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First published online December 11, 2003; 10.1104/pp.103.030734 Plant Physiology 134:361-369 (2004) © 2004 American Society of Plant Biologists A New Abscisic Acid Catabolic Pathway1Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, Saskatchewan, Canada S7N 0W9
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.
The plant hormone S-(+)-abscisic acid (ABA) regulates many aspects of plant growth and development including embryo maturation, seed dormancy, stress responses, and stomatal aperture (Zeevaart and Creelman, 1988  ). The mode of action of ABA is complex and is a very active subject of investigation. Although substantial progress has been made in understanding signaling processes in various plant systems, many questions regarding the pleiotropic responses to ABA remain to be answered (Hetherington, 2001; Ritchie et al., 2002; Xiong et al., 2002). The pathway and regulation of ABA biosynthesis has been much studied in the last few years and is relatively well understood, but there has been far less progress in understanding ABA catabolism (Cutler and Krochko, 1999; Zeevaart, 1999; Taylor et al., 2000; Milborrow, 2001; Schwartz et al., 2003).
ABA can be metabolized by oxidation, reduction, or conjugation (Cutler and Krochko, 1999
Although there are reports that PA and DPA are hormonally inactive (Cutler and Krochko, 1999
We are developing and employing analytical methods for targeted metabolite profiling of plant signaling molecules (Abrams et al., 2003
Detecting a Novel ABA Metabolite As part of a study of ABA metabolism in B. napus, liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS-MS) was used to detect ABA and its metabolites in extracts from both seeds and pericarps from siliques of B. napus and Brassica rapa. In the course of scanning for known oxidized ABA catabolites in extracts of immature seeds and pericarps, a new compound (HPLC retention time, 11.5 min) was detected with an apparent formula weight of 280, isomeric with PA (9.3 min) and 7'-OH ABA (10.4 min; Fig. 1). Under the conditions employed, the retention time of this compound was different from all known ABA metabolites including trans-PA and trans-7'-OH ABA. Daughter ion scans of m/z 279, the apparent [M-H]- of the unknown compound, showed major transitions to m/z 205 and to m/z 139, similar to those of PA and distinct from those of 7'-OH ABA (Fig. 2, A-C).
To determine whether this new compound was an ABA metabolite and, if so, which position of ABA was hydroxylated, feeding experiments were performed using (+)-ABA with deuterium labeling at specific carbon atoms. First, S-(+)-[8',8',8'-2H3]ABA (S-(+)-[8'-2H3]ABA) was fed to immature (green) B. napus siliques. Extracts from [8'-2H3]ABA-treated siliques (separated into pericarps and seeds) were analyzed by LC-MS-MS, scanning for daughter ions from ions with m/z 279, 281, and 282. Peaks with elution time of PA (9.3 min) were observed for endogenous PA (m/z 279) and [2H2]PA (m/z 281), and peaks (11.5 min) were observed for the unknown endogenous metabolite and its corresponding trideutero analog (m/z 282), indicating that the unknown was an ABA metabolite and the position of hydroxylation was not at the 8'-carbon atom. The position of oxidation in this putative hydroxylated ABA metabolite was determined by feeding S-(+)-[5,8',8',8'-2H4]ABA (S-(+)-[5,8'-2H4]ABA), and S-(+)- [8',8',8',9',9',9'-2H6]ABA (S-(+)-[8',9'-2H6]ABA) to green siliques on different B. napus plants either through surface application or by injection. Table I shows the results of LC-MS-MS analysis of metabolites derived from labeled ABA in the feeding study.
In the tissue extract from the [5,8'-2H4]ABA feeding experiment, [2H3]PA (m/z 282; 9.3 min) and [2H4]7'-OH ABA (10.4 min) were detected, as well as the unlabeled putative hydroxylated ABA and its corresponding tetradeutero analog (m/z 283) with emergence times of 11.5 min. From tissue obtained from the [8',9'-2H6]ABA feeding experiment, we detected [2H5]PA (m/z 284, 9.3 min), [2H6]7'-OH ABA (10.4 min), the unlabeled putative hydroxylated ABA, and its corresponding pentadeutero analog (m/z 284, 11.5 min).
