Abscisic Acid localization and metabolism in barley aleurone layers

Aleurone layers of Hordeum vulgare, cv. `Himalaya' took up [¹⁴C]-abscisic acid (ABA) when incubated for various times. Radioactivity accumulated with time in a low speed, DNA-containing pellet accounting for 1.6 to 2.3% of the radioactivity recovered in subcellular fractions at 18 hours. Thin layer chromatography of ethanolic or methanolic extracts of the cytosol, which contained greater than 95% of the radioactivity taken up by layers, revealed that labeled ABA was metabolized to phaseic acid (PA) and 4′-dihydrophaseic acid (DPA) and three polar metabolites Mx₁, Mx₂, and Mx₃. ABA was not metabolized by endosperm, incubated under conditions used for layers, indicating that metabolism was tissue-specific. Layers metabolized [³H]DPA to Mx₁ and Mx₂. ABA, PA, and DPA-methyl ester and epi-DPA-methyl ester inhibited synthesis of α-amylase by layers incubated for either 37 or 48 hours. These layers converted the methyl DPA and epi-methyl-DPA esters to their respective acids. DPA did not inhibit Lactuca sativa germination or root and coleoptile elongation of germinating Hordeum vulgare seeds, or coleoptile elongation of germinating Zea mays seeds.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants

Abscisic acid (ABA), conjugated abscisic acid, phaseic acid (PA), and conjugated phaseic acid were determined by enzyme-linked immunosorbent assay (ELISA) and gas chromatography (GC) in xylem sap of well-watered and drought-stressed sunflower plants. Conjugated ABA and conjugated PA were determined indirectly after chemical or enzymatic hydrolysis. Conjugated ABA was found to be the predominant ABA metabolite in xylem sap. In xylem sap from well-watered plants at least five, and in sap from drought-stressed plants at least six alkaline hydrolysable ABA conjugates were found. One of them corresponds chromatographically (HPLC) with abscisic acid glucose ester (ABAGE). Under drought conditions the concentrations of ABA, alkaline hydrolysable ABA conjugates, -glucosidase hydrolysable ABA conjugates, PA, and conjugated PA increased. After rewatering the drought-stressed plants, the ABA and the conjugated ABA content decreased. The possible function of the ABA conjugates in the xylem sap as a source of free ABA is discussed.     

Differential growth responses to sodium salts involve different abscisic acid metabolism and transport in Prosopis strombulifera

In this work, the response of the halophytic shrub Prosopis strombulifera to lowering an osmotic potential (Ψ_o) to ?1.0, ?1.9, and ?2.6 MPa generated by NaCl, Na₂SO₄, and the iso-osmotic combination of them was studied at 6, 12, and 24 h after reaching such values in the growing media. By analyzing the content of abscisic acid (ABA) and related metabolites and transpiration rates, we observed that ABA content varied depending on type of salt, salt concentration, organ analyzed, and age of a plant. ABA content in leaves was much higher than in roots, presumably because of rapid biosynthesis and transport from roots. Leaves of Na₂SO₄-treated plants had the highest ABA content at Ψ_o ?2.6 MPa (24 h) associated with sulfate toxicity symptoms. Significant content of ABA-glucose ester (ABA-GE) was found in both the roots and leaves, whereas only low content of phaseic acid (PA) and dihydrophaseic acid (DPA). The roots showed high ABA-GE accumulation in all treatments. The highest content of free ABA was correlated with ABA-GE glucosidase activity. The results show that ABA-GE and free ABA work together to create a specific stress signal.     

