Metabolism of gibberellin A1 in germinating bean seeds

Nadeau, Ronn; Rappaport, Lawrence

doi:10.1016/0031-9422(72)85007-6

Cited by 41 publications

(17 citation statements)

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“…During an investigation (8) of the relation between metabolism of [FH]GA, and the induction of a-amylase in barley aleurones, we confirmed previous reports (2,3) that ABA suppresses this induction. In addition, we found that ABA enhances conversion of [ was especially promoted by ABA.…”

supporting

confidence: 89%

“…['H]GA1 was prepared and purified by methods recently described in detail (8). Its specific radioactivity is 43 Ci/mmole, 'This research was supported by United States Public Health Service Grant 12885, National Science Foundation Grant 21241, and North Atlantic Treaty Organization Grant 541.…”

Section: Methodsmentioning

confidence: 99%

“…Details on these procedures have been reported previously (8) and are summarized in Table II (Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

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An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

Nadeau

Rappaport²

1974

Plant Physiol.

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The major metabolite produced during incubation of ['H] ['H]ampho GA1 in barley aleurones begins after a period of 2.5 hours. As judged by degradation studies as well as Sephadex column chromatography, GA1 appears to be linked to a peptide; positions C-3 and C-7 were ruled out as conjugation sites.Among the most studied effects of gibberellins on plant tissues is the induction of certain hydrolytic enzymes in barley aleurone layers (5,7,10,11,12 2 Ernest Sondheimer's research was characterized by his consistent efforts to provide a chemical basis for an understanding of plant hormone action. He understood that simply measuring a change in concentration of a hormone is not sufficient to explain its function in a process. Not only was he concerned with the occurrence of hormones but also with their identity. The dedication of this volume to mark his lamentably early death should serve to focus the attention of those engaged in growth regulator research on the need to attain a fuller comprehension of the role of hormone biosynthesis and turnover in relation to physiological processes. If this occurs his contribution will remain as much more than a footnote in the annals of hormone research. Generally, the incubation procedures folowed were those described by Chrispeels and Varner (3). The buffer was 2 mM in sodium acetate and 20 mm in CaCl, adjusted to pH 4.8. One hundred fifteen isolated aleurone layers were incubated at 25 C in 6 ml of buffer which was 48 ,uM in L'H]GA1 (10 ug, 2.6 X 10' dpm, per 6.0 ml) and 19 FM in ABA (30 ,g per 6 ml). Most of the layers were incubated 24 hr, but nine lots of three layers each were withdrawn at earlier intervals to determine the onset of formation of I (Fig. 1). After 24 hr of incubation, radioactive materials in the remaining layers were extracted with methyl alcohol. The methyl alcohol extract was concentrated by distillation under high vacuum so that the temperature never exceeded 45 C. The concentrate was refined by TLC on ChromAR, using isopropanol-NH,OH-water (8: 1: 1). In this system, I was the only radioactive material remaining at the origin.The chromatographic systems and the RF values of the compounds studied are shown in Table I. Details on these procedures have been reported previously (8) and are summarized in Table II along with the retention times of the compounds under investigation.High voltage paper electrophoresis was done using sheets of Whatman No. 3 MM paper having basic weight = 185 g/m', 0.33 mm thickness, medium flow rate, and smooth surface. In all runs the voltage was 1500 v, and the time was 1.5 hr. Zones were detected by radiochromatogram scanning. Buffers were: pH 10.6, NH4OH (0.065 M); pH 5.9, pyridine-acetic acid-H,O (100:15:900); pH 2.9, pyridine-acetic acid-H.0 (3:100:1900); pH 2.2, 2.5% formic acid-8% acetic acid, with water. RESULTS AND DISCUSSION

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Section: Methodsmentioning

confidence: 99%

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An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

Nadeau

Rappaport²

1974

Plant Physiol.

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“…Both GA20 and GA1 occur in Phaseolus seedlings (2). As the scheme in Figure 6 indicates, GA, can be further metabolized to GA8 (17)(18)(19) and its glucoside (17). As previously mentioned, metabolism of 2,3-[3H]GA2O to any of the above compounds could result in a loss of tritium and could preclude accurate quantitative estimates of conversion by radioactivity measurements.…”

Section: Methodsmentioning

confidence: 98%

Metabolism of [³H]Gibberellin A₂₀ in Light- and Dark-grown Tobacco Callus Cultures

Lance

Durley²,

Reid³

et al. 1976

Plant Physiol.

