Deuterium-labeled phaseic acid and dihydrophaseic acids for internal standards

The concentration of abscisic acid in plants is regulated not only by biosynthesis, but also by metabolism. Abscisic acid is metabolized to phaseic acid via 8'-hydroxyabscisic acid, and phaseic acid is then converted to dihydrophaseic acid and its epimer. A quantitative analysis of these metabolites is important as well as that of abscisic acid to understand changes in the concentration of abscisic acid in plants. However, no internal standards of the metabolites suitable for quantitative analysis have been reported. We prepared 7'-deuterium-labeled phaseic acid with a deuterium content of 86%, using the equilibrium reaction between phaseic acid and 8'-hydroxyabscisic acid. 7'-Deuterium-labeled dihydrophaseic acids were obtained by reducing 7'-deuterium-labeled phaseic acid. The levels of the metabolites in plant organs were determined by using the deuterated metabolites as internal standards.     

Sites of Abscisic Acid Synthesis and Metabolism in Ricinus communis L

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

Transport and metabolism of [214-C]abscisic acid in maize root

The tips of intact maize (cv. LG 11) roots, maintained vertically, were pretreated with a droplet of buffer solution or a bead of anion exchange resin, both containing [2¹⁴-C]abscisic acid (ABA). A significant basipetal ABA movement was observed and two metabolites of ABA (possibly phaseic acid and dihydrophaseic acid) were found. ABA pretreatment enhanced the gravireaction of 10 mm apical root segments kept both in the dark and in the light. The possibility that ABA could be one of the endogenous growth inhibitors produced or released by the cap cells is discussed.Abbreviations ABA abscisic acid - 3,3-DGA 3,3-dimethyl-glutaric acid - DPA dihydrophaseic acid - PA phaseic acid - GCMS gas chromatography-mass spectrometry     

The Determination of Phaseic Acid by Monoclonal Antibody-Based Enzyme immunoassay

A monoclonal antibody PA3-2-B3, IgG₁ (Λ) is described which specifically recognizes phaseic acid and shows very little cross-reactivity (0.14%) with abscisic acid or dihydrophaseic acid (0.88%). Based on this antibody, an enzyme immunoassay was developed which displays a linearity range from 15 pg to 3 ng of phaseic acid. Results obtained with this assay agree with those obtained by gas chromatography-electron capture detection. Using the novel enzyme immunoassay, as well as an established immunoassay for abscisic acid, levels of these two compounds in leaves of Phaseolus vulgaris were determined as a function of plant age, water stress, recovery from stress, and feeding of abscisic acid through the transpiration stream. The production of phaseic acid in a microsomal system from bean leaves was demonstrated. The results show a regulation of the plant's capacity to metabolize abscisic acid to phaseic acid as a function of water stress.     

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     

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.     

Abscisic acid in lichens: variation, water relations and metabolism

For the first time the well known drought stress hormone abscisic acid, which is involved in regulating processes increasing desiccation tolerance in many plant systems, was analysed in lichens. ABA was detected in all 26 species investigated. In contrast to higher plants and liverworts, the ABA content increased after hydration of air dry lichen thalli and decreased in desiccating lichen material. Experiments with Baeomyces rufus (Huds.) Rebent indicated that the mycobiont might be the major site of ABA biosynthesis. After incubation of hydrated lichen thalli with radioactive ABA for up to 72 h no metabolism to phaseic acid and dihydrophaseic acid could be detected. Fluctuations of internal ABA might be a result of ABA release to the external medium.     

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)     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> 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 (7OH 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, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH 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, 7OH 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.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [¹⁴C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).Abbreviations ABA abscisic acid - RIA radioimmunoassay - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography     

Water stress and the catabolism of (±)-abscisic acid by excised leaves of Hordeum vulgare L. cv. Dyan

When excised, light-grown leaves of Hordeum vulgare were fed with (±)-[2-¹⁴C]-abscisic acid and stressed until they had lost 12% of their original fresh weight, marked changes in the distribution of radioactivity between abscisic acid and its catabolites were observed. Wilted leaves were less able than their turgid counterparts to transform (±)-[2-¹⁴C]-abscisic acid into 2-hydroxymethyl abscisic acid, dihydrophaseic acid and water-soluble conjugates of abscisic acid. Water stress had little effect on the production of phaseic acid although refeeding studies with [¹⁴C]-phaseic acid showed that the step from phaseic acid to dihydrophaseic acid was inhibited in wilted leaves. Evidence was obtained which suggested that these changes did not result from dilution of applied, radiolabelled substrate by endogenous abscisic acid. The catabolites of (±)-abscisic acid were identified by capillary gas chromatography-mass spectrometry.     

