8[prime]-Methylene Abscisic Acid (An Effective and Persistent Analog of Abscisic Acid)

We report here the synthesis and biological activity of a new persistent abscisic acid (ABA) analog, 8[prime]-methylene ABA. This ABA analog has one additional carbon atom attached through a double bond to the 8[prime]-carbon of the ABA molecule. (+)-8[prime]-Methylene ABA is more active than the natural hormone (+)-ABA in inhibiting germination of cress seed and excised wheat embryos, in reducing growth of suspension-cultured corn cells, and in reducing transpiration in wheat seedlings. The (+)-8[prime]-methylene analog is slightly weaker than (+)-ABA in increasing expression of ABA-inducible genes in transgenic tobacco, but is equally active in stimulating a transient elevation of the pH of the medium of corn cell cultures. In corn cells, both (+)-ABA and (+)-8[prime]-methylene ABA are oxidized at the 8[prime] position. ABA is oxidized to phaseic acid and (+)-8[prime]-methylene ABA is converted more slowly to two isomeric epoxides. The alteration in the ABA structure causes the analog to be metabolized more slowly than ABA, resulting in longer-lasting and more effective biological activity relative to ABA.     

Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures

Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7[prime]hydroxyABA (7[prime]OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures. A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated. Racemic ABA, (+)-ABA, ([plus or minus])-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ([plus or minus])-7[prime]OHBA, (-)-DHABA, ([plus or minus])-ABA ald, and ([plus or minus])-ABA alc had a moderate effect, and PA was inactive. If the relative cellular water content decreased below 82%, dehydrin gene expression increased. Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance. PA had no effect on the induction of RAB proteins; however, ([plus or minus])- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance. Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance. The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.     

Structural Requirements of Abscisic Acid (ABA) and its Impact on Water Flow During Radial Transport of ABA Analogues Through Maize Roots

The effect of (+) (ABA) and (?)-abscisic acid and nine ABA metabolites, precursors or derivatives on radial water movement through maize roots, was investigated using a suction technique (Freundl and others 1998). (+)-ABA, (+)- and (?)-abscisyl aldehyde, (+)-8?-hydroxymethyl ABA, (+)-8?-methylene, and (+)-8?-acetylene ABA stimulated radial water transport. (?)-ABA, phaseic acid, and (+)-8?-acetylene methyl ABA were ineffective. ELISA analysis for ABA detected and apparent increase of free ABAxyl in xylem sap of excised root systems that were perfused with either (+)-abscisyl aldehyde, (+)-8?-methylene, (+)8?-acetylene-ABA, or ABA-glucose ester. The analogues (+)-8?-hydroxymethyl ABA and (?)-abscisyl aldehyde passed the cortex of maize roots without changing the ABAxyl. The data from this study permit conclusions about the structural requirements for hormonal regulation of hydraulic conductivity.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.     

Role of Endogenous Abscisic Acid in Potato Microtuber Dormancy

Potato (Solanum tuberosum L. cv Russet Burbank) microtubers generated in vitro from single-node explants contained substantial amounts (approximately 250 pmol/g fresh weight) of free abscisic acid (ABA) and were completely dormant for a minimum of 12 weeks. Microtubers that developed in the presence of 10 [mu]M fluridone (FLD) contained considerably reduced amounts (approximately 5-25 pmol/g fresh weight) of free ABA and exhibited a precocious loss of dormancy. Inclusion of exogenous racemic ABA in the FLD-containing medium suppressed the premature sprouting of these microtubers in a dose-dependent manner. At a concentration of 50 [mu]M, exogenous ABA restored internal ABA levels to control values and completely inhibited FLD-induced precocious sprouting. Exogenous jasmonic acid was ineffective in suppressing FLD-induced sprouting. Application of FLD to preformed, fully dormant microtubers also resulted in a reduction in internal ABA content and precocious sprouting. These results indicate that endogenous ABA is essential for the induction and maintenance of potato microtuber dormancy.     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Abscisic acid regulation of heterophylly in Marsilea quadrifolia L.: effects of R-(-) and S-(+) isomers

