Endogenous cytokinin-active ribonucleosides were isolated from the rRNA and tRNA of pea epicotyls (Pisum sativum L., var Alaska) and of wheat germ (Triticum aestivum). The RNA preparations were analyzed for cytokinins by enzymic hydrolysis, ethyl acetate extraction, and Sephadex LH-20 fractionation in several solvents. Tentative identification of the cytokinins was based on cochromatography with synthetic cytokinin standards in several systems and on activity in the tobacco bioassay. Both the rRNA and tRNA from 10 day old pea epicotyls contained ribosylzeatin, isopentenyladenosine, and 2-methylthioribosylzeatin. The latter compound was the most active fraction in the pea rRNA, but was the least active fraction in the tRNA, where isopentenyladenosine activity was predominant. The 2-methylthioribosylzeatin from pea rRNA was identified by gas chromatography-mass spectrometry. Wheat germ rRNA contained cis and trans ribosylzeatin and 2-methylthioribosylzeatin. The tRNA contained isopentenyladenosine in addition. The specific cytokinin activity (activity per A(260) unit) of the tRNA was over forty times that of the rRNA. Significant contamination of the rRNA preparations by cytokinin-containing tRNA is considered unlikely on the basis of quantitative differences in the cytokinin content of the rRNA and tRNA preparations, electrophoretic analysis of rRNA purity and cytokinin analysis of fractionated oligonucleotide digests.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

Northern blot hybridization analysis of polysomal polyadenylated RNA isolated from sunflower crown gall tumor PSCG-15955 demonstrated that a minimum of seven RNAs were transcribed from T-DNA of pTi-15955 from Agrobacterium tumefaciens. The sizes of the T-DNA transcripts were 1.8, 1.6, 1.5, 1.1, 1.0 kilo bases (kb) and two transcripts of 0.8 kb long. The relative abundance of these polyadenylated RNAs varied greatly, the 1.0 kb RNA being the most abundant and the 1.6 kb RNA being the least abundant. Assignment of map locations of the seven polyadenylated RNAs indicated that the conserved region of T-DNA which may play a central role in tumorigenesis contained four RNAs of 1.8, 1.1, 0.8(a) and a portion of 0.8(b) kb long.

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of agrobacterium tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of M r 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five 32 P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the M r 40,000 polypeptide and was not detected in the habituated callus.

Corynebacterium fascians causes fasciation disease or witches’ broom in dicotyledonous plants. The disease is characterized by release of apical dominance and outgrowth of lateral buds, thus giving rise to the characteristic witches’ broom syndrome (Roussaux, 1965). The symptoms of the disease can be duplicated by treatment of seedlings with cytokinins, indicating that the disease may be caused by cytokinins produced by the microbe (Thimann and Sachs, 1966). This work deals with the kinds and quantities of cytokinins released into culture media and present in tRNA of five strains of C. fascians, ranging from highly virulent MW2, moderately virulent Cf2 and Cfl, weakly virulent Cf 15 to avirulent Cfl6. It examines the relationships of the presence of plasmids to cytokinin production and to pathogenicity. It also reports the isolation from highly virulent MW2 of activity of cytokinin synthase that catalyzes the formation of N6-(Δ2-isopentenyl) adenosine-5′-monophosphate (i6 AMP) from 5′-AMP and Δ2-IPP.

