A new abscisic acid catabolic pathway

We report the discovery of a new hydroxylated abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism in Brassica napus siliques. This metabolite reveals a previously unknown catabolic pathway for ABA in which the 9'-methyl group of ABA is oxidized. Analogs of (+)-ABA deuterated at the 8'-carbon atom and at both the 8'- and 9'-carbon atoms were fed to green siliques, and extracts containing the deuterated oxidized metabolites were analyzed to determine the position of ABA hydroxylation. The results indicated that hydroxylation of ABA had occurred at the 9'-methyl group, as well as at the 7'- and 8'-methyl groups. The chromatographic characteristics and mass spectral fragmentation patterns of the new ABA metabolite were compared with those of synthetic 9'-hydroxy ABA (9'-OH ABA), in both open and cyclized forms. The new compound isolated from plant extracts was identified as the cyclized form of 9'-OH ABA, which we have named neophaseic acid (neoPA). The proton nuclear magnetic resonance spectrum of pure neoPA isolated from immature seeds of B. napus was identical to that of the authentic synthetic compound. ABA and neoPA levels were high in young seeds and lower in older seeds. The open form (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid, but not neoPA, exhibited ABA-like bioactivity in inhibiting Arabidopsis seed germination and in inducing gene expression in B. napus microspore-derived embryos. NeoPA was also detected in fruits of orange (Citrus sinensis) and tomato (Lycopersicon esculentum), in Arabidopsis, and in chickpea (Cicer arietinum), as well as in drought-stressed barley (Hordeum vulgare) and B. napus seedlings.     

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)     

Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes

The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.     

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.

     

Analysis of the metabolites of the sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid in Sprague–Dawley rat urine

The sodium salt of 6-hydroxy-5-(phenylazo)-2-naphthalenesulfonic acid (SS-AN), which is a subsidiary color present in Food Yellow No. 5 [Sunset Yellow FCF, disodium salt of 6-hydroxy-5-(4-sulfophenylazo)-2-naphthalenesulfonic acid], was orally administered to Sprague–Dawley rats. Metabolite A, metabolite B, and unaltered SS-AN were detected as colored metabolites in the rat urine. Analysis of the chemical structures showed that metabolite A (major peak) was 6-hydroxy-5-(4-sulfooxyphenylazo)-2-naphthalenesulfonic acid, the sulfuric acid conjugate of SS-AN, and metabolite B (minor peak) was 6-hydroxy-5-(4-hydroxyphenylazo)-2-naphthalenesulfonic acid (SS-PAP), which is a derivative of metabolite A without the sulfuric acid. The colorless metabolites p-aminophenol, o-aminophenol, and aniline present in the urine were analyzed by liquid chromatography–mass spectrometry. The orally administered SS-AN had been metabolized to the colorless metabolites (p-aminophenol 45.3%, o-aminophenol 9.4%, aniline 0.4%) in the 24-h urine samples. Analysis of the colored metabolites by high-performance liquid chromatography with detection at 482 nm indicated the presence of metabolite A (0.29%), SS-PAP (0.01%), and SS-AN (0.02%) were detected in the 24-h urine samples. Approximately 56% of SS-AN was excreted into the urine and the rest is probably excreted into feces.     

Use of the glucosyltransferase UGT71B6 to disturb abscisic acid homeostasis in Arabidopsis thaliana

A glucosyltransferase (GT) of Arabidopsis, UGT71B6, recognizing the naturally occurring enantiomer of abscisic acid (ABA) in vitro, has been used to disturb ABA homeostasis in planta. Transgenic plants constitutively overexpressing UGT71B6 (71B6-OE) have been analysed for changes in ABA and the related ABA metabolites abscisic acid glucose ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxyABA and neo-phaseic acid. Overexpression of the GT led to massive accumulation of ABA-GE and reduced levels of the oxidative metabolites PA and DPA, but had marginal effect on levels of free ABA. The control of ABA homeostasis, as reflected in levels of the different metabolites, differed in the 71B6-OEs whether the plants were grown under standard conditions or subjected to wilt stress. The impact of increased glucosylation of ABA on ABA-related phenotypes has also been assessed. Increased glucosylation of ABA led to phenotypic changes in post-germinative growth. The use of two structural analogues of ABA, known to have biological activity but to differ in their capacity to act as substrates for 71B6 in vitro, confirmed that the phenotypic changes arose specifically from the increased glucosylation caused by overexpression of 71B6. The phenotype and profile of ABA and related metabolites in a knockout line of 71B6, relative to wild type, has been assessed during Arabidopsis development and following stress treatments. The lack of major changes in these parameters is discussed in the context of functional redundancy of the multigene family of GTs in Arabidopsis.     

ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

In plants, metabolism of the plant hormone (+)- S -abscisic acid (ABA) occurs by oxidation at the 8'-carbon on the ABA ring, producing (+)-8'-hydroxy-ABA (8'OH-ABA), which undergoes ring closure to phaseic acid (PA), or at the 7'-carbon producing 7'-hydroxy-ABA (7'OH-ABA). New methods for synthesixing and stabilizing 8'OH-ABA have enabled us for the first time to compare the biological activities of the ABA metabolites. 8'OH-ABA. PA and 7'OH-ABA. Activities were determined in wheat ( Triticum aestivum , cvs Brevor and Clarks Cream) using optically pure natural enantiomers of ABA. 8'OH-ABA, PA, and 7'OH-ABA. Comparison of wheat embryo germination inhibitory activity showed that only the metabolite 7'OH-ABA had significant activity (10 to 40% of effective ABA activity) as a germination inhibitor of wheat embryos. The metabolites had differential effects on ABA-responsive gene expression. Both 8'OH-ABA and PA were effective inducers of an ABA-responsive LEA (late embryogenesis abundant) gene, wheat group 3 LEA , in seedling roots, suggesting that ABA metabolites can maintain and prolong LEA gene expression. The metabolites had only a slight effect on accumulation of an ABA-responsive protein kinase ( PKABA1 ) mRNA or pMA1951 . These results are consistent with the idea that there are several control points in the ABA metabolic pathway and indicate that assessment of ABA signalling responses should include consideration of the biological activities of ABA metabolites.     

Desiccation decreases abscisic acid content in hybrid larch (Larix X leptoeuropaea) somatic embryos

Previous studies indicated that the high endogenous abscisic acid (ABA) content of hybrid larch ( Larix X leptoeuropaea ) somatic embryos was correlated with low germination frequency. However, when dried, the germination rate of the somatic embryos improved. Therefore, our present objective was to study the effect of desiccation on the levels of ABA and its glucose ester metabolite. Cotyledonary somatic embryos were subjected to drying treatments at 4⁰C under relative humidities of 98 and 59% for one week and the levels of both ABA and abscisic acid glucose ester (ABAGE) were followed by enzyme-linked immunoassay (ELISA). During desiccation at 98% relative humidity (RH) both ABA and ABAGE levels decreased in an irregular fashion. Regardless of RH, transient increases in ABA were observed that were paralleled by marked decreases in ABAGE. It is concluded that the desiccation of somatic embryos which leads to a decrease in ABA content, could explain the enhanced germination capacity of such embryos.     

Induction of hydroxycinnamic acid amides and tryptophan by jasmonic acid, abscisic acid and osmotic stress in barley leaves

The effects of jasmonic acid (JA) and abscisic aid (ABA) on secondary metabolism in barley (Hordeum vulgare L.) were investigated. Treatment with JA at 100 microM for 48 h induced accumulation of four compounds in barley primary leaves. The accumulation of these compounds was also observed after treatment with ABA at 100 microM. The induced compounds were identified as p-coumaroylputrescine, p-coumaroylagmatine, p-coumaroyl-3-hydroxyagmatine and tryptophan by spectroscopic methods. The profiles of compounds induced by application of JA and ABA were different. JA exhibited stronger inducing activity for hydroxycinnamic acid amides than ABA, while ABA was more active in tryptophan accumulation. The major hydroxycinnamic acid amides in JA- and ABA-treated leaves were p-coumaroylagmatine and p-coumaroyl-3-hydroxyagmatine, respectively. These differences suggested that JA and ABA act in distinct modes. The induction of these compounds was also observed in leaf segments treated with 1 M sorbitol and glucose. These findings suggested that JA and ABA are involved in accumulation of hydroxycinnamic acid amides and tryptophan in response to osmotic stress in barley.     

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.     

Metabolism of14C-abscisic acid during its IAA-promoted transport in etiolated segments of pea (Pisum sativum L.) epicotyls†

The metabolism of exogenously supplied abscisic acid (ABA) during translocation attracted under the influence of indolyl-3-acetic acid (IAA) was studied in etiolated segments of pea (Pisum sativum L.). After 8 and 24 h 90% and 60% of the ABA, respectively, were found in the segments in unchanged form. Phaseic acid, dihydrophaseic acid and the glucose ester of ABA were found as ABA metabolites. Results indicated that the growth processes initiated by the application of IAA were associated neither with an increased immobilization nor increased metabolization of this growth regulator. † Part II. Influence of Auxin-like Substances upon the Transport of¹⁴C-ABA in Long Pea Epicotyl Segments.     

Metabolism of R,S-[2-14C]abscisic acid by non-stressed and water-stressed detached leaves of wheat (Triticum aestivum L.)