These results suggested that hydroxylation had occurred at the 9'-carbon atom and that the unknown hydroxylated ABA could be either 9'-OH ABA or its ring closed form, which had been given the name epi-PA (Takahashi et al., 1989
The natural product was isolated from a bulk sample of freeze-dried green (immature) seeds and shown to be identical with synthetic neoPA. The purified compound gave a single peak on HPLC on coinjection with synthetic neoPA. The increase in area was proportional to the amount of natural neoPA added (
To determine whether neoPA levels changed during silique development, we analyzed the levels of ABA and the three initial products of its oxidation (note that 8'-OH ABA is converted to PA during sample processing) in siliques at 24 to 25 d after anthesis (seed bloom stage) and 46 d after anthesis (seed physiological maturity stage; Table II). In seeds at 24 to 25 d after anthesis, neoPA was the most abundant of the three oxidized catabolites (0.9 µg g-1 dry weight, corresponding to 3.2 nmol g-1 dry weight in Table II) although seed ABA levels were much higher at 6 µg g-1 dry weight (corresponding to 23 nmol g-1 dry weight as in Table II). Amounts of ABA, PA, neoPA, and 7'-OH ABA were at least 20-fold higher than those at 46 d after anthesis. Amounts of ABA, PA, and neoPA of seeds at 24 to 25 d after anthesis were also at least 50-fold higher than those in their corresponding pericarps. Although ABA levels in pericarps were similar at the two tested stages, at 24 to 25 d after anthesis, there was a higher content of PA and neoPA than at 46 d after anthesis.
As an initial assessment of the hormonal activity of 9'-OH ABA, we compared the ability of the (+)- and (-)-isomers of 9'-OH ABA and neoPA to inhibit germination of Arabidopsis seeds. The (+)-isomer of 9'-OH ABA at a concentration of 10 µM inhibited germination completely; although the level of (+)ABA required to produce the same effect was 1 µM. The (-)-isomer of 9'-OH ABA was weakly active; significant inhibition of germination was apparent only at the highest concentration tested (33 µM; Fig. 4). Neither isomer of neoPA showed significant activity in the assay (Fig. 4).
To test of the ability of 9'-OH ABA to induce gene expression, we compared the ability of ABA, (+) 9'-OH ABA, and neoPA as they affected 3-ketoacyl-CoA synthase (fatty acid elongase [FAE]) expression in microspore-derived embryos of B. napus. 9'-OH ABA (10 µM) was as strong as ABA in inducing FAE gene expression in a little as 6 h, whereas neoPA had a negligible effect, its signal similar to that observed in the untreated T0 control (Fig. 5). This trend continued through the 24-h treatment. After 72 h, the effect of ABA was slightly stronger than that observed in the 9'-OH ABA treatment, the latter staying at the same intensity as that observed after 24 h. As usual, the neoPA was much less active than either the ABA or the 9'-OH ABA analog.
To determine whether ABA 9'-hydroxylation is a pathway that is widespread in plants, the occurrence of neoPA was investigated in a number of plant tissues. We detected neoPA in green Arabidopsis siliques, chickpea (Cicer arietinum) seed and pericarps, tomato (Lycopersicon esculentum) fruits, orange (Citrus sinensis) fruits, drought-stressed barley seedlings, and drought-stressed B. napus seedlings (Table III).