Cytokinin activity in the culture medium of four pathogenic strains of Corynebacterium fascians varied from 168 to 0.4 mug of kinetin (6-furfurylaminopurine) equivalents per liter, as compared to 0.2 in an avirulent control. N(6)-Isopentenyladenine was the predominant cytokinin in the medium of all five strains, and its increased production was correlated with the degree of pathogenicity; however, the virulent strains also produced 8-13 times more cis-zeatin [6-(4-hydroxy-3-methyl-cis-2-butenylamino)purine] than the avirulent strain. The three most virulent strains (with the higher cytokinin contents in the medium) contained a large (M(r) approximately 10(8)) plasmid. The barely virulent strain contained a smaller plasmid. No plasmid was detected in the avirulent control. The total cytokinin content (mole%) and biological activity (mug of kinetin equivalent per mg of tRNA) in tRNA were about the same in all three virulent strains and in the avirulent control. Five cytokinins were isolated from each strain. Four were rigorously characterized as 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-D-ribofuranosylpurin e, 6-(3-methyl-2-butenylamino)-9-beta-D-ribofuranosylpurine, and their 2-methylthio derivatives. The fifth cytokinin was tentatively identified as 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-beta-D-ribofuranosylpur ine. Ribosyl-cis-zeatin was 3-fold higher and N(6)-isopentenyladenosine was correspondingly lower in the plasmid-containing strains than in the plasmidless control. Because the entire syndrome of fasciation caused by infection can be induced with synthetic cytokinins, the disease would appear to be caused by plasmid-induced high rates of cytokinin production by the bacteria.

The distribution of incorporated synthetic cytokinins (N(6)-[8-(14)C]benzyladenine ([8-(14)C]bzl(6)Ade) and N(6)[8-(14)C]furfuryladenine ([8-(14)C]fr(6)Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-(14)C]bzl(6)Ade or [8-(14)C]fr(6)Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-(14)C]bzl(6)A or [8-(14)C]fr(6)A in the rRNA peak after the fractionations. [8-(14)C]fr(6)A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.The distribution of [8-(14)C]fr(6)A moieties in mono- and oligonucleotides derived from combined treatment of [8-(14)C]fr(6)Ade-labeled rRNA with ribonuclease T(1) and pancreatic ribonuclease A was measured by fractionating the digest on a DEAE-cellulose column in the presence of 7 m urea and determining the [8-(14)C]fr(6)A content in each fraction. The [8-(14)C]fr(6)A content in the oligonucleotides varied from 46 to 210 mumol/mol of adenosine (A). The mol ratio of [8-(14)C]fr(6)A to A was three to four times greater for oligonucleotides containing uridine or cytidine ([A](n)Up or [A](n)Cp) than for those containing quanosine ([A](n)Gp).

Kinetin, N(6)-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N(6)-furfuryladenine-8-(14)C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N(6)-furfuryladenosine (fr(6)A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr(6) A moieties. The extent of kinetin incorporation was four times greater than that observed for N(6)-benzyladenine. The radiochemical purity of the recovered fr(6) A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr(6) A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N(6)-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.

The incorporation of the cytokinin N(6)-benzyladenine into tobacco (Nicotiana tabacum) callus tRNA and rRNA preparations isolated from tissue grown on medium containing either N(6)-benzyladenine-8-(14)C or N(6)-benzyladenine-8-(14)C: benzene-(3)H(G) has been examined. N(6)-benzyladenine was incorporated into both the tRNA and rRNA preparations as the intact base. Over 90% of the radioactive N(6)-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 N(6)-benzyladenosine content of the RNA preparation. The distribution of N(6)-benzyladenosine moieties in tobacco callus tRNA fractionated by BD-cellulose chromatography did not correspond to the distribution of ribosylzeatin activity. N(6)-benzyladenosine was released from the rRNA preparation by treatment with venom phosphodiesterase and phosphatase, ribonuclease T(2) and phosphatase, or ribonuclease T(2) and a 3'-nucleotidase. N(6)-benzyladenosine was not released from the RNA preparation by treatment with either ribonuclease T(2) or phosphatase alone or by successive treatment with ribonuclease T(2) and a 5'-nucleotidase. Brief treatment of the rRNA preparation with ribonuclease T(1) and pancreatic ribonuclease converted the N(6)-benzyladenosine moieties into an ethyl alcohol soluble form. On the basis of these and earlier results, the N(6)-benzyladenosine recovered from the tobacco callus RNA preparations appears to be present as a constituent of RNA and not as a nonpolynucleotide contaminant.

The incorporation of (14)C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of (14)C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the (14)C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.