Phosphatidic acid integrates calcium signaling and microtubule dynamics into regulating ABA-induced stomatal closure in Arabidopsis

Specific cellular components have been identified to function in abscisic acid (ABA) regulation of stomatal apertures, including calcium, the cytoskeleton, and phosphatidic acid. In this study, the regulation and dynamic organization of microtubules during ABA-induced stomatal closure by phospholipase D (PLD) and its product PA were investigated. ABA induced microtubule depolymerization and stomatal closure in wide-type (WT) Arabidopsis, whereas these processes were impaired in PLD mutant (pldα1). The microtubule-disrupting drugs oryzalin or propyzamide induced microtubule depolymerization, but did not affect the stomatal aperture, whereas their co-treatment with ABA resulted in stomatal closure in both WT and pldα1. In contrast, the microtubule-stabilizing drug paclitaxel arrested ABA-induced microtubule depolymerization and inhibited ABA-induced stomatal closure in both WT and pldα1. In pldα1, ABA-induced cytoplasmic Ca²⁺ ([Ca²⁺]_cyt) elevation was partially blocked, and exogenous Ca²⁺-induced microtubule depolymerization and stomatal closure were impaired. These results suggested that PLDα1 and PA regulate microtubular organization and Ca²⁺ increases during ABA-induced stomatal closing and that crosstalk among signaling lipid, Ca²⁺, and microtubules are essential for ABA signaling.     

Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum

A cell-free enzyme system capable of metabolizing abscisic acid has been obtained from Eastern Wild Cucumber (Echinocystis lobata Michx.) liquid endosperm. The reaction products were determined to be phaseic acid (PA) and dihydrophaseic acid (DPA) by co-chromatography on thin layer chromatograms as the free acids, methyl esters, and their respective oxidation or reduction products. The crude enzyme preparation was separated by centrifugation into a particulate abscisic acid (ABA)-hydroxylating activity and a soluble PA-reducing activity. The particulate ABA-hydroxylating enzyme showed a requirement for O₂ and NADPH, inhibition by CO, and high substrate specificity for (+)-ABA. Acetylation of short term incubation mixtures gave evidence for the presence of 6′-hydroxymethyl-ABA as an intermediate in PA formation. Determinations of endogenous ABA and DPA concentrations suggest that the ABA-hydroxylating and PA-reducing enzymes are extensively metabolizing ABA in the intact E. lobata seed.     

Impact of Irrigation Strategies on Abscisic Acid and its Catabolites Profiles in Leaves and Berries of Baco noir Grapes

To understand the relationship among soil and plant water status, plant physiology, and the hormonal profiles associated with it, abscisic acid (ABA) and its catabolites [phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy-ABA, 8′-hydroxy-ABA, neophaseic acid, and abscisic acid glucose ester (ABA-GE)] in leaves and berries from wine grape cultivar Baco noir (Folle blanche × Vitis riparia) were analyzed. The experiment was conducted during the growing seasons 2006 and 2007 in an irrigation trial set up in a commercial vineyard located in Niagara-on-the-Lake, ON, Canada. ABA and its metabolites were quantified using liquid chromatography with ion trap combined with electrospray ionization-mass spectrometry. The hormonal profile indicated a direct relationship between the amount of ABA and climatic factors. The ABA varied between 582 and 4,026 ng g^?1 dry matter (DM), DPA between 417 and 562 ng g^?1, and ABA-GE between 337 and 2,764 ng g^?1 DM. At many sampling times PA in the leaves was undetectable, and its highest concentration (260 ng g^?1 DM) was at beginning of July 2007. ABA followed different catabolic pathways depending on the plant water status. ABA was likely catabolized by conjugation to form ABA-GE in treatments at higher water deficit levels, whereas in treatments with high water status, the oxidation pathway leading to DPA or PA was likely preferred. The ABA and ABA-GE concentrations in the berries at harvest showed high correlation with soil and plant water status.     

Copper amine oxidase and phospholipase D act independently in abscisic acid (ABA)-induced stomatal closure in Vicia faba and Arabidopsis