ABA metabolites were first identified and studied either by large-scale isolation (MacMillan and Pryce, 1968 ) or using tracer experiments with radioactive ABA (Milborrow, 1968, 1969, 1970; Tinelli et al., 1973; Lehmann et al., 1983; Boyer and Zeevaart, 1986; Balsevich et al., 1994; Wang et al., 2002). Such studies revealed the existence of the ABA 8'-hydroxylation and 7'-hydroxylation pathways. Using LC-MS-MS we have now discovered a novel ABA 9'-hydroxylation pathway and deduced the structure of the metabolite. The discovery of this novel catabolic pathway was made possible by recent developments in MS, particularly in HPLC-MS-MS. HPLC can be employed to separate components of a complex mixture, which can be directly analyzed with the use of the tandem mass spectrometers by selecting ions with specific mass to charge ratios and by observing fragmentation of the selected ions. The technique of multiple reaction monitoring (MRM) in which the transition of specific parent to fragment ions can be measured allows the analysis of compounds that are not chromatographically resolved. The new metabolite neoPA was first observed in HPLC-MS-MS experiments selecting for the negative ion with m/z 279, corresponding to the molecular weights of the anions of PA and 7'-OH ABA. Such experiments also reveal structural information, especially, as in the present case, when combined with feeding studies with deuterated hormone analogs. It is interesting to speculate about why neoPA had not been observed before. Levels of hormone catabolites in plant tissues are very low, making identification difficult. Radiolabeled ABA has been used to study known catabolites in many plant tissues and processes. Detection of labeled catabolites has been accomplished by thin-layer chromatography (TLC) or by HPLC, using standards to identify known catabolites. It is likely that neoPA and 9'-OH ABA were not previously identified because they can coelute with known metabolites. For example, in a typical HPLC chromatographic system employed for quantitation of ABA catabolites (on a 33 x 4.6 mm C18 column using isocratic acetonitrile-water-acetic acid [15:85:0.07, v/v] solvent system, the retention times of 9'-OH ABA, ABA glucose ester (ABA-GE), PA, neoPA, and trans-ABA are 2.05 min, 2.08 min, 2.16 min, 4.01 min, and 4.03 min), PA, 9'-OH ABA, and ABA-GE elute very close together as do neoPA and trans-ABA. Using typical TLC conditions, the pairs trans-PA and neoPA as well as trans-7'-OH ABA and 9'-OH ABA have similar chromatographic properties. Likely TLC bands or HPLC peaks with low levels of activity not corresponding to known metabolites were attributed to photoisomerization products, metabolism products from radiolabeled unnatural (-)-ABA, or degradation products. As well as these technical problems, previous studies on ABA catabolism may have been performed on tissues in which the levels of neoPA were not high. In this study, we found that levels were higher early in seed development and decreased as the seed matured.
For earlier studies on the importance of ABA turnover in plant physiological processes regulated by ABA, we had synthesized and tested ABA analogs with deuterium atoms on the 8'- and 9'-carbon atoms (Lamb et al., 1996
We previously observed a primary deuterium isotope effect in the oxidation of (+)-ABA to 8'-OH ABA in vivo using suspension cultured maize (Zea mays) cells (Lamb et al., 1996
By analogy with 8'-hydroxylation, it is expected that the first product of 9'-oxidation would be (2Z,4E)-5-[(1R,6S)-1-Hydroxy-6-hydroxymethyl-2,6-dimethyl-4-oxo-cyclohex-2-enyl]-3-methyl-penta-2,4-dienoic acid ((+)-9'-OH ABA), which can cyclize to, and exist in equilibrium with, the closed form neoPA (Fig. 6). Our first indications from studies with the synthetic material are that the open form has greater persistence at physiological pH than 8'-OH ABA. Under the experimental conditions used to isolate the hydroxylated ABA compounds, we found that the open form 9'-OH ABA closed to neoPA. Isolation of the closed form after extensive manipulation does not necessarily indicate that this is the predominant form in plant cells.
Subsequent to our initial identification of neoPA in B. napus siliques, we showed that neoPA occurs in a variety of other plants and tissues (Table III). The widespread occurrence of neoPA in different plants implies that ABA 9'-hydroxylation pathway is significant and general. In the germination assay with Arabidopsis seeds, we observed that the open form of the natural (+)-isomer of 9'-OH ABA is hormonally active (although less so than (+)-ABA), whereas the closed form (neoPA) is inactive (Fig. 4). In the B. napus microspore-derived embryo model system, we were able to demonstrate that (+)-9'-OH ABA is hormonally active on a level equivalent to natural ABA with respect to induction of the 3-ketoacyl-CoA synthase gene, the latter encoding a condensing enzyme essential to the production of erucic and eicosenoic acids in the developing embryo at the (early) torpedo stage. NeoPA (the closed form of (+)-9'-OH ABA) was comparatively far less active (Fig. 5). These findings are analogous to the observations that 8'-OH ABA and the stable analog 8' methylene ABA (Qi et al., 1998
This study and others in which hydroxylated ABAs exhibit significant hormonal activity (Todoroki et al., 1994 In this paper, we have described the discovery of two novel ABA metabolites, 9'-OH ABA and neoPA, arising from a new pathway of ABA metabolism in plants, and we demonstrate that 9'-OH ABA has significant activity in two ABA bioassays. These findings enrich our knowledge of ABA metabolism by revealing hitherto unappreciated complexity, and open new avenues of research in hormone signaling.
