Composition of Combustible Concretions of the Alewife, Alosa pseudoharengus.

Alewives, Alosa pseudoharengus, wash ashore from Onondaga Lake, N.Y., in the form of combustible concretions in which the mnuscles are replaced by calcium salts of fatty acids. In both distribution pattern and total concentration of fatty acids the concretions differ strikingly from normal carcasses. Carbon-13: carbon-12 ratios indicate that the concretions may have formed from lipids of terrestrial or freshwater organisms or from organic pollutants of nonmarine origin, or from lipids and pollutants.

Asymmetry, its importance to the action and metabolism of abscisic Acid.

Unlabeled and (14)C-labeled enantiomorphs of abscisic acid (ABA) were obtained through acetylcellulose chromatography and tested as inducers of abscission, as inhibitors of seed germination, and as antagonists of gibberellic acid-induced synthesis and release of alpha-amylase. The activity of the R isomer was either equal to or less than that of the naturally occurring S form. Greatest differences were in the inhibition of root-related growth. In excised beam axes, although uptake of S-[(14C)]ABA is faster, the internal concentration of R-ABA is higher because of faster conversion of S-ABA to inactive metabolic products. In axes a reversal in chirality is less important to the physiological action of ABA than to its metabolism.

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

Excised embryonic bean axes (Phaseolus vulgaris, var. White Marrowfat) rapidly metabolize 2-(14)C-(+/-)-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.

Activity and metabolism of C-(+/-)-abscisic Acid derivatives.

Several radioactive analogues of abscisic acid have been tested for their growth-inhibitory effects and their metabolism in excised embryonic axes of Phaseolus vulgaris. The compounds tested were the methyl and ethyl esters of 2-(14)C-abscisic acid and the cis- and trans-1',4'-diols of 2-(14)C-abscisic acid. All four compounds cause less growth inhibition than abscisic acid, and all four compounds are converted to abscisic acid in the axes at rates which are sufficient to account for most, if not all, of the observed growth-inhibitory activity. None of the four compounds is metabolized to the extent that abscisic acid is metabolized in the axes, suggesting that the structural requirements for growth-inhibitory activity and metabolism may be similar.

Activity of the asymmetric isomers of abscisic acid in a rapid bioassay.

Highly purified preparations of (+)- and (-)-abscisic acid (ABA) were compared for biological activity in a rapid bioassay based on closure of stomates on excised leaves of Hordeum vulgare. The naturally-occurring (+)-enantiomer was markedly more active. It causes speedier closure of the stomates, as measured by changes in leaf temperature. The differences in the activities of the ABA enantiomers are greater than have yet been reported for any other system.

Structure-activity correlations with compounds related to abscisic acid.

Inhibition of cell expansion of excised embryonic axes of Phaseolus vulgaris was used to evaluate the growth-inhibiting activity of abscisic acid and related compounds. None of the 13 compounds tested was as active as abscisic acid. 4-Hydroxyisophorone, a substance representative of the abscisic acid ring system was essentially inactive; cis, trans-3-methylsorbic acid, a compound resembling the side chain of abscisic acid, had low activity; and cis, trans-beta-ionylideneacetic acid was one-sixth as active. Loss of the ring double bond results in a drastic decrease in biological activity. Comparison of our results with those reported previously leads to the suggestion that the double bond of the cyclohexyl moiety may have an important function in determining the degree of activity of cis, trans-ionylideneacetic acids. Two modes of action are discussed. It seems possible that the ring double bond is involved in covalent bonding in binding of the abscisic acid analogue to macromolecules. This may require formation of an intermediate epoxide. It can also be argued that stereochemical differences between cyclohexane derivatives are important factors in determining the degree of biological activity.

The Metabolism of Hormones during Seed Germination and Dormancy: II. The Metabolism of 8-C-Zeatin in Bean Axes.

8-(14)C-Zeatin is taken up rapidly and is extensively metabolized by excised bean axes during a 12-hour incubation at 26 C. Most of the radioactivity is found in the 80% ethanol soluble fraction and consists of zeatin, zeatin riboside, zeatin-5'-ribotide, as well as corresponding dihydrozeatin derivatives. The characterization of (14)C-dihydrozeatin included crystallization to constant specific radioactivity. No cleavage of the zeatin side chain to adenine, hypoxanthine, their ribosides, or glycylpurine was detected. Dihydrozeatin has been previously isolated from yellow lupin seeds, and our experiments indicate that it can be derived through reduction of the side chain from preexisting cytokinin. While the total amount of zeatin metabolized is not affected by growth-inhibiting concentrations of abscisic acid or cycloheximide, the conversion to dihydrozeatin derivatives is curtailed. Although somewhat less effective than zeatin and zeatin riboside, dihydrozeatin and dihydrozeatin riboside also counteract the abscisic acid-induced growth inhibition.

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, beta-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 mum zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8-(14)C-zeatin and its metabolites, zeatin-9, beta-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-(14)C-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-(14)C-zeatin uptake or conversions.