The plant hormone abscisic acid (ABA) induces a developmental switch in the aquatic fern Marsilea quadrifolia, causing the formation of aerial type characteristics, including the elongation of petioles and roots, a change in leaf morphology, the expansion of leaf surface area, and the shortening of the internodes. A number of ABA-responsive heterophylly (ABRH) genes are induced early during the transition. Using optically pure isomers of ABA, it was found that both the natural S-(+)-ABA and the unnatural R-(-)-ABA are capable of inducing a heterophyllous switch and regulating ABRH gene expression. When dose responses are compared, the unnatural ABA gives stronger morphogenic effects than the natural ABA at the same concentration, it is effective at lower concentrations, and its optimal concentration is also lower compared with the natural ABA. Deuterium-labelled ABA enantiomers were used to trace the fate of applied ABA and to distinguish the applied compound and its metabolites from the endogenous counterparts. In tissues, the supplied (+)-ABA was metabolized principally to dihydrophaseic acid, while the supplied (-)-ABA was converted at a slower rate to 7'-hydroxy abscisic acid. Treatment with either enantiomer resulted in increased biosynthesis of ABA, as reflected in the accumulation of endogenous dihydrophaseic acid. Taken together, these results suggest two distinct mechanisms of action for (-)-ABA: either (-)-ABA is intrinsically active, or its activity is due to the stimulation of ABA biosynthesis.     

Compartmentation and equilibration of abscisic Acid in isolated xanthium cells

The compartmentation of endogenous abscisic acid (ABA), applied (±)-[³H]ABA, and (±)-trans-ABA was measured in isolated mesophyll cells of the Chicago strain of Xanthium strumarium L. The release of ABA to the medium in the presence or absence of DMSO was used to determine the equilibration of ABA in the cells. It was found that a greater percentage of the (±)-[³H]ABA and the (±)-trans-ABA was released into the medium than of the endogenous ABA, indicating that applied ABA did not equilibrate with the endogenous material.     

Inhibitors of abscisic acid 8'-hydroxylase

Structural analogues of the phytohormone (+)-abscisic acid (ABA) have been synthesized and tested as inhibitors of the catabolic enzyme (+)-ABA 8'-hydroxylase. Assays employed microsomes from suspension-cultured corn cells. Four of the analogues [(+)-8'-acetylene-ABA, (+)-9'-propargyl-ABA, (-)-9'-propargyl-ABA, and (+)-9'-allyl-ABA] proved to be suicide substrates of ABA 8'-hydroxylase. For each suicide substrate, inactivation required NADPH, increased with time, and was blocked by addition of the natural substrate, (+)-ABA. The most effective suicide substrate was (+)-9'-propargyl-ABA (K(I) = 0.27 microM). Several analogues were competitive inhibitors of ABA 8'-hydroxylase, of which the most effective was (+)-8'-propargyl-ABA (K(i) = 1.1 microM). Enzymes in the microsomal extracts also hydroxylated (-)-ABA at the 7'-position at a low rate. This activity was not inhibited by the suicide substrates, showing that the 7'-hydroxylation of (-)-ABA was catalyzed by a different enzyme from that which catalyzed 8'-hydroxylation of (+)-ABA. Based on the results described, a simple model for the positioning of substrates in the active site of ABA 8'-hydroxylase is proposed. In a representative physiological assay, inhibition of Arabidopsis thaliana seed germination, (+)-9'-propargyl-ABA and (+)-8'-acetylene-ABA exhibited substantially stronger hormonal activity than (+)-ABA itself.     

Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

The 7[prime]-Methyl Group of Abscisic Acid Is Critical for Biological Activity in Wheat Embryo Germination

Wheat (Triticum aestivum L.) embryo germination is inhibited by natural (S)-(+)-abscisic acid (ABA). In this report we have determined critical structural features of the ABA molecule, particularly the methyl and ketone groups of the ABA ring, required for inhibitory activity. To examine the ring residues a series of new optically active ABA analogs have been synthesized in which the 4[prime]-keto, 7[prime]-, 8[prime]-, or 8[prime]- and 9[prime]-carbons have been replaced with hydrogen atoms. Each of the analogs was tested over a range of concentrations as a germination inhibitor. Enantiomers of the analogs altered at the 4[prime]-keto or 8[prime]- and 9[prime]-methyl groups were active, but less so than ABA. Both enantiomers of 7[prime]-demethylABA were inactive as germination inhibitors. The results show that the 7[prime]-methyl group is absolutely required for activity, but that the other residues are less critical for hormone recognition.     