Metabolism of R,S-[2-¹⁴C]abscisic acid (ABA) was studied in detached leaves of six wheat (Triticum aestivum) cultivars, using non-stressed leaves or leaves water stressed by desiccation to 90% of their original fresh weight. The rate constant of ABA metabolism was similar in nonstressed leaves of all cultivars. Water stress resulted in significantly lower rate constants in two cultivars which accumulated high levels of ABA when stressed, the constants decreasing by a factor of about 1.5. Rate constants for the remainder of the cultivars were not significantly different from those for the non-stressed controls. It was calculated that if decreased metabolism was the mechanism for the accumulation of ABA following water stress the rate constants of metabolism would have to be reduced by a factor of between 25 and 70. The results therefore support the hypothesis that enhanced synthesis rather than reduced degradation is the main process by which ABA levels are elevated following experimentally induced water stress. There were differences between the six cultivars in the products of ABA metabolism. Over the time period studied, oxidation to phaseic acid and dihydrophaseic acid as well as to other unidentified metabolites appeared to be the predominant pathway of ABA metabolism, rather than conjugation to ABA glucose ester and other more polar compounds.Abbreviations ABA abscisic acid - ABAGE abscisic-acid glucose ester - DPA dihydrophaseic acid - PA phaseic acid     

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.     

The Resolution by HPLC of RS-[2-14C]Me 1', 4'-cis-Diol of Abscisic acid and the Metabolism of (-)-R- and (+)-S-Abscisic Acid

The R- and S-enantiomers of racemic [2-¹⁴C]Me 1', 4'-cis-diolof abscisic acid have been separated by high performance liquidchromatography on an optically-active Pirkle column. R-[2-¹⁴C]-and S-[2-¹⁴C]abscisic acids, formed from the Me 1', 4'-cis-diolby oxidation and alkyline hydrolysis were fed to tomato shootsand the extracts analysed by reversed phase high performanceliquid chromatography. R-[2-¹⁴C]abscisic acid formed mainlythe abscisic acid glucose ester (ABAGE), abscisic acid l'-glucoside(ABAGS) and an uncharacterized conjugate. Dihydrophaseic acid4'-B-D-glucoside, the major metabolite of RS-abscisic acid intomato shoots, was found to be derived virtually exclusivelyfrom the natural, S-abscisic acid. Phaseic acid and conjugatesof abscisic acid were also found as products of the naturallyoccurring enantiomer. The resolution method was used to measurethe relative proportions of R and S enantiomers in the freeacid liberated from conjugates formed from RS-[2-¹⁴C]ABA fedto shoots. The ratios show an excess of the R-enantiomer: 5.8:1, ABAGE; 29.4: 1, ABAGE; 8.3: 1 for an uncharacterized conjugateand 6.1: 1 for the residual free [2-¹⁴C]ABA. Key words: ABA, HPLC, Tomato     

12(R)-hydroxyeicosatrienoic acid: a vasodilator cytochrome P-450-dependent arachidonate metabolite from the bovine corneal epithelium

When corneal microsomes were incubated with arachidonic acid in the presence of an NADPH-generating system, two biologically active metabolites of arachidonic acid were formed. The structure of one of the metabolites, compound C, was previously reported to be 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid and was found to be a potent inhibitor of the Na+/K+-ATPase in the cornea. The second metabolite, compound D, was found to be a potent vasodilator as well as having the property of stimulating protein influx into the aqueous humor of the eye. Following purification of compound D by thin layer chromatography and high pressure liquid chromatography, it was found to lack a UV chromophore in contrast to the previously reported cytochrome P-450-dependent metabolite. Mass spectrometric analysis using positive and negative ionization modes was carried out on derivatized compound D that had been synthesized from a mixture of labeled [( 5,6,8,9,11,12,14,15-2H8]) and unlabeled arachidonic acid incubated with corneal microsomes. The novel arachidonate metabolite had abundant fragment ions consistent with compound D being a monooxygenated derivative of arachidonic acid with a hydroxyl substituent at carbon 12 of the eicosanoid backbone; only seven deuterium atoms from [2H8]arachidonate were retained in the structure. Oxidative ozonolysis yielded a product indicating that the double bonds in metabolite D resided between carbons at positions 8 and 9 and positions 14 and 15 of the 20-carbon chain. Compound D was therefore characterized as 12-hydroxy-5,8,14-eicosatrienoic acid. Model compounds were synthesized from dimethyl malate with the hydroxy at the 12 position with both the R and S absolute configuration and with all double bonds of the cis configuration. Only the 12(R) isomer was found to be a potent vasodilator and to increase aqueous humor protein concentration, suggesting that the biologically active compound D was 12(R)-hydroxy-5,8,14-(Z,Z,Z)-eicosatrienoic acid. As this compound possesses proinflammatory properties, it may play a role in the wound-healing processes of corneal injury.     

