Approximately 20 to 25% of the cytokinin benzyladenine (BA) taken up by soybean tissues in culture is converted to a stable, long-lived derivative which contains BA as part of its structure. This derivative may be metabolically related to 6-benzylamino 9-,B-D-ribofuranosylpurine 5'-monophosphate (BAMP). In in vivo incubations of 2 hours or less, we recover only BA, benzyladenosine, and BAMP. Benzyladenosine never accounts for more than 10% of the total radioactivity while BAMP builds up to about 20% of the total within 2 to 4 hours. After this period it begins to disappear, and a new, unidentified substance arises at a rate which roughly parallels the loss of BAMP. After about 48 hours this substance, which has good cytokinin activity, accounts for some 20 to 25% of the total radioactivity and persists at this level for at least 60 days. In the meantime the remainder of the BA, as well as benzyladenosine and BAMP, disappear completely. In addition, evidence is presented which suggests that the urea cytokinins are not active as such but first are metabolically transformed into other substances. Despite the increasing amount of evidence demonstrating the presence of molecules with cytokinin activity in the transfer RNA of a wide variety of organisms (see review by Key [12]), the biological roles of these substances remain obscure. Particularly puzzling is the finding (2) that, even in plant tissues which require an exogenous cytokinin source for in vitro growth, molecules with potent cytokinin activity exist as naturally occurring constituents of the tRNA. One is led to believe that the exogenous cytokinin has at least one biological role quite distinct from the cytokinin in situ.To investigate this role, one needs to know the short term metabolic fate of a cytokinin in a tissue which requires an exogenous source of these substances in order to grow. There are, unfortunately, very few sufficiently detailed studies of the low molecular weight metabolites of the cytokinins in the literature. McCalla et al. (15) investigated the soluble metabolites of 6-benzylaminopurine in Xanthium leaves and concluded that the riboside was the major metabolite. In addition, small amounts of what was apparently the 5'-ribonucleotide

The cytokinin, 6-benzylaminopurine, is converted to its 7-glucoside in intact seedlings, organ slices, and tissue cultures from several plants. The ribonucleoside and 5'-ribonucleotide appear transiently, and the general metabolic sequence seems to be nearly identical in the four plant species thus far studied. The glucoside persists for long periods in plant tissues, while all other forms of the cytokinin are rapidly metabolized and disappear within a few hours. A procedure for the isolation in pure form of the glucoside is described.

Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N0-(A2-isopentenyl) adenosine and N'-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N'-(A'-Isopentenyl)- Hall [14]). This hydroxylated derivative appears to be restricted to the tRNA of higher plants. In addition, the 2-methylthio derivatives of both the parent compound (z.2-iA) and the hydroxylated derivative have been isolated from the tRNA of wheat germ (6).The trans-isomer of ribosylzeatin (trans-RZ) and/or its free base and nucleotide have been found as free compounds in chicory roots (5) (8) showed that the A2-iA of the tRNA of this tissue is synthesized by attachment of a A2-isopentenyl side chain to the preformed tRNA. The requirement for the exogenous cytokinin does not seem to relate directly to this biosynthetic mechanism. In search, therefore, of leads that bear on the mechanism of action of cytokinins we decided to compare the A2-iA content of an autonomous line derived from the Wisconsin 38 strain of tobacco pith callus tissue (11) that grows well in the absence of exogenously added cytokinins. We have found that the tRNA of this strain also contains A2-iA and cis-RZ in its tRNA, but in contrast to the cytokinin-requiring strain the autonomous tissue contains a significant level of A2-iA in the free state. MATERIALS AND METHODSTissue Culture Strains. Normal tobacco pith tissue has an absolute requirement of added cytokinins for growth (11). Occasionally this normal tissue spontaneously loses its cytokinin requirement. Such autonomous strains of tissue presumably meet their cytokinin requirement through an alteration in some metabolic process, although the mechanism(s) of this transformation is not known.The normal cytokinin dependent tobacco pith tissue (Nicotiana tabacum [L] var. Wisconsin 38) and the derived autonomous strain used in this study were gifts from J. E. Fox of the University of Kansas. The normal pith tissue was grown on a previously described (11) basal medium containing naphthaleneacetic acid (1 mg per liter) and 6-furfurylaminopurine (kinetin) (1 mg per liter). The autonomous strain was grown on the same basal medium without the growth factors. Tissues were harvested after 4 to 5 weeks growth in the dark.Bioassay. Aliquots of column fractions to be bioassayed for cytokinin activity were added to 25-ml flasks and dried in vacuo over P20,. A basal medium (11), complete except for cytokinin, was added (10 ml per flask), and the flasks were autoclaved at 18 lb, 121 C for 5 min. A cytokinin requiring strain of soybean cotyledon tissue (Glycine max [L.] Merril, var. Acme) was planted (0.5 mg pieces, three per flask), and after 30 days incubation in the dark at room temperature, the increase in fresh weight was compared to controls grown in the presence (...

By use of a radioactive labeling technique N-(nebularin-6-ylcarbamoyl)threonine has been detected in plant transfer RNA. Derivatives of this nucleoside promote cell division of a cytokinin-requiring soya bean tissue.

Several ureidopurine derivatives have been tested for cytokinin activity in a soya bean callus assay system. N-(Purin-6-ylcarbamoyl)threonine (Ade-CO-Thr), a naturally occurring component of tRNA, is inactive in the assay. Analogues of Ade-CO-Thr having unmodified hydrocarbon side chains are active; optimal activity is associated with tert-butylureidopurine. In the homologous straight chain series n-butylureidopurine is the most active. Introduction of −COOH and/or −OH groups to analogues with otherwise active alkyl chains negates activity.The phenylureidopurine (PUP) derivative is barely active itself, though ortho-halogenated derivatives (o-chloro-, o-fluoro-PUP) are the most potent ureidopurines known. Biological activity decreases rapidly as the electronegative halide is moved from the ortho to the meta position, and lost entirely in the para position. Replacement of the ortho halide with a non-electronegative group (methyl) greatly reduces activity. Molecular models reveal that Ade-CO-Thr and o-chloro-PUP have nearly identical side-chain bulk. The carboxyl groups of Ade-CO-Thr and the chlorine atom of o-chloro-PUP occupy the same position relative to the electronegative carboxyl oxygen of the urea bridge. It is proposed that Ade-CO-Thr has a growth regulatory role in the intact organism, but that exogenous Ade-CO-Thr may be unable to reach the active site.

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Tenn., 1965. (8) Fractional coordinates, bond distances, bond angles, and observed and calculated structure factors are available as a supplement to publication.To obtain a microfiche copy of the supplement to publication, contact the

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