ABA, ABA Catabolites, and Deuterium-Labeled Analogs
The following chemicals were prepared as described previously: (+)ABA (Balsevich et al., 1994
Brassica napus cv Quantum plants were grown in a greenhouse under natural light supplemented with artificial light and heating to provide a 16-h light (25°C)/8-h night (18°C) cycle. Drip irrigation (Netafim Irrigation Inc., East Hamilton, Fresno, CA) of Plant-Prod 24-10-20 (Plant Products Co. Ltd., Brampton, ON, Canada) at 165 mg nitrogen L-1 was used for each young plant/seedling once a day for 2 min (rate of application 80-100 mL min-1); increased to 2 min twice daily for mature plants; and later increased to 4 to 6 min twice daily (a total of 320-600 mL d-1). The humidity was maintained at 45% to 65%. Only the primary and two secondary inflorescences were retained on each plant. Siliques were separated into seeds and pericarps, weighed, and frozen in liquid nitrogen before lyophilization. For large-scale purification and identification of ABA metabolites, B. napus cv AC EXCEL plants grown at the experimental farm of the Saskatoon Research Centre, Agriculture and Agri-Food Canada were collected between September 12 and 17, 2002. The branches with green siliques were shipped on dry ice and stored at -20°C. Green seeds were isolated from pericarps and frozen immediately in liquid nitrogen. The lyophilized seeds were mixed and stored for extraction. Other plant materials were: a green chickpea (Cicer arietinum L. cv Kabula Yuma) capsule from a greenhouse-grown plant with the seed separated from the remainder of the fruit; a portion of the peel and fruit of a Navel orange (Citrus sinensis L. Osbeck); green siliques and green leaves of Arabidopsis ecotype Columbia; garden-grown green cherry tomatoes (Lycopersicon esculentum Mill cv Favorita); mature rice (Oryza sativa L. cv Nipponbare) leaves; 10-d-old B. napus cv Quantum seedlings, which were air-dried to lose 10% to 15% of fresh weight and then placed in a sealed plastic bag for 5 h; 10-d-old barley (Hordeum vulgare cv Himalaya) seedlings, which were air-dried to lose 10% to 15% of fresh weight and then placed in a sealed plastic bag for 5 h.
Deuterium-labeled ABA S-(+)-[8'-2H3]ABA (1 and 10 µg silique-1), S-(+)-[5,8'-2H4]ABA (33 µg silique-1), and S-(+)-[8',9'-2H6]ABA (33 µg silique-1) were dissolved in methanol at 100 µg µL-1 and then diluted with 1% (v/v) detergent MERGE to the desired concentrations. The ABA solutions were applied to immature green siliques either through surface application or injection at 20 µL silique-1. Tissues were harvested after 48 h and immediately frozen in liquid nitrogen before lyophilization.
