, respectively, of the carbon assimilated during the light period is deposited in the leaves for export during the night period. Taking into account the carbon consumption in the source leaves by dark respiration, it is evaluated that rates of assimilate export during the light period from spinach and barley leaves [38 and 42 atom C · (mg Chl)^–1 · h^–1] are reduced in the dark period to 16 atom C · (mg Chl)^–1 · h^–1 in both species. The calculated C/N ratios of the photoassimilates exported during the dark period were 0.029 and 0.015 for spinach and barley leaves, respectively.This work was supported by the Deutsche Forschungsgemeinschaft. We thank Dr. Dieter Heineke for stimulating discussions and Mrs. Petra Hoferichter and Mrs. Marita Feldkämper for their technical assistance.     

Localization of wheat-germ agglutinin in developing wheat embryos and those cultured in abscisic acid

The time course of appearance of wheat-germ agglutinin (WGA) in the various embryonic tissues during embryogenesis in Triticum aestivum L. was studied by sensitive immunofluorescence and peroxidase-antiperoxidase detection systems. The radicle, root cap and coleorhiza first accumulated WGA in early Stage II (8-10 d post-anthesis) prior to the main period of embryo growth, while WGA was found in the epiblast and coleoptile in early and late State III, respectively. Stage III is characterized by maximum embryo growth, followed by desiccation which occurs in Stage IV. When Stage-II embryos were precociously germinated in the absence of abscisic acid (ABA) no WGA was detected in the coleoptile and epiblast of the young seedlings. In the presence of ABA, Stage-II embryos did not germinate but WGA precociously accumulated in the coleoptile and epiblast. The levels and distribution of WGA in the resulting embryo resembled those in a fully mature, dry embryo (Stage V). Barley possesses a seed lectin similar to WGA, but it is never detected in coleoptiles. Some but not all of the barley cultivars tested were found to accumulate lectin in this organ of mature embryos when treated with ABA. Thus, ABA appears to be involved in the highly regulated temporal and spatial expression of WGA during embryogenesis in cereals.Abbreviations ABA abscisic acid - DIC differential interference contrast - PAP peroxidase-antiperoxidase - WGA wheat-germ agglutinin     

Isolation and characterization of a barley mutant with abscisic-acid-insensitive stomata

A barley (Hordeum vulgare L.) mutant (cool) with leaf transpiration unaffected by the application of 1 mM abscisic acid (ABA) was isolated from the population of M2 seedlings using thermography (electronic visualization, and quantitation of the temperature profiles on the surface of the leaves). Stomata of the mutant plants were insensitive to exogenously applied ABA, darkness, and such desiccation treatments as leaf excision and drought stress. The evaporative cooling of the leaves of the cool barley was always higher than that of the wild-type barley, even without ABA application, indicating that the diffusive resistance of the mutant leaves to water loss was always lower. Guard-cell morphology and stomatal density as well as ABA level and metabolism were seemingly unaltered in the mutant plants. In addition, gibberellin-induced -amylase secretion and precocious embryo germination in the mutant barley was inhibited by ABA to the same extent as in the wild-type barley.Abbreviations ABA (±) cis-trans abscisic acid - GA gibberellin     

