Abscisic Acid Glucose Ester Research Articles

Abscisic acid metabolism and lipid accumulation of a cell suspension culture of Lesquerella fendleri

Lesquerella fendleri (commonly known as “Fendler’s bladderpod” or “yellowtop”) is a member of the Brassicaceae and is an important seed oil-producing plant. The lipid profile of L. fendleri seed indicates potential for producing a high quality replacement for castor oil. In this work, characterization of the lipid content of a suspension cell culture, derived from seedlings of L.fendleri, is provided. Under the described suspension cell culture conditions, 16:0, 18:1Δ9, 18:2 Δ9, Δ12 and 18:3 Δ9, Δ12, Δ15 fatty acids were found to accumulate in the cells, while 16:0, 26:0 and 28:0 fatty acids were predominant in the culture medium. Subsequently, the effect of application of abscisic acid (ABA), which modulates lipid accumulation, was assessed. Exogenously applied ABA was taken up by the cells and metabolized via the conjugation pathway, resulting in the accumulation of ABA-glucose ester. Preliminary tests demonstrate the cell line is responsive to exogenous ABA, resulting in increased cellular lipid content and increased accumulation of lipids in the culture medium. This novel L. fendleri suspension culture presents a valuable model system for efficient characterization of mechanisms associated with ABA-induced accumulation of lipids.

Genetic association mapping identifies single nucleotide polymorphisms in genes that affect abscisic acid levels in maize floral tissues during drought

In maize, water stress at flowering causes loss of kernel set and productivity. While changes in the levels of sugars and abscisic acid (ABA) are thought to play a role in this stress response, the mechanistic basis and genes involved are not known. A candidate gene approach was used with association mapping to identify loci involved in accumulation of carbohydrates and ABA metabolites during stress. A panel of single nucleotide polymorphisms (SNPs) in genes from these metabolic pathways and in genes for reproductive development and stress response was used to genotype 350 tropical and subtropical maize inbred lines that were well watered or water stressed at flowering. Pre-pollination ears, silks, and leaves were analysed for sugars, starch, proline, ABA, ABA-glucose ester, and phaseic acid. ABA and sugar levels in silks and ears were negatively correlated with their growth. Association mapping with 1229 SNPs in 540 candidate genes identified an SNP in the maize homologue of the Arabidopsis MADS-box gene, PISTILLATA, which was significantly associated with phaseic acid in ears of well-watered plants, and an SNP in pyruvate dehydrogenase kinase, a key regulator of carbon flux into respiration, that was associated with silk sugar concentration. An SNP in an aldehyde oxidase gene was significantly associated with ABA levels in silks of water-stressed plants. Given the short range over which decay of linkage disequilibrium occurs in maize, the results indicate that allelic variation in these genes affects ABA and carbohydrate metabolism in floral tissues during drought.

Short-term cold stress in two cultivars of Digitaria eriantha: effects on stress-related hormones and antioxidant defense system

The two cultivars of Digitaria eriantha: cv. Sudafricana (a cold-sensitive cultivar) and cv. Mejorada INTA (a cold-resistant cultivar) were exposed to low temperature and compared in terms of the involvement of abscisic acid (ABA) and catabolites, jasmonates, and antioxidant defense in cold tolerance. Cold stress caused a greater ABA increase in cv. Mejorada INTA than in cv. Sudafricana. In both cultivars abscisic acid glucose ester and dihydrophaseic acid were the most abundant catabolites. Cold treatment decreased JA in leaves of both cultivars. In cv. Sudafricana, 12-hydroxyjasmonate (12-OH-JA) decreased and 12-oxo-phytodienoic acid increased. In regard to antioxidant defense, both cultivars increased the non-protein thiols in response to cold stress. However, reduced glutathione (GSH) level was higher in leaves of cv. Mejorada INTA than cv. Sudafricana. Cold-treated leaves of cv. Sudafricana increased thiobarbituric acid-reactive substances (TBARS), but cv. Mejorada INTA leaves showed no such increase. Superoxide dismutase activity decreased and ascorbate peroxidase activity increased in cold-treated leaves of cv. Sudafricana. No significant change of these enzymes was observed for cv. Mejorada INTA. The cold tolerance of cv. Mejorada INTA could be related to JA, 12-OH-JA and GSH high basal contents, ABA increase, and TBARS stability after cold treatment.