Freeze-dried tissues were extracted and prepared by the same procedures as described previously (Zhou et al., 2003
LC-ESI-MS-MS
Analyses for these experiments were performed on two LC-ESI-MS-MS systems. The HP1100 series binary HPLC system (Agilent, Palo Alto, CA) with a Quattro LC mass spectrometer (Micromass, Manchester, UK) has been described previously (Zhou et al., 2003
Electron Impact MS
Methyl neoPA (M
Methyl PA (M
For large-scale extraction, 158 g of lyophilized seeds was extracted once with acetone:water:acetic acid (80:19:1, v/v; 1.5 L). The filtrate volume was reduced to about 300 mL under reduced pressure using a rotary evaporator and was partitioned against hexane (3 x 300 mL). The aqueous phase was freeze-dried. Then the dry extract was dissolved in 5% (w/v) NaHCO3 (450 mL) and washed with ethyl acetate (3 x 450 mL). The aqueous phase was adjusted to pH 2.6 with 3 M HCl and extracted with ethyl acetate (5 x 450 mL). The combined organic fractions were washed with aqueous NaCl (180 g of NaCl, 1 L of water), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The filtrate (440 mg) was dissolved in methanol (5 mL) and diluted to 50 mL with 1% (v/v) acetic acid. The solution was passed through a 50-mL filter (0.22-µm cellulose acetate Tube Top Filter, Corning Incorporated, Corning, NY). A 50-µL aliquot was removed for LC-MS-MS. The remainder of the clarified extract was loaded on a 35 cc Oasis HLB cartridge (6 g of sorbent; Waters) preconditioned with methanol (50 mL) followed by 1% (v/v) acetic acid (100 mL). After loading the sample, the cartridge was washed with 1% (v/v) acetic acid (50 mL) and eluted with 50-mL portions of solvent with increasing proportions of methanol. A 50-µL aliquot of each fraction was removed for LC-MS-MS. NeoPA was found in the fraction of methanol:water:acetic acid (60:39:1, v/v). This fraction was dried under reduced pressure using a rotary evaporator and reconstituted in 1 mL of acetonitrile:water:acetic acid (15:84:1, v/v). Subsequently, this fraction was loaded on two normal-phase silica gel plates (20 x 20 cm, 250-µm layer thickness; Silica gel 60 F254, EM Science, Gibbstown, NJ) and developed with ethyl acetate:chloroform:acetic acid (100:50:7.5, v/v). The relative retention (RF value) for each compound was ABA = 0.68; neoPA = 0.60; PA = 0.52; 9'-OH-ABA = 0.43; 7'-OH-ABA = 0.36; DPA = 0.21; ABA-GE = 0.04. The band corresponding to neoPA, identified by LC-MS-MS, was excised, and the silica was extracted with ethyl acetate: methanol (100:5, v/v; 10 mL). The neoPA fraction was dried and then reconstituted in acetonitrile:water:acetic acid (20:79:1, v/v) and clarified with a 0.2-µm nylon filter (Costar Spin-X HPLC Micro Centrifuge filter, Corning). The sample was further purified by semipreparative HPLC (25 cmx10 mm, 5 µm; Supelcosil LC-18 HPLC column, Supelco, Belletone, PA). The mobile phase was acetonitrile:water:acetic acid (20:80:0.07, v/v) that ran at a flow rate of 5 mL min-1. The separation was carried out on a 1100 series HPLC (Hewlett-Packard, Palo Alto, CA) equipped with a guard column (5 cmx4.6 mm; Pelliguard LC-18, Supelco) and a HP 1040 photodiode array detector (Hewlett-Packard) at 262 nm. NeoPA had a retention time of 15 min with
For each replicate assay, 50 seeds of Arabidopsis (ecotype Columbia) were placed on two sheets of filter paper (70 mm; No. 1, Whatman, Clifton, NJ) in a petri plate (100 x 15 mm) wetted with 2 mL of sterile water containing the additives indicated. Plates were sealed with Parafilm and incubated at 24°C, with a 16-h/8-h light/dark cycle for the duration of the experiment (7 d). The number of seeds germinated in each plate was recorded twice each day. ABA and ABA analogs were added from ethanolic stock solutions, and control plates contained ethanol at the highest amount in the test plates. The results were quantified by a weighted germination index (Walker-Simmons, 1988
B. napus cv Hero plants were grown in controlled environment growth chambers and microspores were isolated and cultured according to the methods described previously for B. napus cv Reston (Zou et al., 1995
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permissions will be the responsibility of the requestor.
We thank Dr. G.F.W. Rakow and Mr. Don Rode for permission to collect B. napus siliques from Agiculture and Agri-Food Canada field plots, Jenny Yang for help with large-scale seed isolation, and Drs. Irina Zaharia and Garth Abrams for helpful discussions. Received July 25, 2003; returned for revision September 2, 2003; accepted October 3, 2003.
1 This work was supported by Dow AgroSciences and Natural Sciences and Engineering Research Council of Canada (strategic grant no. 234754-2000 to S.R.A.). This paper is National Research Council of Canada number 45,271.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030734. * Corresponding author; e-mail sue.abrams{at}nrc-cnrc.gc.ca; fax 306-975-4839.