Abscisic Acid Structure-Activity Relationships in Barley Aleurone Layers and Protoplasts (Biological Activity of Optically Active,Oxygenated Abscisic Acid Analogs)

Optically active forms of abscisic acid (ABA) and their oxygenated metabolites were tested for their biological activity by examining the effects of the compounds on the reversal of gibberellic acid-induced [alpha]-amylase activity in barley (Hordeum vulgare cv Himalaya) aleurone layers and the induction of gene expression in barley aleurone protoplasts transformed with a chimeric construct containing the promoter region of an albumin storage protein gene. Promotion of the albumin storage protein gene response had a more strict stereochemical requirement for elicitation of an ABA response than inhibition of [alpha]-amylase gene expression. The naturally occurring stereoisomer of ABA and its metabolites were more effective at eliciting an ABA-like response. ABA showed the highest activity, followed by 7[prime]-hydroxyABA, with phaseic acid being the least active. Racemic 8[prime]-hydroxy-2[prime],3[prime]-dihydroABA, an analog of 8[prime]-hydroxyABA, was inactive, whereas racemic 2[prime],3[prime]-dihydroABA was as effective as ABA. The differences in response of the same tissue to the ABA enantiomers lead us to conclude that there exists more than one type of ABA receptor and/or multiple signal transduction pathways in barley aleurone tissue.     

Abscisic acid metabolism in Ceratocystis coerulescens

The fungus Ceratocystis coerulescens Bakshi (strain RWD 390) has been shown to produce the plant hormone, abscisic acid (ABA). The production of ABA in defined liquid medium during a culture period of 50 days was measured by gas-liquid chromatography. A considerable accumulation of ABA occurred in the stationary phase. Maximum ABA contents were 3.5 ng ml⁻¹ in culture media and 218 ng (g dry weight)⁻¹ in mycelial extracts.
The ABA-metabolizing capability of the fungus was investigated. Dihydrophaseic acid, and phaseic acid, ABA metabolites in higher plants, were not present in cultures of Ceratocystis coerulescens . When [2-¹⁴C]-ABA was fed to the fungus, the formation of [2-¹⁴C]- 2-trans , 4- trans -ABA and a second metabolite, less polar than ABA, was observed. This suggests a different metabolic pathway of ABA in the fungus.     

Evidence for an Extracellular Reception Site for Abscisic Acid in Commelina Guard Cells

The phytohormone abscisic acid (ABA) triggers stomatal closing as a physiological response to drought stress. Several basic questions limit an understanding of the mechanism of ABA reception in guard cells. Whether primary ABA receptors are located on the extracellular side of the plasma membrane, within the intracellular space of guard cells, or both remains unknown. Furthermore, it is not clear whether ABA must be transported into guard cells to exert control over stomatal movements. In the present study, a combination of microinjection into guard cells and physiological assays of stomatal movements have been performed to determine primary sites of ABA reception in guard cells. Microinjection of ABA into guard cells of Commelina communis L. resulted in injected cytosolic concentrations of 50 to 200 [mu]M ABA and in additional experiments in lower concentrations of approximately 1 [mu]M ABA. Stomata with ABA-loaded guard cells (n > 180) showed opening similar to stomata with uninjected guard cells. The viability of guard cells following ABA injection was demonstrated by neutral red staining as well as monitoring of stomatal opening. Extracellular application of 10 [mu]M ABA inhibited stomatal opening by 98% at pH 6.15 and by 57% at pH 8.0. The pH dependence of extracellular ABA action may suggest a contribution of an intracellular ABA receptor to stomatal regulation. The findings presented here show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. Furthermore, these data provide evidence that a reception site for ABA-mediated inhibition of stomatal opening is on the extracellular side of the plasma membrane of guard cells.     