Sulphate as a xylem-borne chemical signal precedes the expression of ABA biosynthetic genes in maize roots

Recent reports suggest that early sensing of soil water stress by plant roots and the concomitant reduction in stomatal conductance may not be mediated by root-sourced abscisic acid (ABA), but that other xylem-borne chemicals may be the primary stress signal(s). To gain more insight into the role of root-sourced ABA, the timing and location of the expression of genes for key enzymes involved in ABA biosynthesis in Zea mays roots was measured and a comprehensive analysis of root xylem sap constituents from the early to the later stages of water stress was conducted. Xylem sap and roots were sampled from plants at an early stage of water stress when only a reduction in leaf conductance was measured, as well as at later stages when leaf xylem pressure potential decreased. It was found that the majority of ABA biosynthetic genes examined were only significantly expressed in the elongation region of roots at a later stage of water stress. Apart from ABA, sulphate was the only xylem-borne chemical that consistently showed significantly higher concentrations from the early to the later stages of stress. Moreover, there was an interactive effect of ABA and sulphate in decreasing maize transpiration rate and Vicia faba stomatal aperture, as compared to ABA alone. The expression of a sulphate transporter gene was also analysed and it was found that it had increased in the elongation region of roots from the early to the later stages of water stress. Our results support the suggestion that in the early stage of water stress, increased levels of ABA in xylem sap may not be due to root biosynthesis, ABA glucose ester catabolism or pH-mediated redistribution, but may be due to shoot biosynthesis and translocation to the roots. The analysis of xylem sap mineral content and bioassays indicate that the anti-transpirant effect of the ABA reaching the stomata at the early stages of water stress may be enhanced by the increased concentrations of sulphate in the xylem which is also transported from the roots to the leaves.

Profiles of Abscisic Acid and Its Catabolites in Developing Merlot Grape (Vitis vinifera) Berries

The plant hormone abscisic acid (ABA) is a signaling molecule involved in grape berry development and ripening. To obtain a more comprehensive understanding of ABA synthesis and catabolism in developing grape berries, ABA and catabolite profiles of Merlot grapes grown in a commercial vineyard in the southern Okanagan Valley in British Columbia, Canada, were measured using HPLC–MS/MS in multiple-reaction monitoring mode. While pulp and skin of preveraison berries contained low to moderate concentrations of ABA, these tissues had high concentrations of the ABA catabolite dihydrophaseic acid (DPA), indicating that large quantities of ABA had been produced and catabolized early in berry development. In the pulp and skin, two peaks of ABA were found, the first at two weeks preveraison and the second at late veraison, which was followed by a decline in ABA as the berries ripened. Abscisic acid glucose ester (ABA-GE) in the pulp and skin rose just before veraison, and then declined after veraison. In developing seeds similar trends were observed, but with initial high levels of both ABA and DPA and with ABA-GE remaining high into ripeness. ABA applied to berries at veraison had a half life of one week and resulted in a 69% increase in DPA concentration. However, this constituted only a small increase in the total ABA and catabolite pool. The treatment had no effect on berry maturation rate, basic juice composition, or skin and seed tannins, but did increase the anthocyanin content of skins by 7% at berry maturity. These observations indicate that little ABA was taken into the berry and metabolized, while the majority of it remained on the surface.

Profiling ABA metabolites in Nicotiana tabacum L. leaves by ultra-performance liquid chromatography–electrospray tandem mass spectrometry