Abrams SR, Nelson K, Ambrose SJ (2003) Deuterated abscisic acid analogs for mass spectrometry and metabolism studies. J Label Compd Radiopharm 46: 273-283[CrossRef] Balsevich JJ, Cutler AJ, Lamb N, Friesen LJ, Kurz EU, Perras MR, Abrams SR (1994) Response of cultured maize cells to (+)-abscisic acid, (-)abscisic acid, and their metabolites. Plant Physiol 106: 135-142[Abstract] Boyer GL, Zeevaart JAD (1986) 7'-Hydroxy (-)-R-abscisic acid: a metabolite of feeding (-)-R-abscisic acid to Xanthium strumarium. Phytochemistry 25: 1103-1105[CrossRef][Web of Science] Chiwocha SDS, Abrams SR, Ambrose SJ, Cutler AJ, Loewen M, Ross ARS, Kermode AR (2003) A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds. Plant J 35: 405-417[CrossRef][Web of Science][Medline] Cutler AJ, Krochko JE (1999) Formation and breakdown of ABA. Trends Plant Sci 4: 472-478[CrossRef][Web of Science][Medline] Cutler AJ, Rose PA, Squires TM, Loewen MK, Shaw AC, Quail JW, Krochko JE, Abrams SR (2000) Inhibitors of abscisic acid 8'-hydroxylase. Biochemistry 39: 13614-13624[Medline] Hampson CR, Reaney MJT, Abrams GD, Abrams SR, Gusta LV (1992) Metabolism of (+)-abscisic acid to (+)-7'-hydroxyabscisic acid by bromegrass cell cultures. Phytochemistry 31: 2645-2648[CrossRef] Hetherington AM (2001) Guard cell signaling. Cell 107: 711-714[CrossRef][Web of Science][Medline] Hill RD, Liu JH, Durnin D, Lamb N, Shaw A, Abrams SR (1995) Abscisic acid structure-activity relationships in barley aleurone layers and protoplasts. Plant Physiol 108: 573-579[Abstract]
Krochko JE, Abrams GD, Loewen MK, Abrams SR, Cutler AJ (1998) (+)-Abscisic acid 8'-hydroxylase is a cytochrome P450 monooxygenase. Plant Physiol 118: 849-860 Lamb N, Wahab N, Rose PA, Shaw AC, Abrams SR, Cutler AJ, Smith PJ, Gusta LV, Evan B (1996) Synthesis, metabolism and biological activity of a deuterated analogue of the plant hormone S-(+)-abscisic acid. Phytochemistry 41: 23-28 Lehmann H, Preiss A, Schmidt J (1983) A novel abscisic acid metabolite from cell suspension cultures of Nigellas damascena. Phytochemistry 22: 1277-1278[CrossRef] Lehmann H, Schwenen L (1988) Nigellic acid: an endogenous abscisic acid metabolite from Vicia faba leaves. Phytochemistry 27: 677-678[CrossRef] MacMillan J, Pryce RJ (1968) Phaseic acid, a putative relative of abscisic acid, from seed of Phaseolus multiflorus. Chem Commun 124-126 Milborrow BV (1968) Identification and measurement of (+)-abscisic acid in plants. In F Wightman, G Setterfield, eds, Biochemistry and Physiology of Plant Growth Substances. The Runge Press Ltd., Ottawa, pp 1531-1545 Milborrow BV (1969) Identification of "Metabolite C" from abscisic acid and a new structure for phaseic acid. Chem Commun 966-967
Milborrow BV (1970) The metabolism of abscisic acid. J Exp Bot 21: 17-29
Milborrow BV (2001) The pathway of biosynthesis of abscisic acid in vascular plants: a review of the present state of knowledge of ABA biosynthesis. J Exp Bot 52: 1145-1164 Nelson LAK, Shaw AC, Abrams SR (1991) Synthesis of (+)-, (-)-, and (+/-)-7'-hydroxy abscisic acid. Tetrahedron 47: 3259-3270[CrossRef]