Response of Cultured Maize Cells to (+)-Abscisic Acid, (-)-Abscisic Acid,and Their Metabolites

The metabolism and effects of (+)-S- and (-)-R-abscisic acid (ABA) and some metabolites were studied in maize (Zea mays L. cv Black Mexican Sweet) suspension-cultured cells. Time-course studies of metabolite formation were performed in both cells and medium via analytical high-performance liquid chromatography. Metabolites were isolated and identified using physical and chemical methods. At 10 [mu]M concentration and 28[deg] C, (+)-ABA was metabolized within 24 h, yielding natural (-)-phaseic acid [(-)-PA] as the major product. The unnatural enantiomer (-)-ABA was less than 50% metabolized within 24 h and gave primarily (-)-7[prime]-hydroxyABA [(-)-7[prime]-HOABA], together with (+)-PA and ABA glucose ester. The distribution of metabolites in cells and medium was different, reflecting different sites of metabolism and membrane permeabilities of conjugated and nonconjugated metabolites. The results imply that (+)-ABA was oxidized to (-)-PA inside the cell, whereas (-)-ABA was converted to (-)-7[prime]-HOABA at the cell surface. Growth of maize cells was inhibited by both (+)- and (-)-ABA, with only weak contributions from their metabolites. The concentration of (+)-ABA that caused a 50% inhibition of growth of maize cells was approximately 1 [mu]M, whereas that for its metabolite (-)-PA was approximately 50 [mu]M. (-)-ABA was less active than (+)-ABA, with 50% growth inhibition observed at about 10 [mu]M. (-)-7[prime]-HOABA was only weakly active, with 50% inhibition caused by approximately 500 [mu]M. Time-course studies of medium pH indicated that (+)-ABA caused a transient pH increase (+0.3 units) at 6 h after addition that was not observed in controls or in samples treated with (-)-PA. The effect of (-)-ABA on medium Ph was marginal. No racemization at C-1[prime] of (+)-ABA, (-)-ABA, or metabolites was observed during the studies.     

12S,19- and 12S,20-dihydroxyeicosanoids: novel 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid metabolites formed by hydroxylation and reduction in murine lymphocytes

Murine spleen cells and purified B lymphocytes oxidized arachidonic acid via the lipoxygenase pathway. The major metabolite of both the whole spleen and enriched B lymphocytes was 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. A novel metabolite was observed that did not have an absorbance from 210 to 400 nm, indicating the absence of a conjugated double bond system. The new metabolite was converted to the methyl ester, reduced by platinum oxide, derivatized to the trimethylsilyl ether, and analyzed by gas chromatography-mass spectrometry. A major and a minor component were observed in the analysis of the new compound. The major component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-19. The minor component had major diagnostic ions indicating the presence of hydroxyl groups at C-12 and C-20. The new metabolites are characterized as a mixture of 12S,19- and 12S,20-dihydroxyeicosanoids presumably formed by hydroxylation and reduction of one or more double bonds of 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid. These metabolites were formed predominantly with whole spleen lymphocytes but could be detected at longer incubation times or by using 12S-hydroxy-5,8-cis-10-trans-14-cis-eicosatetraenoic acid as the starting substrate with highly enriched B lymphocytes.     

The biological and structural similarity between lunularic acid and abscisic acid

Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced alpha-amylase induction in embryoless barley seeds at 120 microM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 microM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.     

Extracellular beta-glucosidase activity in barley involved in the hydrolysis of ABA glucose conjugate in leaves

Abscisic acid conjugate concentrations increased in barley xylem sap under salinity, whereas it remained at a low level in the intercellular washing fluid (IWF) of barley primary leaves (Hordeum vulgare cv. Gerbel). Here it is shown that IWF contains beta-glucosidase activity which releases abscisic acid (ABA) from the physiologically inactive ABA-glucose conjugate pool in the leaf apoplast. The following data support this conclusion and give the first biochemical and physiological characterization of the extracellular glucosidase activity in barley. Free ABA was released by the incubation of ABA glucose ester with IWF. The product exhibited the retention time of authentic ABA upon separation by thin layer chromatography and was identified by ABA-ELISA. p-Nitrophenol-beta-D-glucopyranoside (pNPG) was used as the substrate for beta-glucosidases. The K(M)(pNPG) was 1.8 mmol l(-1). The activity was affected by ABA glucopyranoside in a competitive type of inhibition with a K(I) of 400 micromol l(-1). Various hormone conjugates were compared with respect to their inhibitory effect on beta-glucosidase activity. Inhibition was highest for the ABA glucopyranoside and the zeatin riboside, but insignificant for ABA methyl ester and zeatin-9-beta-D-glucoside. The specific activity of the beta-glucosidase was 16-fold greater in IWF as compared to crude leaf extracts confirming its extracellular compartmentation. The activity of beta-glucosidase was strongly increased after growth in hydroponic medium supplemented with NaCl. The data support the hypothesis that the glucose conjugate is a long-distance transport form of ABA.