The role of cis-carotenoids in abscisic acid biosynthesis

Evidence has been obtained which is consistent with 9-cis-neoxanthin being a major precursor of abscisic acid (ABA) in higher plants. A mild, rapid procedure was developed for the extraction and analysis of carotenoids from a range of tissues. Once purified the carotenoids were identified from their light-absorbance properties, reactions with dilute acid, high-performance liquid chromatography R_ts, mass spectra and the quasiequilibria resulting from iodine-catalysed or chlorophyllsensitised photoisomerisation. Two possible ABA precursors, 9-cis-neoxanthin and 9-cis-violaxanthin, were identified in extracts of light-grown and etiolated leaves (of Lycopersicon esculentum, Phaseolus vulgaris, Vicia faba, Pisum sativum, Cicer arietinum, Zea mays, Nicotiana plumbaginifolia, Plantago lanceolata and Digitalis purpurea), and roots of light-grown and etiolated plants (Lycopersicon, Phaseolus and Zea). The 9,9-di-cisisomer of violaxanthin was synthesised but its presence was not detected in any extracts. Levels of 9-cis-neoxanthin and all-trans-violaxanthin were between 20- to 100-fold greater than those of ABA in light-grown leaves. The levels of 9-cis-violaxanthin were similar to those of ABA but unaffected by water stress. Etiolated Phaseolus leaves contained reduced amounts of carotenoids (15–20% compared with light-grown leaves) but retained the ability to synthesise large amounts of ABA. The amounts of ABA synthesised, measured as increases in ABA and its metabolites phaseic acid and dihydrophaseic acid, were closely matched by decreases in the levels of 9-cis-neoxanthin and all-trans-violaxanthin. In etiolated seedlings grown on 50% D₂O, deuterium incorporation into ABA was similar to that into the xanthophylls. Relative levels of carotenoids in roots and light-grown and etiolated leaves of the ABA-deficient mutants, notabilis, flacca and sitiens were the same as those found in wild-type tomato tissues.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PA phaseic acid - t trans - Xan xanthoxin - flc flacca - not notabilis - sit sitiens The authors would like to thank the following for their help and advice: G. Britton (Department of Biochemistry, University of Liverpool, UK), B.H. Davies (Department of Biochemistry, University of Wales, Aberystwyth), P. Molnar, J. Szabolcs, D.C. Walton (Department of Biology, Suny, Syracuse, N.Y., USA), and Mr. J.K. Heald for his expert operation of the mass spectrometer. A.D.P. was supported initially by a Science and Engineering Research Council CASE award with Shell Biosciences, Sittingbourne, Kent, UK, and later by a Agricultural and Food Research Council (AFRC) grant. M.J.B. received a NATO fellowship. The mass spectrometer and HPLC-photodiode-array detector were purchased with funds provided by the AFRC.     

Effect of abscisic acid on the cell cycle in the growing maize root

The mechanism by which the rate of cell proliferation is regulated in different regions of the root apical meristem is unknown. The cell populations comprising the root cap and meristem cycle at different rates, proliferation being particularly slow in the quiescent centre. In an attempt to detect the control points in the cell cycle of the root apical meristem of Zea mays L. (cv. LG 11), quiescent-centre cells were stimulated to synthesise DNA and to enter mitosis either by decapping or by immersing intact roots in an aqueous 3,3-dimethyl-glutaric acid buffer solution. From microdensitometric and flow-cytometric data, we conclude that, upon immersion, the G₂ phase of the cell cycle of intact roots was shortened. However, when 50 M abscisic acid (ABA) was added to the immersion buffer, parameters of the cell cycle were restored to those characteristic of intact roots held in a moist atmosphere. On the other hand, decapping of primary roots preferentially shortened the G₁ phase of the cell cycle in the quiescent centre. When supplied to decapped roots, ABA reversed this effect. Therefore, in our model, applied ABA retarded the completion of the cell cycle and acted upon the exit from either the G₁ or the G₂ phase. Immersion of roots in buffer alone seems to trigger cells to more rapid cycling and may do so by depleting the root of some ABA-like factor.Abbreviations ABA cis-abscisic acid - DGA 3,3-dimethyl-glutaric acid - DAPI 4,6-diamidino-2-phenylindole - LI labelling index We thank Pierre Zaech of the Ludwig Institute, Epalinges, Switzerland, for expert assistance in flow cytometry and Dr. Jean-Marcel Ribaut of our Institute for providing data on exodiffusion and metabolism of ABA.     

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (K_a=1.3x10⁹l mol^-1). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10^-12 mol ABA, the measuring range extends to 20x10^-12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.Abbreviations ABA abscisic acid - BHT 2.6-di-t-butyl-4-methyl phenol - TLC thin-layer chromatography - HSA human serum albumin Part 7 in the Series: Use of Immunoassay in Plant Science     

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with ¹⁴C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 M abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.Abbreviations ABA abscisic acid - FW fresh weight - IAA indole acetic acid - pd membrane potential difference - 1× perfusing solution (see methods) - H⁺ electrochemical proton gradient - pd solute-induced maximum depolarization of the membrane     

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