We have developed a simple method for extracting and purifying (+)-abscisic acid (ABA) and eight ABA metabolites – phaseic acid (PA), dihydrophaseic acid (DPA), neophaseic acid (neoPA), ABA-glucose ester (ABAGE), 7′-hydroxy-ABA (7′-OH-ABA), 9′-hydroxy-ABA (9′-OH-ABA), ABAaldehyde, and ABAalcohol – before analysis by a novel technique for these substances, ultra-performance liquid chromatography–electrospray ionisation tandem mass spectrometry (UPLC–ESI-MS/MS). The procedure includes addition of deuterium-labelled standards, extraction with methanol–water–acetic acid (10:89:1, v/v), simple purification by Oasis ® HLB cartridges, rapid chromatographic separation by UPLC, and sensitive, accurate quantification by MS/MS in multiple reaction monitoring modes. The detection limits of the technique ranged between 0.1 and 1 pmol for ABAGE and ABA acids in negative ion mode, and 0.01–0.50 pmol for ABAGE, ABAaldehyde, ABAalcohol and the methylated acids in positive ion mode. The fast liquid chromatographic separation and analysis of ABA and its eight measured derivatives by UPLC–ESI-MS/MS provide rapid, accurate and robust quantification of most of the substances, and the low detection limits allow small amounts of tissue (1–5 mg) to be used in quantitative analysis. To demonstrate the potential of the technique, we isolated ABA and its metabolites from control and water-stressed tobacco leaf tissues then analysed them by UPLC–ESI-MS/MS. Only ABA, PA, DPA, neoPA, and ABAGE were detected in the samples. PA was the most abundant analyte (ca. 1000 pmol/g f.w.) in both the control and water-stressed tissues, followed by ABAGE and DPA, which were both present at levels ca. 5-fold lower. ABA levels were at least 100-fold lower than PA concentrations, but they increased following the water stress treatment, while ABAGE, PA, and DPA levels decreased. Overall, the technique offers substantial improvements over previously described methods, enabling the detailed, direct study of diverse ABA metabolites in small amounts of plant tissue.

Enhanced determination of abscisic acid (ABA) and abscisic acid glucose ester (ABA-GE) in Cistus albidus plants by liquid chromatography–mass spectrometry in tandem mode

An improved, quick and simple method for the extraction and quantification of the phytohormones (+)-abscisic acid (ABA) and its major glucose conjugate, abscisic acid glucose ester (ABA-GE) in plant samples is described. The method includes the addition of deuterium-labeled internal standards to the leaves at the beginning of the extraction for quantification, a simple extraction/centrifugation process and the injection into the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS-MS) system in multiple reaction monitoring mode (MRM). Quality parameters of the method (detection limits, repeatability, reproducibility and linearity) have been studied. The objective of this work is to show the applicability of this method for quantifying the endogenous content of both ABA and ABA-GE in Cistus albidus plants that have been grown during an annual cycle under Mediterranean field conditions. Leaf samples from winter plants have low levels of ABA which increase in spring and summer showing two peaks that corresponded to April and August. These increases are coincident with the high temperature and solar radiation and the low RWC and RH registered along the year. On the other hand, the endogenous levels of ABA-GE increase until maximum values in July just before the ABA content reaches its highest concentration, decreasing in August and during autumn and winter. Our results suggest that the method is useful for quantifying both compounds in this plant material and represents the advantage of a short-time sample preparation with a high accuracy and viability.

Tomato mutants sensitive to abiotic stress display different abscisic acid content and metabolism during germination

AbstractWe report the determination of abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA) and ABA glucose ester (ABA-GE), in non-dormant dry and imbibed seeds of tomato (Solanum lycopersicumMill.) cv. Moneymaker (wild type), and itstss1,tss2andtos1mutants. High ABA in dry seeds may originate from ABA accumulation in the sheath tissue, which was in contact with an ABA-containing medium, the endocarpus. The highest germination percentages at 72 h, observed intss1andtss2, coincided with minimal ABA content. Wild-type and mutant seeds showed different ABA and catabolic patterns, and these were correlated with their sensitivity to abiotic stress. Whereas dry seeds showed a high basal ABA, imbibed seeds showed higher ABA metabolite content, particularly DPA. The dramatic decrease of ABA following seed imbibition suggests an activation of ABA catabolism during the early stages of the germination process. The observed variation of ABA metabolites among dry and imbibed seeds ofSolanum lycopersicumcv. Moneymaker and itstss1,tss2andtos1mutants shows that ABA metabolism is differentially regulated in these genotypes.

