Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobhum Japoncum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Meff.), was grown in defined medium.Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found.Total cytokinin activity In the B. Japoncum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. Japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.Cytokinin production in several plant-associated microbes has been well characterized (13,14,23,33). These include the gall-forming phytopathogenic bacteria, Agrobacterium tumefaciens and Pseudomonas savastanoi, and the causative agent of witches broom, Corynebacterium fascians. In all of these organisms, the disease symptoms are correlated with cytokinin production (20,22,23). Almost thirty years ago, Arora et al. (3) suggested that the action of rhizobia in nodule formation might likely involve the supply or activation of several different growth factors. Despite considerable evidence for the production of cytokinins by Rhizobium and for the involvement of cytokinins in legume nodulation, neither the nature of cytokinin production by Rhizobium nor the role of these cytokinins in the Rhizobium-legume symbiosis has been determined. 'A portion of this research was presented in preliminary form at the annual meeting of the American Society of Plant Physiologists, St. Louis, 1987. There are reports that at least some strains of Rhizobium produce cytokinins in culture, though they have not been quantitated or rigorously identified. Three cytokinin-active compounds were detected in the culture filtrate ofRhizobium leguminosarum, one of which cochromatographed with i6Ade2 (9). Phillips and Torrey (27) also reported cytokininlike...

Azotobacter vinelandii OP was grown to stationary phase in defined medium. The cell-free culture medium was analyzed for cytokinin content by XAD-2 and Sephadex LH-20 chromatography, thin-layer chromatography, tobacco callus bioassay, and enzyme immunoassay. Three cytokinin-active fractions were detected and tentatively identified as trans-zeatin, isopentenyladenosine, and isopentenyladenine. The total cytokinin activity was equivalent to 0.75 jig of kinetin per liter.

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...

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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...