The effects of abscisic Acid on growth and nucleic Acid synthesis in excised embryonic bean axes.

Abscisic acid (ABA) is an effective inhibitor of cell elongation in excised embryonic bean axes whether added prior to or after the initiation of cell elongation. Zeatin partially reverses this growth inhibition. ABA inhibits (32)P incorporation into ribosomal RNA, transfer RNA, and DNA but not into the tenaciously bound fraction of elongating axes in a manner resembling 5-fluorouracil, a compound which does not inhibit axis growth. The methylated albumin on kie-selguhr elution profiles of nucleic acids obtained from axes treated with either ABA, 5-fluorouracil, or a combination of the two are similar, and zeatin treatment has little apparent effect on these results. Our results suggest that the inhibition of growth in the axes by ABA is not due to its inhibition of DNA synthesis.ABA (1.9 x 10(-5)m) inhibits growth by 30 and 70% within 1 and 2 hours, respectively, after its addition to elongating axes. Its kinetics of inhibition are similar to those obtained with cycloheximide, and both compounds are more effective than 8-azaadenine. Based on these results, it is suggested that one possible effect of ABA may be at the level of translation.

Abscisic Acid levels and seed dormancy.

Dormant seeds from Fraxinus species require cold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhibit germination of excised nondormant embryos and that this can be reversed with a combination of gibberellic acid and kinetin. Using Milborrow's quantitative "racemate dilution" method the ABA concentration in 3 types of Fraxinus seed and pericarp were determined. While ABA was present in all tissues, the highest concentration was found in the seed and pericarp of dormant F. americana. During the chilling treatment of F. americana the ABA levels decreased 37% in the pericarp and 68% in the seed. The ABA concentration of the seed of the nondormant species, F. ornus, is as low as that found in F. americana seeds after cold treatment. Experiments with exogenously added ABA solutions indicate that it is unlikely that the ABA in the pericarp functions in the regulation of seed dormancy. However, the ABA in the seed does seem to have a regulatory role in germination.

Biological and chemical properties of the epidioxide isomer of abscisic Acid and its rearrangement products.

The growth inhibitory activity of the epidioxide (II), a precursor in the synthesis of abscisic acid (ABA), has been confirmed with additional assay systems. Under physiological conditions the epidioxide is rearranged to give ABA and an isomer of ABA which has probably the structure V. This major product has very low, if any, biological activity. The biological activity of the epidioxide is explained by its partial conversion (about 20%) to ABA. The reaction rate was enhanced by heavy metal ions and decreased by EDTA. At pH 12.5, the decomposition of the epidioxide is slower than it is near neutrality and ABA is the predominant product. In the biological systems studied the activity of the epidioxide can be accounted for by nonenzymatic conversion to ABA.

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-(14)C-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.Dormant intact seeds also metabolize (S)-2-(14)C-abscisic acid during stratification at 5 C or incubation at 25 C. The metabolites appear to be similar to those observed in excised embryos although by 12 days of stratification a fourth metabolite is observed. More than 90% of the (14)C-abscisic acid was metabolized after 26 days of stratification at 5 C or after 12 days of incubation at 25 C. Stratification at 5 C leads to the breaking of dormancy while incubation at 25 C does not.

Metabolically active glucosides in oleaceae seeds: I. The effects of germination, growth, and hormone treatments.

The seeds of six woody species of Oleaceae representing three genera, contain high concentrations of water-soluble glucosides, with major absorption maxima below 240 nanometers. In Fraxinus americana seeds three of these compounds, designated GL-3, GL-5, and GL-6, account for almost 10% of the dry weight. They are found in the endosperm and embryo but not in the pericarp. While the level of GL-5 is not particularly influenced by the physiological state of the embryo, that of GL-3 and GL-6 decreases as a result of germination and growth during a 10-day period. As the concentrations of GL-3 and GL-6 decrease, new ultraviolet-absorbing compounds are formed. The changes in the concentration of the ultraviolet-absorbing glucosides during cold temperature after-ripening, prior to germination, are small. When germination of dormant embryos is induced with gibberellic acid, the concentrations of GL-3 and GL-6 decrease in a manner similar to that observed with nondormant embryos. In the presence of abscisic acid no losses of GL-3 and GL-6 were observed. It is suggested that GL-3 and GL-6 fulfill some definite functions in the germination and growth of F. americana embryos, and that gibberellic acid and abscisic acid can exert a regulatory effect on the metabolism of these glucosides.

Germination of Excised Fraxinus ornus Embryos With and Without Phleomycin.

In excised embryos of Fraxinus ornus, the first macroscopic sign of germination is a curvature near the root apex. Histological studies show that this curvature is due to cell elongation on the convex side and is accompanied by cell division. Metabolic changes, manifested by the disintegration of protein bodies and tissue differentiation, also occur during this time. In embryos treated with phleomycin cell division is completely inhibited but elongation, root hair formation, disintegration of protein bodies, and differentiation are still detectable. Phleomycin also drastically inhibits the formation of chlorophylls and the fresh weight increase of embryos.