Phytohormones and their metabolites during long shoot development in Douglas-fir following cone induction by gibberellin injection

Changes in plant hormones and metabolites in long-shoot stems of interior Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco) during cone induction by gibberellic acid (GA) treatment were analyzed by high performance liquid chromatography-electrospray ionization tandem mass spectrometry in multiple reaction monitoring mode. A mixture of GA(4) and GA(7), including small amounts of GA(3) and GA(1), was stem-injected into each tree in amounts of 0, 4, 40 or 400 mg. One week after injection, concentrations of GA(4), GA(7) and GA(3) were elevated in all GA-treated samples. The ratio of GA(4) to GA(7) decreased significantly at Week 3. Absolute concentrations of all gibberellins declined sharply at Week 3 after GA application. After 5 weeks, GA(1) and GA(4) were below detection limits in all samples, and GA(7) and GA(3) were found only in the samples from trees treated with 40 or 400 mg of GA. Endogenous indole-3-acetic acid (IAA) concentrations increased following GA injection, and peaked at Week 2 or Week 3 in the trees treated with 40 or 400 mg GA, respectively. Injection of 400 mg of GA brought about a twofold increase in IAA concentration compared with control values. Injection of 40 and 400 mg of GA caused significant increases in stem dry mass in Week 5. Seed orchard data revealed that injection of either 40 or 400 mg GA enhanced female cone formation, whereas male cone formation was enhanced only by 400 mg GA. Slight decreases in concentrations of abscisic acid (ABA) and isopentenyl adenosine were observed after GA application. No significant changes were detected in the concentrations of ABA metabolites except for a slight decrease in the concentration of 7'-hydroxy ABA. The concentration of ABA declined during the growing season and the concentration of ABA glucose ester increased correspondingly.

Long-distance signalling of abscisic acid (ABA): the factors regulating the intensity of the ABA signal

Abscisic acid (ABA) is a stress signal, which moves in the xylem from the roots to the aerial parts of the plant, where it regulates stomatal movement and the activity of shoot meristems. Root growth-promoting microorganisms in the rhizosphere, lateral ABA flows in the root cortex across apoplastic barriers, ABA redistribution in the stem, leaf apoplastic pH values, and the action of beta-glucosidases, both in the apoplast and the cytosol of the mesophyll, play an important role in the regulation of signal intensity. The significance of abscisic acid glucose ester as a long-distance stress signal is discussed.

New developments in abscisic acid perception and metabolism

Abscisic acid is a powerful signaling molecule that accumulates in response to abiotic stress. However, no potential receptors that could perceive this increase in abscisic acid had been identified until recent reports of three abscisic acid binding proteins: the nuclear protein Flowering Time Control Locus A, the chloroplast protein Magnesium Protoporphyrin-IX Chelatase H subunit, and the membrane-associated protein G Protein Coupled Receptor 2. Abscisic acid metabolism also has a new and prominent component with the identification of a beta-glucosidase capable of releasing biologically active abscisic acid from inactive abscisic acid-glucose ester in a stress-inducible manner. These observations refocus our attention on the metabolism underlying abscisic acid accumulation, sites of abscisic acid perception, and delivery of abscisic acid to those sites.

Parasitism of seed of Douglas fir (Pseudotsuga menziesii) by the seed chalcid, Megastigmus spermotrophus, and its influence on seed hormone physiology

Parasitism of Douglas fir (Pseudotsuga menziesii) megagametophytes by the seed chalcid, Megastigmus spermotrophus Wachtl, occurs naturally after pollination but before fertilization. In the absence of fertilization, the presence of insect larvae within the megagametophyte prevents abortion and the storage tissue continues to develop as if the seed had been fertilized. We investigated the effect of parasitism on the metabolism of abscisic acid (ABA), auxins, cytokinins, and gibberellins during early development of Douglas fir seeds. Hormones and hormone metabolites of infested and uninfested megagametophytes with or without pollination were analyzed by HPLC–ESI/MS/MS. At 1 week after Megastigmus introduction, the insect’s presence stimulated ABA accumulation in unpollinated megagametophytes compared to unpollinated, unparasitized megagametophytes. In pollinated material, parasitism did not stimulate ABA accumulation compared to levels present in unparasitized megagametophytes. In all four treatments, the metabolism of ABA occurred primarily through conjugation to the ABA glucose ester (ABAGE), while the 7′-, 8′- and, 9′-hydroxylation pathways were only minor. ABAGE levels declined with time in all treatments and this occurred to a greater extent in pollinated, parasitized megagametophytes, suggesting that the insect’s presence induced the dramatic decrease in ABAGE. Although there were temporal variations in the auxin, cytokinin, and gibberellin profiles of parasitized megagametophytes, the profiles were generally similar to those of unparasitized megagametophytes. Our results suggest that failure of parasitized megagametophytes to abort may be due to the insect inducing similar hormone profiles to those present during normal development of Douglas fir seed.