Recent evidence has demonstrated that both copper amine oxidase (CuAO; EC 1.4.3.6) and phospholipase D (PLD; EC 3.1.4.4) are involved in abscisic acid (ABA)-induced stomatal closure. In this study, we investigated the interaction between CuAO and PLD in the ABA response. Pretreatment with either CuAO or PLD inhibitors alone or that with both additively led to impairment of ABA-induced H₂O₂ production and stomatal closure in Vicia faba. ABA-stimulated PLD activation could not be inhibited by the CuAO inhibitor, and CuAO activity was not affected by the PLD inhibitor. These data suggest that CuAO and PLD act independently in the ABA response. To further examine PLD and CuAO activities in ABA responses, we used the Arabidopsis mutants cuaoζ and pldα1. Ablation of guard cell-expressed CuAOζ or PLDα1 gene retarded ABA-induced H₂O₂ generation and stomatal closure. As a product of PLD, phosphatidic acid (PA) substantially enhanced H₂O₂ production and stomatal closure in wide type, pldα1, and cuaoζ. Moreover, putrescine (Put), a substrate of CuAO as well as an activator of PLD, induced H₂O₂ production and stomatal closure in WT but not in both mutants. These results suggest that CuAO and PLD act independently in ABA-induced stomatal closure.     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure     

Abscisic Acid and Its Catabolites in Leaves and Berries of Chardonnay are Affected by Water Status

The biosynthetic pathway of abscisic acid (ABA) is well known. The aim of this study was to investigate the relationship among various ABA catabolites in leaves and berries of Chardonnay grapevines grown under various irrigation regimes. An irrigation trial was set up in one vineyard, located in Niagara-on-the-Lake, ON, Canada, consisting of seven treatments: control (non-irrigated), plus three water levels (100, 50, and 25 % of estimated crop evapotranspiration) combined with two irrigation imposition times (fruit set, veraison). No irrigation occurred prior to treatment imposition. ABA, phaseic acid (PA), dihydrophaseic acid (DPA), 7′-hydroxy-ABA, 8′-hydroxy-ABA, neophaseic acid, and ABA glucose ester (ABA-GE) were quantified in leaves and berries by HPLC–MS. ABA was likely catabolized by conjugation to form ABA-GE in treatments under high levels of water deficit, while in treatments with high water status, the oxidation pathway leading to DPA or PA predominated. Concentrations of ABA and its catabolites therefore reflected vine water status, whereby the specific ABA catabolic pathways in leaves and berries were determined by water status level. Hormonal profiles suggested a direct relationship between ABA and vine water status. The concentration of ABA in Chardonnay may explain why and how white cultivars adapt to drought stress versus red cultivars.

     

Abscisic acid synthesis and metabolism in barley leaves and protoplasts

Protoplasts isolated from barley (Hordeum vulgare L. cv. Clipper) leaves contained abscisic acid (ABA). The ABA content of these protoplasts did not change when they were incubated for up to 6 h in media of decreasing osmotic potential. There was a substantial, but transient, increase in ABA in barley leaf segments during protoplast isolation. The magnitude of this increase was inversely dependent on the osmotic potential of the isolation medium. Maximum ABA content was recorded after 2 h of exposure to the sorbitol-containing medium. The subsequent decline was due to conversion of ABA to phaseic acid (PA) and to other metabolites.Barley mesophyll protoplasts were not able to metabolise ABA, PA or any of the other metabolites formed from ABA by intact leaf tissue.     

Sites of Abscisic Acid Synthesis and Metabolism in Ricinus communis L

The sites of abscisic acid (ABA) synthesis and metabolism in Ricinus communis L. were investigated by analyzing the levels of ABA and its two metabolites phaseic acid (PA) and dihydrophaseic acid (DPA) in the shoot tips, mature leaves, and phloem sap of stressed and nonstressed plants.     

Abscisic Acid Metabolism in Relation to Water Stress and Leaf Age in Xanthium strumarium

Intact plants of Xanthium strumarium L. were subjected to a water stress-recovery cycle. As the stress took effect, leaf growth ceased and stomatal resistance increased. The mature leaves then wilted, followed by the half expanded ones. Water, solute, and pressure potentials fell steadily in all leaves during the rest of the stress period. After 3 days, the young leaves lost turgor and the plants were rewatered. All the leaves rapidly regained turgor and the younger ones recommenced elongation. Stomatal resistance declined, but several days elapsed before pre-stress values were attained.

