The Metabolism of Hormones during Seed Germination and Dormancy

Abstract

8-14C-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 "4C-dihydrozeatin included crystallization to constant specific radioactivity. No labe-led hormone. In this paper we report on the metabolism of 8-'4C-zeatin in axes excised from beans, a tissue where germination does not involve the breaking of dormancy.Excised axes from seeds of Phaseolus vulgaris imbibe water rapidly, go through a lag phase, and then initiate fresh weight increase at a linear rate (16). At 26 C the growth phase starts 4 to 5 hr after imbibition and continues for at least 10 hr. This growth is inhibited by ABA and while exogenously added zeatin does not affect the normal growth rate, it does lead to a partial reversal of the ABA-induced inhibition (18). Because of these relations, we studied the metabolism of 8-`4C-zeatin alone and in the presence of growth-inhibiting concentrations of ABA in order to determine whether ABA affects zeatin metabolism. The effect of cycloheximide, a growth inhibitor in bean axes (17) and a general inhibitor of protein synthesis, on 8-'4C-zeatin metabolism was also determined. MATERIALS AND METHODS8-'4C-Zeatin. This compound was synthesized by the procedure of Shaw et al. (15). 6-Chloropurine-8-14C, 4.3 mg (Calbiochem, nominal specific radioactivity 3.6 mc/mmole) and 7.3 mg of trans-4-amino-2-methyl-2-buten-1-ol sulfate were dissolved in 0.27 ml of 1-butanol and 0.03 ml of triethylamine. This mixture was heated in a sealed tube at 130 C for 90 min, cooled, and concentrated. The radioactive product was isolated and purified by pap-r chromatography with solvent systems a, b, and c, Table I. The isolated material was chromatographically homogeneous by radiographic and ultraviolet criteria and has the same RF values and absorption spectrum as unlabeled zeatin. The yield was 70%, and the specific radioactivity was 2.95 mc/mmole based on a molar extinction coefficient, E27Onm = 16,500.Zeatin-9,f/-riboside was synthesized by the procedure of Shaw et al. (15) and (RS)-dihydrozeatin-9,pB-riboside was prepared from the above compound by catalytic hydrogenation (9). That the dihydrozeatin-9, f3-riboside was free of zeatin-9,,4-riboside could be established by mass spectrometry. 2iP' and 2iPA were synthesized by condensation of 3-methyl-2-butenylamine with 6-chloropurine and its riboside respectively (8). 6-Glycylpurine was prepared from 6-chloropurine and glycine (3). RF values for the above and other purines used in this study are shown in Table I.Tissue Incubation. Information on the growth characteris-'Abbreviations: MAK: methylated albumin kieselguhr; 2iP: 6-(3-methyl-2-butenylamino)purine; 2iPA: 6-(3-methyl-2-butenylamino)-9-P-D-ribofuranosylpurine.

The Metabolism of Hormones during Seed Germination and Release from Dormancy

“…From Table IV (8). In the bean axes, more than 50% conversion to dihydrozeatin derivatives was found (8).…”

“…In this reaction, crystalline trans-4-amino-2-methyl-2-butene-1-ol hemisulfate, m.p. 110 to 111 C, and 6-chloropurine-8-'4C (Calbiochem) are condensed in 1-butanol and triethylamine, and the product is purified by paper chromatography in several solvent systems (8). After publication of our earlier data, Letham and Young (3) claimed that the procedure of Shaw et al is not readily adaptable to the synthesis of radioactive zeatin and leads to a mixture of isomers difficult to purify by paper chromatography.…”

“…Additional RF values for these and related compounds in five solvent systems are given in the earlier paper (8).…”

“…The embryos were ground with 80% ethanol in a mortar at 0 C, centrifuged at 15000g for 5 min, and the supernatants from three extractions were pooled. The methods used for the separation, counting, and characterization of the 80% ethanolsoluble components were the same as those used with the products obtained from excised bean axes (8). Solvent A is 1-butanol-acetic acid-water (12:3:5); solvent B is water-saturated 1-butanol on 0.03 M, pH 8.4, borate impregnated paper.…”

“…The previous report in this series (8) dealt with the metabolism of 8-"C-zeatin during a 12-hr incubation with excised bean axes. In that tissue, in which early growth is due primarily to cell elongation, the main conversion products were zeatin ribonucleoside, zeatin-5'-ribonucleotide, dihydrozeatin and its ribonucleoside, and 5'-ribonucleotide.…”