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     

The Metabolism of Hormones during Seed Germination and Release from Dormancy: III. The Effects and Metabolism of Zeatin in Dormant and Nondormant Ash Embryos

Zeatin and zeatin-9, β-ribonucleoside enhance the germination of dormant ash embryos. While the first macroscopic signs of germination appear only after about 72 hours, 12 hours of exposure to 50 μm zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8-¹⁴C-zeatin and its metabolites, zeatin-9, β-ribonucleoside, the 5′-mono and the suspected di- and triphosphates, accumulate against a concentration gradient. Zeatin ribonucleoside is about as effective as zeatin in enhancing embryo germination, yet the internal 8-¹⁴C-zeatin level is lower by a factor of about 50 when the ribonucleoside is fed. The physiological effects of zeatin and abscisic acid on the germination of ash embryos are antagonistic. There is, however, no evidence that abscisic acid has a significant effect on 8-¹⁴C-zeatin uptake or conversions.     

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.     

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.     

Metabolism of 2-C-(+/-)-abscisic Acid in excised bean axes

Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-¹⁴C-(±)-abscisic acid to two compounds, M-1 and M-2, which have very low growth-inhibitory activity. Chemical tests indicate the M-1 and M-2 are not previously described abscisic acid metabolites. M-2 accumulates in the axes and evidence is presented for the hypothesis that abscisic acid → M-1 → M-2. Zeatin, which partially reverses the abscisic acid-mediated growth inhibition of axes, neither decreases abscisic acid uptake nor causes any major changes in its metabolism. It was observed that axes transferred from abscisic acid-containing solutions to buffer resume control rates of fresh weight increase while still containing considerable quantities of abscisic 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.     

Membrene turnover in imbibed and dormant embryos of the wild oat (Avena fatua L.)

A comparative study of protein synthesis has been carried out with embryos excised from dormant (D) and non-dormant (ND) caryopses of the wild oat. Although D embryos imbibed in water or ND embryos imbibed in abscisic acid do not germinate, they incorporate [¹⁴C]leucine into TCA-insoluble material for the first 48 h as readily as embryos that do germinate (ND embryos imbibed in water, or D embryos imbibed in gibberellic acid). Pulsechase experiments with [¹⁴]leucine show that in both D and ND embryos the proteins associated with the membranes undergo turnover. The rates of decay of incorporated radioactivity are similar in both dormant and germinating embryos up to 98 h following embryo excision. Fractionation of the membrane proteins in SDS-polyacrylamide gels indicates that the different polypeptides have different rates of turnover. It is concluded that membrane proteins in imbibed D embryos are in a state of constant turnover, and that this is a part of the replacement processes necessary to maintain the integrity of hydrated cells. The continuation of such synthetic events could account for long term survival of dormant Avena fatua in the imbibed state.Abbreviations CCRSE cytochrome relative stain equivalents - D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid GA₃     

An autoradiographic study of the effect of the plant hormone abscisic acid on nucleic acid and protein metabolism

Summary Abscisic acid maintains embryos in a state of dormancy and inhibits the incorporation of H³uridine and H³thymidine but not the incorporation of H³leucine. Ribosomes present in imbibed but dormant embryos do not become associated into polysomes until actual germination of the embryos. Protein synthesis still occurs in embryos when RNA synthesis is inhibited and therefore stable m-RNA must be present in dormant embryos. It is concluded that abscisic acid maintains dormancy by inhibiting the production of specific types of m-RNA, and therefore the formation of specific proteins. The activity of abscisic acid is antagonistic to the effect of gibberellic acid in dormancy.     

Abscisic Acid Metabolism by a Cell-free Preparation from Echinocystis lobata Liquid Endoserum

A cell-free enzyme system capable of metabolizing abscisic acid has been obtained from Eastern Wild Cucumber (Echinocystis lobata Michx.) liquid endosperm. The reaction products were determined to be phaseic acid (PA) and dihydrophaseic acid (DPA) by co-chromatography on thin layer chromatograms as the free acids, methyl esters, and their respective oxidation or reduction products. The crude enzyme preparation was separated by centrifugation into a particulate abscisic acid (ABA)-hydroxylating activity and a soluble PA-reducing activity. The particulate ABA-hydroxylating enzyme showed a requirement for O₂ and NADPH, inhibition by CO, and high substrate specificity for (+)-ABA. Acetylation of short term incubation mixtures gave evidence for the presence of 6′-hydroxymethyl-ABA as an intermediate in PA formation. Determinations of endogenous ABA and DPA concentrations suggest that the ABA-hydroxylating and PA-reducing enzymes are extensively metabolizing ABA in the intact E. lobata seed.     