The Catabolism of (+/-)-Abscisic Acid by Excised Leaves of Hordeum vulgare L. cv Dyan and Its Modification by Chemical and Environmental Factors

Excised light-grown leaves and etiolated leaves of Hordeum vulgare L. cv Dyan catabolized applied (±)-[2-¹⁴C]abscisic acid ([±]-[2-¹⁴C]ABA) to phaseic acid (PA), dihydrophaseic acid (DPA), and 2′-hydroxymethyl ABA (2′-HMABA). Identification of these catabolites was made by microchemical methods and by combined capillary gas chromatographymass spectrometry (GC-MS) following high dose feeds of nonlabeled substrate to leaves. Circular dichroism analysis revealed that 2′-HMABA was derived from the (−) enantiomer of ABA. By selecting tissue samples in which endogenous catabolites were undetectable by gas chromatography, it was possible to identify unequivocally ABA catabolites by GC-MS without the need to employ deuteriated substrate to distinguish the (±)-ABA catabolites from the same endogenous compounds. Refeeding studies were used to confirm the catabolic route. The methyl ester of (±)-[2¹⁴C]-ABA was hydrolyzed efficiently by light-grown leaves of H. vulgare. Leaf age played a significant role in (±)-ABA catabolism, with younger leaves being less able than their older counterparts to catabolize this compound. The catabolism of (±)-ABA was inhibited markedly in water-stressed Hordeum leaves which was characterized by a decreased incorporation of label into 2′-HMABA, DPA, and conjugates. The specific, mixed function oxidase inhibitor, ancymidol, did not inhibit, dramatically, (±)-ABA catabolism in light-grown leaves of Hordeum whereas the 80s ribosome, translational inhibitor, cycloheximide, inhibited this process markedly. The 70s ribosome translational inhibitors, lincomycin and chloramphenicol, were less effective than cycloheximide in inhibiting (±)-ABA catabolism, implying that cytoplasmic protein synthesis is necessary for the catabolism of (±)-ABA in Hordeum leaves whereas chloroplast protein synthesis plays only a minor role. This further suggests that the enzymes involved in (±)-ABA catabolism in this plant are cytoplasmically synthesized and are `turned-over' rapidly, although the enzyme responsible for glycosylating (±)-ABA itself appeared to be stable.     

Induction of a wheat Em promoter by ABA and optically pure ABA analogs in white spruce (Picea glauca) protoplasts

In white spruce ( Picea glauca ) protoplasts, abscisic acid (ABA) and optically pure ABA analogs induced expression of a reporter gene under regulation of a wheat ABA-responsive promoter. A fusion of a 650 bp promoter fragment from the wheat Em gene promoter and the Escherichia coli uidA sequence encoding β -glucuronidase (GUS) was linked in the plasmid pBM 113Kp. Expression of the Em-uidA fusion varied among 6 white spruce genotypes. Protoplasts from 4-day-old embryogenic suspension cultures gave the highest GUS activity relative 10 other stages in the 7-day growth cycle of suspension cultures. Racemic ABA [R.S-(±)-ABA] induced a significant increase of protoplast GUS activity over background at a concentration of 1 × 10⁻⁵ M , but maximum GUS activity was found at 1 × 10⁻³ M , ABA stereochemistry had a significant effect on gene expression. The natural isomer of ABA [S-(+)-ABA] was an effective inducer at a concentration as low as 1 × 10⁻⁷ M , but a concentration of greater than 1 × 10⁻⁴ M was required for induction by [R-(—)-ABA]. Moreover, analogs with the same configuration at C-l¹ as that of natural ABA were more effective for induction of expression from the Em-uidA . insert at 1 × 10⁻⁴ M than were their enamiomers. Plasnud pBI511. carrying the chloramphenicol acety] transferase (CAT) gene driven by the constitutively expressed, tandemly duplicated cauliflower mosaic virus 35S promoter, was co-electroporated with pBM113Kp for monitoring Ihe influence of addition of exogenous ABA or ABA analogs on heterologous gene expression in protoplasts. CAT activity was not significantly affected by the presence or absence of ABA or the analogs used.     