Abscisic acid (ABA) and phaseic acid (PA) levels rose in all the leaves after the mature ones wilted. ABA-glucose ester (ABA-GE) levels increased to a lesser extent, and the young leaves contained little of this conjugate. PA leveled off in the older leaves during the last 24 hours of stress, and ABA levels declined slightly. The young leaves accumulated ABA and PA throughout the stress period and during the 14-hour period immediately following rewatering. The ABA and PA contents, expressed per unit dry weight, were highest in the young leaves. Upon rewatering, large quantities of PA appeared in the mature leaves as ABA levels fell to the pre-stress level within 14 hours. In the half expanded and young leaves, it took several days to reach pre-stress ABA values. ABA-GE synthesis ceased in the mature leaves, once the stress was relieved, but continued in the half expanded and young leaves for 2 days.

Mature leaves, when detached and stressed, accumulated an amount of ABA similar to that in leaves on the intact plant. In contrast, detached and stressed young leaves produced little ABA. Detached mature leaves, and to a lesser extent the half expanded ones, rapidly catabolized ABA to PA and ABA-GE, but the young leaves did not. Studies with radioactive (±)-ABA indicated that in young leaves the conversion of ABA to PA took place at a much lower rate than in mature ones. Leaves of all ages rapidly conjugated PA to PA-glucose ester. Furthermore, when half expanded leaves were stressed on the intact plant, their rate of ABA catabolism was enhanced, an effect not observed in the young leaves.

In conclusion, young leaves on intact Xanthium plants produce little stress-induced ABA themselves, but due to import and a low rate of catabolism accumulate more ABA and PA than mature leaves.

     

Analysis of Phytohormone Profiles during Male and Female Cone Initiation and Early Differentiation in Long-shoot Buds of Lodgepole Pine

In lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone initiation and gender differentiation are site-specific in long-shoot buds, with female cones in the distal portion and male cones in the proximal portion. By using high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI–MS/MS) in multiple-reaction monitoring (MRM) mode, cytokinins, indole-3-acetic acid (IAA), abscisic acid (ABA), and their selected metabolites were investigated in developing long-shoot buds from multiple genotypes. Spatially, higher concentrations of trans-zeatin riboside (t-ZR) and dihydrozeatin riboside (dhZR) existed in the distal parts of long-shoot buds, whereas concentrations of isopentenyl adenosine (iPA), IAA, ABA glucose ester (ABA-GE), and phaseic acid (PA) were higher in the proximal parts in all investigated genotypes. In long-shoot buds of genotypes with a history of high female cone yield, concentrations of t-ZR and the ratio of zeatin-type to isopentenyl-type cytokinins were higher in the entire buds, whereas dhZR or IAA was higher in either the distal or the proximal part, respectively. In low female cone yielding genotypes, concentrations of c-ZR, iPA, ABA-GE, and PA were higher in both of the parts. Temporally, concentrations of several hormone-related compounds showed obvious changes in late June and late July, prior to male and female cone bud differentiation. This study reveals that the local hormonal status in a long-shoot bud at specific developmental stages may play an important role in gender determination and cone yield.     

Delayed leaf senescence by exogenous lyso-phosphatidylethanolamine: Towards a mechanism of action

Exogenous application of the lysophospholipid, lyso-phosphatidylethanolamine (LPE) is purported to delay leaf senescence in plants. However, lyso-phospholipids are well known to possess detergent-like activity and application of LPE to plant tissues might be expected to rather elicit a wound-like response and enhance senescence progression. Since phosphatidic acid (PA) accumulation and leaf cell death are a consequence of wounding, PA- and hormone-induced senescence was studied in leaf discs from Philodendron cordatum (Vell.) Kunth plants in the presence or absence of egg-derived 18:0-LPE and senescence progression quantified by monitoring both lipid peroxidation (as the change in malondialdehyde concentration), and by measuring retention of total chlorophyll (Chl_a+b) and carotenoids (C_c+x). Only abscisic acid (ABA) stimulated lipid peroxidation whereas ABA, 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene (ETH), and 16:0–18:2-PA stimulated loss of chloroplast pigments. Results using primary alcohols as attenuators of the endogenous PA signal confirmed a role for PA as an intermediate in both ABA- and ETH-mediated senescence progression. Exogenous 18:0-LPE did not appear to influence senescence progression and was unable to reverse hormone-induced senescence progression. However, when supplied together with 16:0–18:2-PA at 1:1 (mol:mol), activity of phosphatidylglycerol (PG) hydrolase, chlorophyllase (E.C. 3.1.1.14), and progression of leaf senescence were negated. This apparent anti-senescence activity of exogenous 18:0-LPE was associated with induction of the pathogenesis-related protein, extracellular acid invertase (Ac INV, E.C. 3.2.1.26) suggesting that 18:0-LPE like 16:0–18:2-PA functions as an elicitor.     