Qi Q, Rose PA, Abrams GD, Taylor DC, Abrams SR, Cutler AJ (1998) (+)-Abscisic acid metabolism, 3-ketoacyl-coenzyme A synthase gene expression, and very long-chain monounstaurated fatty acid biosynthesis in Brassica napus embryos. Plant Physiol 117: 979-987 Ritchie SM, Swanson SJ, Gilroy S (2002) From common signaling components to cell specific responses: insights from the cereal aleurone. Physiol Plant 115: 342-351[CrossRef][Medline] Rose PA, Cutler AJ, Irvine NM, Shaw AC, Squires TM, Loewen MK, Abrams SR (1997) 8'-Acetylene ABA: an irreversible inhibitor of ABA 8'-hydroxylase. Bioorg Med Chem Lett 7: 2543-2546[CrossRef]
Schmitz N, Abrams SR, Kermode AR (2000) Changes in abscisic acid content and embryo sensitivity to (+)-abscisic acid during the termination of dormancy of yellow cedar seeds. J Exp Bot 51: 1159-1162
Schwartz SH, Qin X, Zeevaart JAD (2003) Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol 131: 1591-1601
Takahashi S, Oritani T, Yamashita K (1989) Synthesis of (+)-methyl phaseate and its isomer from (-)-
Taylor IB, Burbidge A, Thompson AJ (2000) Control of abscisic acid synthesis. J Exp Bot 51: 1563-1575 Tinelli ET, Sondheimer E, Walton DC (1973) Metabolites of 2-14C-abscisic acid. Tetrahedron Lett 2: 139-140 Todoroki Y, Hirai N, Koshimizu K (1994) 8'- and 9'-Methoxyabscisic acids as antimetabolic analogs of abscisic acid. Biosci Biotech Biochem 58: 707-715 Walker-Simmons M (1988) Enhancement of ABA responsiveness in wheat embryos by high temperature. Plant Cell Environ 11: 769-775[CrossRef]
Wang Z, Mambelli S, Setter TL (2002) Abscisic acid catabolism in maize kernels in response to water deficit at early endosperm development. Ann Bot 90: 623-630 Xiong L, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell Suppl 14: S165-S183 Zeevaart JAD (1999) Abscisic acid metabolism and its regulation. In PJJ Hooykaas, MAK Hall, R Libbenga, eds, Biochemistry and Molecular Biology of Plant Hormones. Elsevier Science. Amsterdam, pp 189-207 Zeevaart JAD, Creelman RA (1988) Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol Plant Mol Biol 39: 439-473[CrossRef][Web of Science] Zhou R, Squires TM, Ambrose SJ, Abrams SR, Ross ARS, Cutler AJ (2003) Rapid extraction of ABA and its metabolites for liquid chromatography-tandem mass spectrometry analysis. J Chromatogr A 1010: 75-85[Medline] Zou J, Abrams GD, Barton DL, Taylor DC, Pemeroy MK, Abrams SR (1995) Induction of lipid and oleosin biosynthesis by (+)-abscisic acid and its metabolites in microspore-derived embryos of Brassica napus L. cv Reston. Plant Physiol 108: 563-571[Abstract] This article has been cited by other articles:
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ABSTRACT
RESULTS
-amylase activity, although not as effectively as ABA (Hill et al., 1995
max = 265 nm). The 1H-NMR spectra of the natural and synthetic material were identical (
, (+)-ABA; 
, (+)-9'-OH ABA; 
, (-)-9'-OH ABA; 
, (+)-neoPA; 
, (-)-neoPA.
153; [8'-2H3]ABA, m/z 266 
7.67 (d, J = 16.0 Hz, 1H, C-4), 6.01 (d, J = 16.0 Hz, 1H, C-5), 5.84 (s, 1H, C-2), 3.80 (dd, J = 2.5, 9.0 Hz 1H, C9'), 3.76 (d, J = 9.0 Hz, 1H, C-9'), 2.83 (d, J = 17.5 Hz, 1H, C-3'), 2.81 (d, J = 2.5, 17.5 Hz, 1H, C-5'), 2.26 (dd, J = 1.5, 17.5 Hz, 1H, C-3'), 2.19 (dd, J = 1.5, 17.5 Hz, 1H, C-5'), 1.95 (s, 3H, C-6), 1.10 (s, 3H, C-7'), 0.91 (s, 3H, C-8'). The NMR spectrum was obtained on a BrukerAvance DRX 500 MHz NMR (Bruker Biospin, Rheinstetten, Germany) equipped with a CryoProbe accessory (Bruker Biospin AG, Fällanden, Switzerland). 
-pinene. Agric Biol Chem 53: 2711-2718