A Quick Release Mechanism for Abscisic Acid

The hormone abscisic acid regulates development and survival of plants in response to environmental stresses. now demonstrate that in response to stress, abscisic acid can be made available via polymerization of a beta-glucosidase enzyme. This enzyme is reported to hydrolyze glucose-conjugated abscisic acid, thus increasing active abscisic acid concentrations.

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.

The etr1‐2 mutation in Arabidopsis thaliana affects the abscisic acid, auxin, cytokinin and gibberellin metabolic pathways during maintenance of seed dormancy, moist‐chilling and germination

In Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moist-chilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moist-chilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.

Preparation of deuterated abscisic acid metabolites for use in mass spectrometry and feeding studies

Four deuterium-labelled ABA catabolites of high isotopic purity were prepared for hormone profiling studies using HPLC-ESI-MS-MS. The catabolites, namely [7′,7′,7′-d3]-PA, [7′,7′,7′-d3]-DPA, [5,8′,8′,8′-d4]-7′-hydroxyABA and [4,5,8′,8′,8′-d5]-ABA glucose ester were prepared either by biotransformation or chemical synthesis starting with labelled ABA. The labelled compounds are employed as internal standards for plant hormone profiling by liquid chromatography–electrospray–tandem mass spectrometry. Copyright © 2005 John Wiley & Sons, Ltd.

Response to abscisic acid application and hormone profiles in spring Brassica napus seed in relation to secondary dormancy

The plant hormone abscisic acid (ABA) has been implicated in the inception and maintenance of seed dormancy, while gibberellins promote dormancy breakage and germination in some species. We investigated whether osmotic stress induced secondary dormancy in Brassica napus L. is associated with changes in ABA sensitivity and metabolism, as well as changes in gibberellin levels. Seeds of two genotypes, one with low dormancy potential (LDP) and one with high dormancy potential (HDP) for secondary dormancy, were exposed to a dormancy-inducing osmotic treatment for up to 4 weeks and then germinated in the presence of increasing ABA concentrations. Even at relatively high concentrations of supplied ABA, germination of LDP seed was not inhibited, while relatively low ABA concentrations inhibited the germination of HDP seed after osmotic treatment. Fluridone was highly effective in suppressing secondary dormancy development in HDP seed, but had no effect on germinability in LDP seed. Despite the lack of differences in nonosmotically treated seed, ABA and ABA-glucose ester accumulated to higher levels, and gibberellin A1 accumulated to lower levels, in HDP relative to LDP seed by the end of the osmotic treatment. Our findings indicate an association among ABA sensitivity, biosynthesis and accumulation, and secondary dormancy potential in B. napus seed.Key words: abscisic acid (ABA), Brassica napus, fluridone, induced dormancy, osmotic stress, sensitivity.

Endogenous levels of abscisic acid in various organs of <em>Vitis vinifera</em> L. (cv. Cabernet Sauvignon) between flower buds separated and grape closed stages

<p>Free abscisic acid (ABA) and bound abscisic acid (ABA-GE: ABA glucose ester) contents and growth were investigated in leaves, internodes, flowers buds, flowers and berries of <em>Vitis vinifera</em> L. cv. Cabernet Sauvignon between flower buds separated and grape closed stages. The ABA levels in leaves and in internodes decrease when the growth was hight and increases when the growth become smaller or stopped. What is more, ABA was accumulated hightly in the flowers (at 50% of flowering) and then appears to be a good marker of flowering. ABA contents decreased at berry setting and till grape closed stage. In leaves, internodes, flowers buds and flowers, ABAGE and ABA show an inverse development and then suggests an interconversion phenomenon between theses two forms. Two most importants points were discuted in this paper: 1) The role of ABA in the controle of growth and development of the organs studied and (2) The translocation of abscisic acid between the organs studied and the role of ABA-GE in this transport.</p>

Purification and Characterization of a Barley Aleurone Abscisic Acid-binding Protein

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.

Dormancy termination of western white pine ( Pinus monticola Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7'-hydroxy ABA (7'OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC-tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7'OH ABA and ABA-GE also accumulated during moist chilling; however, 7'OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7'OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.