Cytokinins: Formation of the nucleoside‐5′‐triphosphate in tobacco and Acer cells

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...

Section: <>mentioning

confidence: 99%

Kinetin Incorporated into Tobacco Callus Ribosomal RNA and Transfer RNA Preparations

Murai¹,

Taller²,

Armstrong³

et al. 1977

“…The conversion of bzl'Ade and the naturally occurring cytokinin i'A to the corresponding nucleoside triphosphates by suspension cultures of tobacco and Acer pseudoplatanus has been reported recently (13), and may provide a pathway for the incorporation of bzl6Ade into RNA. Evidence from earlier studies (17,26) suggests that the formation of nucleoside triphosphate derivatives of cytokinins may be a normal feature of cytokinin metabolism in plant tissues.…”

Section: Discussionmentioning

confidence: 95%

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

Armstrong¹,

Murai²,

Taller³

et al. 1976

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...

“…The biological functions of zeatin metabolites (such as 0-and N-glucosides [4,5,8,24], lupinic acid [11,21,25], glucosylribosylzeatin [29], nucleoside and nucleotides [10,28] (16). P. vulgaris x P. acutifolius crosses result in embryos which develop to the cotyledonary stage but also do not reach maturity.…”

Section: Discussionmentioning

confidence: 99%

Cytokinin Metabolism in Phaseolus Embryos

Lee¹,

Mok²,

Mok³

et al. 1985

The metabolism of trans-18-'4Cjzeatin was examined in embryos of Phaseolus vulgaris cv Great Northern (GN) and P. lunatus cv Kingston (K) in an attempt to detect genetic variations in organized plant tissues. Metabolites were fractionated by HPLC, and identified by chemical and enzymic tests Metabolism of '4CiZeatin. Immature embryos at three developmental stages (measuring 3, 6, and 9 mm in length) were dissected from the pods under sterile condition. The embryos corresponded to late heart and early and mid cotyledonary stages at the respective length. ['4C]Zeatin (0.05 MCi, 0.002 umol) dissolved in 250 ,d of H20 was applied aseptically to 250 mg of immature embryos. The vials were sealed and maintained at 27°C in the dark for 2, 4, and 8 h. In addition, embryos were incubated with radioactively labeled zeatin dissolved in 0.05 M Tris-HCI (pH 6.0) buffer with all other conditions identical to those described above. To determine the amount of radioactivity recovered at time 0, ['4C]zeatin was applied and metabolites extracted immediately. This determination was made for the three sizes of embryos of both genotypes. Each experiment was repeated at least once and averages of two experiments are presented.To extract metabolites, the embryos were homogenized with a Tissuemizer equipped with a Microprobe Shaft (Tekmar) in 2.5 parts (v/w) of cold 95% ethanol. Cell debris was removed by successive filtration through Whatman paper No. 1 and Millipore filters (0.45 ,um). The ethanol extract was taken to dryness in vacuo at 35°C, redissolved in 4 ml of 50% (v/v) ethanol, and centrifuged at 23,500g for 20 min. The supernatant was condensed in vacuo to 100 Ml with a speed vac concentrator (Savant). The sample was then analyzed directly by HPLC. A Beckman Model 110 dual pump HPLC system with a prepacked column of reversed-phase C18 (Ultrasphere ODS 5 gm, 4.6 x 250 mm; Altex) was used. The aqueous buffer consisted of 0.2 M acetic acid, adjusted to pH 3.5 with TEA. Samples were eluted with a 635 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from