Incorporation of Cytokinin N⁶-Benzyladenine into Tobacco Callus Transfer Ribonucleic Acid and Ribosomal Ribonucleic Acid Preparations

Abstract

The incorporation of the cytokinin N6-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N6-benzyladenine-8_-4C or N6 -benzyladenine -8 14C: benzene-3H(G) has been examined. N6-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N6 -benzyladenosine recovered from the RNA preparations was associated with the rRNA. Purification of the crude rRNA by either MAK chromatography or Sephadex G-200 gel filtration had no effect on the N6-benzyladenosine content of the RNA preparation. The distribution of N6-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N6-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T2 and phosphatase, or ribonuclease T2 and a 3'-nucleotidase. N6 -benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T2 or phosphatase alone or by successive treatment with ribonuclease T2 and a 5'-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T, and pancreatic ribonuclease converted the N6-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N6 -benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.The apparent incorporation of the cytokinin N6-benzyladenine (bzl6Ade)3 into the soluble RNA of cytokinin-dependent 1 This work was supported in part by National Science Foundation Research Grant BMS72-02226 A02 to F. S.2 Present address: Department of Botany and Plant Pathology, Oregon State University, Corvallis, Ore. 97331.3 Abbreviations: bzl6Ade: N6-benzyladenine or 6-benzylaminopurine; bzl6A: N6-benzyladenosine or 6-benzylamino-9-j3-D-ribofuranosylpurine; '4C-bzl6Ade: N6-benzyladenine-8-14C; 3H:'4C-bzl6Ade: N6-benzyladenine -8 -14C: benzene-3H(G); i6A: N6-(6'-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9-,3-D-ribofuranosylpurine; cio6A: cis-ribosylzeatin or 6-(cis-4-hydroxy-3-methyl-2-butenylamino)-9-,6-D-ribofuranosylpurine; ms2io6A: methylthioribosylzeatin or 6-(4-tobacco tissue cultures was reported by Fox in 1966 (7). The subsequent discovery of the natural occurrence of the highly active cytokinin N6-(A2-isopentenyl)adenosine (i6A) and related compounds as constituents of tRNA molecules focused attention on the possibility that cytokinins might function by being incorporated into RNA molecules (11,19,24,25,28). It now appears established that cytokinins do not serve as direct precursors of the structurally identical nucleosides in tRNA. The i6A moieties in tRNA are synthesized by the transfer of the isopentenyl group from z2-isopentenylpyrophosphate (A2-IPP) to specific adenosine residues in preformed tRNA molecules (12).The enzyme that cat...

Presence and source of free isopentenyladenosine in yeasts.

“…While tRNA has been the prime candidate for cytokinin generation by polynucleotide turnover, N6-modified adenosines have been found as rare components of some ribosomal RNAs (42)(43)(44)(45)(46)(47), and the direct incorporation of exogenous cytokinins into tRNA, rRNA, and polyadenylylated mRNA has been reported in tobacco cells (33,(42)(43)(44)(45)(47)(48)(49). This incorporation is relatively low and may result from transcriptional errors in the presence of high levels of the adenosine derivatives (43).…”

Metabolism of benzyladenine by excised embryos of Picea abies

“…This aspect was not investigated in this study. Small quantities of BA have, however, been shown to be incorporated into poiynucleotidies in other systems (Walker et al 1974, Armstrong et al 1976. The fact that the major metabolite, which persisted in Picea embryos, gave chromatographic evidence indicating that it was BA-nucleotide, clearly indicates that more attention must be given to this aspect or at least to the fact that BA action may be mediated via its nucleotide.…”

Short‐Term Metabolism of Radioactive Kinetin during Lettuce Seed Germination

“…A long standing controversy concerns the functional involvement of cytokinins in RNA (Kende and Tavares 1968, Key 1969, Burrows 1975). Recent studies of cytokinin metabolism using tissue culture have demonstrated incorporation of intact cytokinins into RNA (Fox and Chen 1967, Walker et al 1974, Armstrong et al 1976). This incorporation followed long periods of incubation.…”