Separation and Quantification of Abscisic Acid and its Metabolites by High-performance Liquid Chromatography

A technique for the separation and quantitative estimation ofabscisic acid and its metabolic products, phaseic acid and dihydrophaseicacid by high performance liquid chromatography (HPLC) is described.The efficiency of two HPLC columns, LiChrosorb RP-18 and LiChrosorbNH₂ is compared. The method has been tested using extracts fromtomato plants and some of the results are presented. Abscisic acid, dihydrophaseic acid, phaseic acid, chromatography, tomato, Lycopersicon esculentum, Mill     

A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid     

Metabolism of abscisic acid and the occurrence of epi-dihydrophaseic acid in Phaseolus vulgaris

When (±)-abscisic acid-[2-¹⁴C] or (±)-abscisic acid-[4′-¹⁸O] was fed to bean (Phaseolus vulgaris) shoots, phaseic acid (PA) and dihydrophaseic acid (DPA) were the major metabolites, while epi-dihydrophaseic acid (epi-DPA) appeared as a minor metabolite. In the acidic fraction the amount of epi-DPA ranged from 18 to 42% of the DPA content, in the conjugated form from 50 to 200%. The content of endogenous epi-DPA amounted to only 1–2% of that of the DPA. These data indicate that the applied abscisic acid is not metabolised in a manner identical with that of the endogenous material. DPA and epi-DPA were shown to be formed separately from PA and could not be inter-converted either by the extraction conditions employed or when fed to bean shoots during short term experiments.     

Identification of gibberellins A17, A25, A45, abscisic acid,phaseic acid,and dihydrophaseic acid in seeds of Pyrus communis

Gibberellin A₁₇, abscisic acid, and 4′-dihydrophaseic acid were identified by GC-MS of derivatized extracts from both immature and mature seeds of pear. Immature seeds also contained phaseic acid, gibberellins A₂₅ and A₄₅, and two presumed mono-hydroxylated derivatives of GA₄₅, one of which was tentatively identified as 3β-hydroxy-GA₄₅. Several presumed metabolites of abscisic acid were detected in both mature and immature seeds.     

The Metabolism of Hormones during Seed Germination and Dormancy: IV. The Metabolism of (S)-2-C-Abscisic Acid in Ash Seed

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-¹⁴C-abscisic acid (ABA) under a variety of conditions. Both dormant and stratified embryos rapidly metabolize abscisic acid to phaseic acid, dihydrophaseic acid, and an unidentified polar metabolite apparently derived from dihydrophaseic acid. Although the stratified embryos may have an increased capacity to metabolize abscisic acid, our calculations suggest that such an increased capacity would probably not be physiologically significant.     

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

A highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quantification by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7'-hydroxy-abscisic acid (7'-OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA)1, GA3, GA4, and GA7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 33 degrees C instead of 23 degrees C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 33 degrees C. GA1 and GA3 were detected during germination, and levels of GA1 increased during early post-germinative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 23 degrees C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is significantly different from that of germinating seeds.     

A comparison of some methods for the purification of abscisic acid and its metabolites from plants extracts

The use of small columns containing charcoal and cellulose for the purification of abscisic acid and its metabolites from plant extracts is described. These columns have high capacity and the sample processing time is short. The efficiencies of recovery of abscisic acid, abscisic acid glucose ester, dihydrophaseic acid and an unidentified highly polar metabolite of abscisic acid are about 90%. The method is shown to be superior to other published methods using commercial pre-packed cartridges.     

Hormonal regulation of oil accumulation in Brassica seeds: metabolism and biological activity of ABA, 7'-, 8'- and 9'-hydroxy ABA in microspore derived embryos of B. napus

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.     

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

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