Metabolites of 2-14C abscisic acid

105

The in vivo feeding of 2-'4C-ABA to embryonic bean axes (Phaseolus vulgaris L.) has established PA2 and DPA as products in one ABA metabolic pathway (21,23). The accumulation of DPA in bean seeds (24), as well as the continuous formation of PA and DPA in water-stressed leaves (5), indicates that this pathway has an active role in regulating total ABA concentration within the plant. The decreased activity of PA in bioassay systems for growth inhibition (9, 22), abscission promotion (1, 4), and stomatal closure (7) suggests that the DPA pathway has a role in regulating the hormonal activity of ABA. The recent observation that PA inhibits photosynthesis, while ABA does not, indicates that the DPA pathway may also play a role in the activation of physiological activities (7). The actual role which the DPA pathway plays in regulating ABA levels requires the investigation of the enzymes involved. The goal of the present investigation was to establish a cell-free enzyme system capable of metabolizing ABA by the DPA pathway.

Section: Resultsmentioning

confidence: 93%

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mentioning

confidence: 99%

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

Gillard

Walton²

1976

105

“…Although PA and DPA are now known as the major ABA metabolites in many systems (20,26,27,30) and a cell-free preparation that can degrade ABA into PA and DPA has been successfully isolated from 'Eastern Wild' cucumber endosperm (7), these findings do not rule out the possibility of an "interim" metabolite role for bound ABA. The bound ABA peak on day 30 ( Fig.…”

mentioning

confidence: 99%

Abscisic Acid Accumulation in Developing Seeds of Phaseolus vulgaris L

Hsu¹

1979

Free and bound abscisic acid (ABA) in the pod, seed coat, and embryo were determined separately throughout seed develpment of Phaseolus vulgans L. cv. 'Taylor's Horticultural.' An internal standard method of gas-lquid chromatography was used for ABA quantification. In the embryo, two peaks of free ABA occurred at days 22 (1.18 micrograms per gram or 5.5 micromolar) and 28 (1.74 micrograms per gram or 12 micromolar); and a single peak of bound ABA at day 30. In the seed coat, there was one peak of free ABA at day 22 and only small amounts of bound ABA. Very small amounts of ABA were detected in the pod at any stage of development. In cv. PI 226895, in which seed development is more rapid than in 'Taylor's Horticultural,' the embryo ABA peaks occur on days 20 and 26. The timing of the ABA peak in the embryo, and the concentration attained, are consistent with previous reports on the natural pattern of RNA synthesis and with ABA inhibition of RNA synthesis in developing bean fruit.Developing fruits and seeds have long been known as a rich source of plant hormones (14). In many species, changes of hormone content throughout development of the reproductive parts have been determined. From the accumulated data, there appears to be a general pattern of high growth promoter content in early development followed by high inhibitor, mainly ABA, content later in development (1,6,14). This late appearance of ABA has been thought to have important implications in fruit maturation (2), abscission (3), or seed dormancy (5).This report is an analysis of the change in ABA content during reproductive development of Phaseolus vulgaris, with a discussion of its correlation with seed weight changes and other related processes. P. vulgaris was chosen because of the large body of information on reproductive development (9, 24), macromolecular metabolism (21, 23), and genetics (28). Clarkson Chemical Co., Williamsport, Pa.) was suspended in chloroform and packed in a 5-ml glass disposable pipette which was plugged with a piece of glass wool at the outlet. ABA extracts were dissolved in chloroform and loaded into the column. The column was eluted first with 10 ml of chloroform to wash off some impurities. It was next eluted with 10 ml of chloroform-methanol (90:10). This fraction contained ABA, and was saved for derivatization. A small scale method was used to generate diazomethane which derivatized ABA into its methyl ester (19).A Hewlett-Packard model 1610A gas chromatograph equipped with a flame ionization detector and an automatic peak area integrator was used throughout ABA quantification. An XE-60 column was used for all samples. OV-I and OV-17 columns were used to verify the authenticity of any suspect ABA peaks. A suspect peak was confirmed as c-t ABA if it co-chromatographed with authentic c-t ABA in all three columns. Operating conditions were: column temperature, 200 C; injector and detector temperature, 250 C; carrier gas (N2) flow rate, 40 ml/min.Tetraphenyl ethylene was used as an internal standard to facilitate th...

“…Previous work with two ABA metabolites from bean seed had shown them to be phaseic acid and dihydrophaseic acid (10). Because o f their imilar srolubilities and chromator raphic mobilities the metabolites from ash seed were compared with the 2 bean metabolites.…”

Section: Resultsmentioning

confidence: 99%

“…Because o f their imilar srolubilities and chromator raphic mobilities the metabolites from ash seed were compared with the 2 bean metabolites. GLC-mass spectrometry had been used for the identification of phaseic acid from bean seed (10), and this technique was also used to identify M-1 from ash seed as phaseic acid. The M-2 from ash seed has the same chemical reactivity and chromatographic mobility as dihydrophaseic acid (Table II).…”

Section: Resultsmentioning

confidence: 99%

The Metabolism of Hormones during Seed Germination and Dormancy

Sondheimer¹,

Galson²,

Tinelli³

et al. 1974

Embryos from dormant and stratified Fraxinus americana seed were incubated with (S)-2-"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-VC-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 "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.The plant growth regulator ABA prevents germination when applied to embryos of Fraxinus ornus, a nondormant ash species, and to F. americana embryos from seeds whose dormancy has been overcome by stratification (5). The concentration of ABA in the seeds and pericarp of F. americana has been shown to decrease during the course of stratification to approximately that found in the nondormant F. ornus (6). These latter results suggested the possibility of ABA metabolism during the stratification process. While ABA has been shown to be metabolized rapidly in several other plants including tomato and bean (4, 11), its metabolism in dormant tissues has not been reported. We were interested in the extent to which ABA is metabolized by dormant seeds, the identity of the metabolites and in the possibility that the stratification process might result in an increased capacity for ABA metabolism. In order to answer these 'Eva Galson, Eugene Tinelli, and Daniel Walton dedicate this paper to the memory of our colleague and friend, Ernest Sondheimer. We shall sadly miss his companionship, creativity, and infectious enthusiasm for research.questions about ABA metabolism, dormant and stratified excised F. americana embryos and endosperm were incubated with 2-"`C-ABA. 2-w"C-ABA metabolism was also followed in intact seeds during a stratification period at 5 C sufficient to break dormancy, and during an incubation period at 25 C which does not result in the breaking of domancy. All of this work was done with the naturally occurring (S) enantiomer of ABA, and in some cases with the (R) enantiomer also. Relatively large scale incubations of embryos with (RS)-2-uC-ABA were also done in order to obtain sufficient quantities of metabolites for identification by chemical and spectral methods.MATERIALS AND METHODS Short Term Incubations. F. americana seeds (1971 crop from Herbst Bros., Brewster, N.Y.) were depericarped by hand and then imbibed in tap water for 24 hr. After the embryos were excised, they were shaken for 24 hr in 25-ml round b...