Changes in ABA turnover and sensitivity that accompany dormancy termination of yellow-cedar (Chamaecyparis nootkatensis) seeds.

Yellow-cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds exhibit prolonged coat-imposed dormancy following their dispersal from the parent plant. Analyses were undertaken using S-(+)-[(3)H] abscisic acid (ABA) to monitor the capacity of embryos to metabolize ABA following their isolation from seeds subjected to various dormancy-breaking and control treatments. Radiolabelled phaseic acid (PA) and dihydrophaseic acid (DPA) were detected in embryos and, to a greater extent in the surrounding media, by 48 h regardless of whether the embryos had been excised from seed previously subjected to only a 3 d soak or to a full dormancy-breaking treatment. Of the two enantiomers of ABA, only the natural S-(+)-ABA effectively inhibited germination of isolated embryos. A metabolism-resistant synthetic ABA analogue S-[8',8',8',9',9',9']-hexadeuteroabscisic acid, S-(+)-d6-ABA, consistently slowed the germination rate of excised embryos to a greater extent than that caused by natural S-(+)-ABA. The deuterium-labelled ring methyl groups of the analogue made it more resistant to oxidation by yellow-cedar embryos and thus rendered the analogue more persistent and possessing greater activity. With increasing time of exposure to moist chilling, yellow-cedar embryos became increasingly insensitive to both ABA and to the analogue. Subjecting seed to chemical treatments (GA(3) in combination with 1-propanol) prior to moist chilling strongly enhanced the germinability of whole seeds. This treatment also had a relatively greater impact on ABA metabolism than did moist chilling alone, as indicated by a greater capacity of S-(+)-d6-ABA to inhibit the germination of embryos as compared to S-(+)-ABA. Moist chilling was most critical for reduced ABA sensitivity of embryos. A change in the embryo's ability to metabolize ABA and reduced embryo sensitivity to ABA are two factors associated with dormancy termination of whole seeds of yellow cedar; a change in only one of these factors is insufficient to elicit high germinability.     

Competitive Inhibition of Abscisic Acid-Regulated Gene Expression by Stereoisomeric Acetylenic Analogs of Abscisic Acid

The properties of two enantiomeric synthetic acetylenic abscisic acid (ABA) analogs (PBI-51 and PBI-63) in relation to ABA-sensitive gene expression are reported. Using microspore-derived embryos of Brassica napus as the biological material and their responsiveness to ABA in the expression of genes encoding storage proteins as a quantitative bioassay, we measured the biological activity of PBI-51 and PBI-63. Assays to evaluate agonistic activity of either compound applied individually showed a dose-dependent increase in napin gene expression on application of PBI-63. Maximal activity of about 40 [mu]M indicated that PBI-63 was an agonist, although somewhat weaker than ABA. PBI-63 has a similar stereochemistry to natural ABA at the junction of the ring and side chain. In contrast, PBI-51 showed no agonistic effects until applied at 40 to 50 [mu]M. Even then, the response was fairly weak. PBI-51 has the opposite stereochemistry to natural ABA at the junction of the ring and side chain. When applied concurrently with ABA, PBI-63 and PBI-51 had distinctly different properties. PBI-63 (40 [mu]M) and ABA (5 [mu]M) combined gave results similar to the application of either compound separately with high levels of induction of napin expression. PBI-51 displayed a reversible antagonistic effect with ABA, shifting the typical ABA dose-response curve by a factor of 4 to 5. This antagonism was noted for the expression of two ABA-sensitive genes, napin and oleosin. To test whether this antagonism was at the level of ABA recognition or uptake, ABA uptake was monitored in the presence of PBI-51 or PBI-63. Neither compound decreased ABA uptake. Treatments with either PBI-51 or PBI-63 showed an effect on endogenous ABA pools by permitting increases of 5- to 7-fold. It is hypothesized that this increase occurs because of competition for ABA catabolic enzymes by both compounds. The fact that ABA pools did not decrease in the presence of PBI-51 suggests that PBI-51 must exert its antagonistic properties through direct competition with ABA at a hormone-recognition site.