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.     

Abscisic Acid Translocation and Metabolism in Soybeans following Depodding and Petiole Girdling Treatments

It was found earlier that depodding and girdling treatments which obstruct translocation, result in increased leaf AbA levels and partial stomatal closure. In the present work (±) [2-¹⁴C]abscisic acid (AbA) was introduced into leaves and the mass, and radioactivity of AbA and AbA-metabolites were analyzed following translocation obstruction to determine whether the increased AbA was due to higher rates of synthesis, or lower rates of catabolism or export. The (±) [2-¹⁴C]AbA was introduced into soybean (Merr.) leaves by injection into the petiole region. AbA and AbA-metabolites (phaseic acid [PA], dihydrophaseic acid [DPA], AbA-conjugate, and an unknown metabolite) were separated with preparative high performance liquid chromatography. Methyl esters of AbA (free and that released after hydrolysis of AbA-conjugate), PA and DPA were determined with gas chromatography using electron capture detection.     

Metabolism of C-Maltose in Avena fatua Seeds During Germination

Non-dormant and dormant seeds of Avena fatua metabolize ¹⁴C-maltose in different ways: in non-dormant seeds, ¹⁴C-maltose administered to the endosperm is readily converted to sucrose in the scutellum and translocated to the embryo; in dormant seeds, little sucrose is synthesized from ¹⁴C-maltose, and maltose and glucose tend to accumulate in the endosperm. It is suggested that biosynthesis of sucrose is essential for effective transport of the endosperm reserve to the embryonic axis in germinating seeds.     

Promoting Effect of Fusicoccin on Seed Germination

The effects of fusicoccin on the germination of dormant, light-requiring or abscisic acid-inhibited seeds has been investigated. (1) Fusicoccin (10^?6M) induces germination in dormant wheat seeds (Triticum durum cv. Cappelli; 1972 crop) and stimulates it in seeds already relieved from dormancy (1971 crop), with an effect similar to that of gibberellic acid. (2) Fusicoccin (1.5 × 10^?6M) is more active than the two phytohormones gibberellic acid and benzyladenine and than white light in stimulating light-requiring lettuce seeds (Lactuca sativa cv. Grand Rapids) to germinate. Germination of radish seeds (Raphanus sativus) is also accelerated by fusicoccin, while benzyladenine and gibberellic acid are less active in this material. (3) Fusicoccin (1.5 × 10^?5M) removes almost completely the inhibitory effect of abscisic acid on germination of radish and lettuce seeds, whereas benzyladenine (10^?4M) and gibberellic acid (3 × 10^?4M) remove the inhibition only partially. The possible relationship between these results and previous information on growth by cell enlargement is discussed in terms of the mechanism of action of fusicoccin as compared with natural hormones.     

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.     

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

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

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     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Membrane turnover in imbibed dormant embryos of the wild oat (Avena fatua L.)

Germinating non-dormant (ND) embryos of wild oat incorporate [³H]glycerol into phospholipid, and a 250% increase in total extractable phospholipid occurs within 72 h. During germination, leveles of phosphatidyl inositol showed the greatest change, increasing approximately 5-fold.Imbibed dormant (D) embryos of the wild oat also incorporate [³H]gycerol into phospholipids, but there is no net synthesis. A continuous turnover of membrane phospholipids could be demonstrated in pulse chase experiments, and although the proportions of most phospholipids does not change, there was a decrease of 50% in phosphatidyl serine.The half-life of [³H]glycerol in the extracted phospholipids of D and ND embryos varies between 35 and 57 h, and in membrane fractions separated on sucrose density gradients the half-lives vary between 26 and 56 h.D embryos induced to germinate with GA and ND embryos in which germination is repressed by ABA show similar phospholipid changes to ND and D embryos respectively, with the exception that the proportion of phosphatidyl serine remained unchanged in the ND-ABA embryos.It is concluded that the continual turnover of membranes of imbibed dormant embryos is consistent with the maintenance of cellular integrity determining the longevity of the seed under natural conditions.Abbreviations D dormant - ND nondormant - ABA abscisic acid - GA gibberellic acid (GA₃)     

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