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.

A bstract. Dormant seeds from Fraxinus species require oold-temperature after-ripening prior to germination. Earlier, we found that abscisic acid (ABA) will inhilbit 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 tisisues, the highest concentration was found in the seed and, perioarp of dormant F. americana. During the chililing treatment of F. americana the ABA levels decreased 37 N% 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 adided 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.Abscisic acid (ABA) (1) has been implicated in the regulation of leaf abscission, senescence, growth inhibition, and bud and seed dormancy. Up to now most of the studies have dealt with effects of exogenously added ABA. The few isolation studies that have been reported do not permit a quantitative correlation between endogenous ABA levels and a change in the physiological state of the test plant.WNe are studying physiological and biochemical aspects of seed germination with dormant and nondormlant Fraxinus (ash) seeds. Villiers and \Vare-ing (11) have slhown that the germination behavior of excised embryos from this genus depends on their previous hiistorv. Embryos from cold-temperature after-ripened seeds will germinate rapidly when placed in moist chambers at room temperature, while those from untreated seeds show only very limited germination during a 10 day period. In Fraxinits excelsior L., germination of dormant embryos can be indtuced either by leaching or with gibberellic acid, GA3 (11). WVe found that we can prevent germination in nondormant Fraxini(s antericana L.aind Fraxinus ormus L. embryos with exogenously added (RS)-ABA. Furthermore the ABA-induced inhibition is partially reversedl by combinations of GA, anid kinetini (9).It is generally accepte(1 thiat resuilts obtained e.xclusively witlh exogenously applied growth substances are insuifficienit to establish a regulatory role for these substances in the intact organism. A minimum requirement for such a role would be to show the presence of the hormones in the intact plant and to demonstrate a correlation between hormonal concentration levels and physiological states. With the elegant "racemate dilution" method developed by Miliborrow (7) it is now possible to obtain quantitative values for the ABA concentration in plants. With

Zeatin and zeatin-9, ,f-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 uM zeatin is as effective as continuous incubation. There must be barriers against transport out of the embryos since 8-&4C-zeatin and its metabolites, zeatin-9,,8-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-'4C-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-'4C-zeatin uptake or conversions.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. In bean axes, exogenously added zeatin has no effect on growth but does partially reverse the ABA-induced inhibition of fresh weight increase.We have now conducted a parallel study with dormant ash embryos in which exogenously added zeatin has pronounced physiological effects, leading to extended root development, fresh weight increase, and synthesis of chlorophyll. These effects of zeatin are antagonized by ABA (6). 8-14C-Zeatin was fed, its early conversion products identified, and the effects of ABA on the uptake and metabolism determined.