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The growth of tobacco callus in culture (previously shown to contain gibberelin [GAJ-like substances), and its abilit to metabolize 13HI-GA20 were examined. Growth rates, in the presence and absence of exogenously applied GA, were examined in light and dark conditions. Dark-grown callus grew at a much faster rate than light-grown and [3HIGA2o was metabolized much more rapidly in darkness than in light.[3HIGA, was identified by combined gas-liquid chromatography/mass spectrometry as a major product of 13H1GA2o, and was found to be a more potent promoter of tobacco callus growth than GA20.In an earlier paper (11), evidence was presented showing that tobacco callus contains endogenous gibberellins (GAs), the levels of which varied with cultural treatments such as light or darkness or the concentration of cytokinin in the medium. On this and other evidence, it was postulated that endogenous GAs might play a role in controlling growth of tobacco callus cells.Variations in endogenous GA levels could arise through modifications of rate of biosynthesis and/or utilization (i.e. metabolism). Extraction of plant tissue at any one point in time or even at a number of times provides information only on GA pool size, and will often reveal little about over-all GA metabolism. This paper describes an investigation of the metabolism of [3H]GA20

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“…Distribution of radioactivity on the strips was determined with a Packard 7201 radiochromatogram scanner, and the chromatograms were then cut into 10 or more equal segments. These were placed in liquid scintillation vials together with 5 ml of counting fluid, and the radioactivity was termined by counting to 1% error.…”

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Selective Binding of [ ³ H]Gibberellin A ₁ by Protein Fractions from Dwarf Pea Epicotyls

Stoddart

Breidenbach

Nadeau

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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Studies of the regulatory action of animal steroid hormones have revealed specific receptor proteins present in higher concentration in the target tissues. The behavior of the hormone-receptor complex, subsequent to binding, has been clearly characterized in the uterine response to estradiol (1). Similar data have been obtained on the association between a metabolite of a-ecdvsone and the crustacean hepatopancreas receptor (2). Attempts to date have failed to demonstrate an analogous receptor system for gibberellin in plants, although in etiolated dwarf peas, labeled GA3 accumulated most in tissues that provided maximal growth response (3). Results from further fractionation of the hormone-enriched zones, with the objective of localizing the receptor site(s), were inconclusive (4). The specific radioactivity of the labeled gibberellin used in the studies with pea was at least three orders of magnitude lower than the values reported for the hormones used in the animal systems, and the low level of binding encountered in animal tissues indicates that low specific radioactivity was probably the basic limitation in the plant investigations.Nadeau and Rappaport (5) recently submitted methods for preparing and purifying tritiated GA1 of high specific radio. Growth, Preparation, and Treatment of Epicotyls. "Progress No. 9" dwarf peas (Asgrow Seed Co., 1972 harvest, lot no. 37055) were rinsed repeatedly with distilled water to remove the bulk of the captan (Orthocide) fungicidal dressing, and were then imbibed, with aeration, for 12 hr at laboratory temperature. The imbibed seeds were sown in trays containing moistened vermiculite, and were allowed to grow, without further watering, for 5 days, in an enclosed dark cabinet at 250. Then the epicotyls, which were 3-3.5 cm long, were cut just above the point of insertion into the cotyledon, and trimmed to a uniform length of 2.5 cm, measured from the apex of the hook. All operations to this point were carried out either in darkness or weak green light. Groups of 30 epicotyls were placed in 5-ml beakers each containing 1.0 ml of solution containing 58 nM labeled hormone; beakers were placed. in the dark or under red light. The entire volume of the solution was taken up during a subsequent 12-hr period.Preparation of Test Solutions. [1,2-3H Igibberellin Al ([3H]GA1), with a specific radioactivity of 43 Ci/mmole (2.6 X 108 dpm/ug) 1, was prepared and purified by the method of Nadeau and Rappaport (5), and dispensed from a stock solution of MeOH-EtOAc (1:1 v/v) containing 3.58 X 105 cpm/ /diter. Portions of this solution were reduced to drvness in a stream of nitrogen, and the hormone was then redissolved in the requisite amount of distilled water to provide a final concentration of 20 ng/ml (2 X 106 cpm/ml, 58 nM).The 16 Tissue samples in the weight range of 2.5-3.0 g were ground at 20 with a pestle and mortar in 1.0 ml of 10 mM Trismaleate buffer, pH 6.5, containing 2 mM reduced glutathione, per g of fresh weight. The fluid extracted from the resultant paste by squeezing thro...

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Metabolism of gibberellin A1 in germinating bean seeds

Cited by 41 publications

References 8 publications

An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

Metabolism of [³H]Gibberellin A₂₀ in Light- and Dark-grown Tobacco Callus Cultures

Selective Binding of [ ³ H]Gibberellin A ₁ by Protein Fractions from Dwarf Pea Epicotyls

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Metabolism of gibberellin A1 in germinating bean seeds

Cited by 41 publications

References 8 publications

An Amphoteric Conjugate of [3H]Gibberellin A1 from Barley Aleurone Layers

An Amphoteric Conjugate of [3H]Gibberellin A1 from Barley Aleurone Layers

Metabolism of [3H]Gibberellin A20 in Light- and Dark-grown Tobacco Callus Cultures

Selective Binding of [ 3 H]Gibberellin A 1 by Protein Fractions from Dwarf Pea Epicotyls

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An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

An Amphoteric Conjugate of [³H]Gibberellin A₁ from Barley Aleurone Layers

Metabolism of [³H]Gibberellin A₂₀ in Light- and Dark-grown Tobacco Callus Cultures

Selective Binding of [ ³ H]Gibberellin A ₁ by Protein Fractions from Dwarf Pea Epicotyls