Abscisic Acid metabolism by source and sink tissues of sugar beet

The fate of exogenously applied, labeled abscisic acid (±)-(ABA) was followed in source leaves and taproot sink tissues of sugar beet (Beta vulgaris cv AH-11). The objective was to determine if differential pathways for ABA metabolism exist in source and sink tissues. Tissue discs were incubated for up to 13 hours in a medium containing 1 micromolar labeled ABA. At various time intervals, samples were taken for metabolite determination by reverse-phase high performance liquid chromatography. The labeled metabolites were identified by retention times using an online scintillation counter.

Dihydrophaseic acid (DPA) aldopyranoside, DPA, phaseic acid (PA), ABA glucose ester (ABA-GE), and two unidentified compounds were recovered from both tissues. An additional unidentified metabolite was also present in root tissue. Leaf tissue discs exhibited a higher capacity for ABA conjugation, and root discs showed a greater preference for ABA catabolism to PA and DPA. After 4 to 5 hours, ABA incorporation into the various metabolites was proportional to the external ABA concentration in both tissues. But the internal ABA pool size was independent of external concentrations below 10⁻⁶ molar. These results suggested that rates of ABA metabolism was proportional to the rates of uptake in both tissues.

     

para-Aminobenzoic Acid Is a Precursor in Coenzyme Q6 Biosynthesis in Saccharomyces cerevisiae

Coenzyme Q (ubiquinone or Q) is a crucial mitochondrial lipid required for respiratory electron transport in eukaryotes. 4-Hydroxybenozoate (4HB) is an aromatic ring precursor that forms the benzoquinone ring of Q and is used extensively to examine Q biosynthesis. However, the direct precursor compounds and enzymatic steps for synthesis of 4HB in yeast are unknown. Here we show that para-aminobenzoic acid (pABA), a well known precursor of folate, also functions as a precursor for Q biosynthesis. A hexaprenylated form of pABA (prenyl-pABA) is normally present in wild-type yeast crude lipid extracts but is absent in yeast abz1 mutants starved for pABA. A stable ¹³C₆-isotope of pABA (p- amino[aromatic-¹³C₆]benzoic acid ([¹³C₆]pABA)), is prenylated in either wild-type or abz1 mutant yeast to form prenyl-[¹³C₆]pABA. We demonstrate by HPLC and mass spectrometry that yeast incubated with either [¹³C₆]pABA or [¹³C₆]4HB generate both ¹³C₆-demethoxy-Q (DMQ), a late stage Q biosynthetic intermediate, as well as the final product ¹³C₆-coenzyme Q. Pulse-labeling analyses show that formation of prenyl-pABA occurs within minutes and precedes the synthesis of Q. Yeast utilizing pABA as a ring precursor produce another nitrogen containing intermediate, 4-imino-DMQ₆. This intermediate is produced in small quantities in wild-type yeast cultured in standard media and in abz1 mutants supplemented with pABA. We suggest a mechanism where Schiff base-mediated deimination forms DMQ₆ quinone, thereby eliminating the nitrogen contributed by pABA. This scheme results in the convergence of the 4HB and pABA pathways in eukaryotic Q biosynthesis and has implications regarding the action of pABA-based antifolates.     

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.