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

Abstract

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 Most of the layers were incubated 24 hr, but nine lots of three layers each were withdrawn at earlier intervals to determine the onset of formation of I (Fig. 1).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

Gibberellins: synthesis, compartmentation and physiological process

“…We incubated barley ( Hordeum v u l g a r e ) protoplasts and v according to the methods of Nadeau & R appaport (1974 a). Probable occurrence of GAX has been reported in Hordeum vulgare (Faull et al 1974) and we have studied the metabolism of pHJGAj in barley aleurone layers and half seeds ,1974aStolp et al 1973Stolp et al ,1977)*…”

Determination of Endogenous Gibberellins in Germinating Barley by Combined Gas Chromatography-Mass Spectrometry

“…Brooks and Martin (1) also reported similar results. The metabolism of gibberellins in barley is not clearly understood, but the reduction in GA 3 added to barley may have been the result of its conversion to other compounds, such as GAs (23,27,34) or glycosides (6,23,28), which exhibit different mobility patterns at the purification step. As shown in Fig.…”

Metabolism of Tritiated Gibberellins in d-5 Dward Maize

“…It is also possible, however, that CHI affects the system indirectly by interfering with the production of ATP and NADH, both of which could be required for [3H]GA1 metabolism. Cycloheximide has been reported to inhibit respiration and energy transfer in some plant systems (2,8), possibly by the effects of inhibition of protein synthesis on the enzymes involved; however, this inhibition is less likely in the green tissues (9) (13,21) (Fig. 6) (4).…”

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Gibberellin metabolism in plants involves conversion of a GA to other 11,[17][18][19][20], or to biologically active or inactive conjugated derivatives such as GA-glucosides (1,4,13,21,23,24). Since metabolism results in modification of the types and levels of GAs, it could be a major factor controlling plant growth and development.

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“…Unless otherwise stated, the specific radioactivities of these compounds were [3H]GA3, 13 Ci/mmole; ['H]GA1, 14 43 Ci/mmole, was prepared as described previously (26). [ second nearly fully expanded, and the third with less than 3 cm of lamina protruding from the leaf sheaths of the older leaves.…”