Kinetin Incorporated into Tobacco Callus Ribosomal RNA and Transfer RNA Preparations

Abstract

Kinetin, N6-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) caUus RNA isolated from rapidly growing tissue cultured in the presence of N6-furfuryladenine-8-14C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N6-furfuryladenosine (frA) in the rRNA and tRNA preparations from the tobacco cafus. The rRNA contained over 90% of these fr6A moieties. The extent of kinetin incorporation was four times greater than that observed for N6-benzyladenine. The radiochemical purity of the recovered fr6A was confirmed by three successive chromatographic prifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 elated with water). A cytokinin-active ribonadeoside with elation volumes corresponding to frWA was isolated from the tobacco caflus rRNA preparation. This compound was analyzed by gasliquid chromatogrphy and rtorously chacterized as N6-furfuyladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.The occurrence, biosynthesis, and function of the cytokininactive ribonucleosides in tRNA have been examined in detail in Escherichia coli (9,14,16). N6-(A2-Isopentenyl)adenosine and ms2i6A3 have been identified as cytokinin-active ribonucleosides from E. coli tRNA. They have been found to be located next to the 3'-end of the anticodon of tRNA species responding to codons beginning with uridine. The cytokinin i6A is synthesized by the transfer of the isopentenyl moiety from A2-isopentenyl pyrophosphate to the specific adenosine residues in this location N6-furfuryladenosine or 6-furfurylamino-9-f>-D-ribofuranosylpurine; i6A: N6-(A2-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9-,D3--ribofuranosylpurine; ms2i6A: 6-(3-methyl-2-butenylamino)-2-methylthio-9-f3-D-ribofuranosylpurine; c-io6A: 6-(cis-4-hydroxy-3-methyl-2-butenylamino)-9-,-D-ribofuranosylpurine; t-io6A: 6-(trans-4-hydroxy-3-methyl-2-butenylamino)-9-,-E.nribofuranosylpurine; c-ms2io6A: 6-(cis-4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-/-E>-ribofuranosylpurine; t-ms2io6A: 6-(trans-4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-3-n-ribofuranosylpurine; CTAB: cetyltrimethylammonium bromide; TEAB: tetraethylammonium bromide; DEAE-cellulose: diethylaminoethyl-cellulose; 8HQ: 8-hydroxyquinoline; NSA: naphthalene-1,5-disulfonic acid disodium salt.in preformed tRNA molecules. Evidence that the cytokininactive components of tRNA function to insure proper codonanticodon interaction of the mRNA-tRNA complex on ribosomes has also been obtained in in vitro E. coli systems.The subsequent identification of cytokinins in tRNA preparations from various plant sources stimulated interest in the possibility that the hormonal regulation of plant growth and development by cytokinins might be due to their control of the synthesis and/or function of particular tRNA species (16). The exact location of the cytokinin moieties in nucleotide sequences of particular plant tRNA species...

Endogenous Cytokinins in the Ribosomal RNA of Higher Plants

“…Similarly, tRNA contamination could not be ruled out when weak cytokinin activity corresponding to io6A was found in tobacco callus rRNA grown in the presence of isopentenyladenine (16). Cytokinin activity has also been detected in fractionated hydrolysates of highly purified tobacco mosaic virus RNA (24).The incorporation of the cytokinins BA and kinetin into tobacco cell RNA has been found to occur preferentially into ribosomal RNA, that is, the concentration of incorporated cytokinin is three times greater in rRNA than in the corresponding tRNA (2,18,26). The physiological significance of such incorporation is not known, but these data renewed interest in the possible occurrence of endogenous cytokinins in plant rRNA.…”

“…The incorporation of the cytokinins BA and kinetin into tobacco cell RNA has been found to occur preferentially into ribosomal RNA, that is, the concentration of incorporated cytokinin is three times greater in rRNA than in the corresponding tRNA (2,18,26). The physiological significance of such incorporation is not known, but these data renewed interest in the possible occurrence of endogenous cytokinins in plant rRNA.…”

Distribution of Incorporated, Synthetic Cytokinins in Ribosomal RNA Preparations from Tobacco Callus

“…It has been shown that bzl6Ade is incorporated as the intact molecule into both the tRNA (18) and the rRNA (2), and both cytokinins have been recovered as the corresponding ribonucleosides to about three times greater extent per A2eo unit from the rRNA than from the tRNA preparations (2,14). It is not certain whether the observed incorporation of cytokinins is the result of specific events involved in the mechanism of cytokinin action or merely the result of transcriptional errors.…”

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ABSTRACTThe distribution of incorporated synthetic cytokinins (N6-jS-14CIbenzyl-adenine and Evidence has been presented that the synthetic cytokinins N6-benzyladenine (bzl6Ade)3 and N6-furfuryladenine (fr6Ade, kinetin) art incorporated into RNA of cytokinin-dependent tobacco callus tissue (2,14, 18). It has been shown that bzl6Ade is incorporated as the intact molecule into both the tRNA (18) and the rRNA (2), and both cytokinins have been recovered as the corresponding ribonucleosides to about three times greater extent per A2eo unit from the rRNA than from the tRNA preparations (2, 14).

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“…The distribution of fr6A moieties in the major rRNA components and in oligonucleotides obtained by partial enzymic digestion of the rRNA preparation has been examined. , and rRNA was isolated as described (2,14).…”