N⁶-(Δ²-Isopentenyl)adenosine: Its Occurrence as a Free Nucleoside in an Autonomous Strain of Tobacco Tissue

Abstract

Cytokinins from both the free nucleoside pool and the transfer RNA have been isolated and identified in a habituated strain of tobacco pith callus (Nicotiana tabacum [L] var. Wisconsin 38). The transfer RNA of this strain contains both N0-(A2-isopentenyl) adenosine and N'-(4-hydroxy-3-methylbut-2-cis-enyl) adenosine. The trans-hydroxylated derivative is absent from the transfer RNA of this dark-grown tissue. N'-(A'-Isopentenyl)- Hall [14]). This hydroxylated derivative appears to be restricted to the tRNA of higher plants. In addition, the 2-methylthio derivatives of both the parent compound (z.2-iA) and the hydroxylated derivative have been isolated from the tRNA of wheat germ (6).The trans-isomer of ribosylzeatin (trans-RZ) and/or its free base and nucleotide have been found as free compounds in chicory roots (5) (8) showed that the A2-iA of the tRNA of this tissue is synthesized by attachment of a A2-isopentenyl side chain to the preformed tRNA. The requirement for the exogenous cytokinin does not seem to relate directly to this biosynthetic mechanism. In search, therefore, of leads that bear on the mechanism of action of cytokinins we decided to compare the A2-iA content of an autonomous line derived from the Wisconsin 38 strain of tobacco pith callus tissue (11) that grows well in the absence of exogenously added cytokinins. We have found that the tRNA of this strain also contains A2-iA and cis-RZ in its tRNA, but in contrast to the cytokinin-requiring strain the autonomous tissue contains a significant level of A2-iA in the free state. MATERIALS AND METHODSTissue Culture Strains. Normal tobacco pith tissue has an absolute requirement of added cytokinins for growth (11). Occasionally this normal tissue spontaneously loses its cytokinin requirement. Such autonomous strains of tissue presumably meet their cytokinin requirement through an alteration in some metabolic process, although the mechanism(s) of this transformation is not known.The normal cytokinin dependent tobacco pith tissue (Nicotiana tabacum [L] var. Wisconsin 38) and the derived autonomous strain used in this study were gifts from J. E. Fox of the University of Kansas. The normal pith tissue was grown on a previously described (11) basal medium containing naphthaleneacetic acid (1 mg per liter) and 6-furfurylaminopurine (kinetin) (1 mg per liter). The autonomous strain was grown on the same basal medium without the growth factors. Tissues were harvested after 4 to 5 weeks growth in the dark.Bioassay. Aliquots of column fractions to be bioassayed for cytokinin activity were added to 25-ml flasks and dried in vacuo over P20,. A basal medium (11), complete except for cytokinin, was added (10 ml per flask), and the flasks were autoclaved at 18 lb, 121 C for 5 min. A cytokinin requiring strain of soybean cotyledon tissue (Glycine max [L.] Merril, var. Acme) was planted (0.5 mg pieces, three per flask), and after 30 days incubation in the dark at room temperature, the increase in fresh weight was compared to controls grown in the presence (...

Biosynthesis of Cytokinins in Cytokinin-Autotrophic Tobacco Callus

“…Both cytokinins have been reported earlier as constituents of tRNA in cytokinin-dependent (4) and cytokinin-autotrophic (5) tobacco callus. N6-A2-isopentenyladenosine was not found but has been reported for another cytokinin-autotrophic strain of tobacco callus (6). The presence of ribosylzeatin was suggested by biological activity in the region corresponding to the elution of the authentic compound but could not be confirmed in the radioactivity profiles.…”

Cytokinins: Metabolism and Biological Activity of N6-(Δ2-Isopentenyl)adenosine and N6-(Δ2-Isopentenyl)adenine in Tobacco Cells and Callus

“…Uptake parameters are not readily accessible with callus grown on agar medium. We studied uptake parameters with tobacco 3 Our first report indicated the presence of inosine in this fraction after treatment with calf intestinal alkaline phosphatase (from BoehringerMannheim). In fact, this preparation contained some adenosine-deaminase activity as a contaminant.…”

“…The aim of the present study was to ascertain the metabolic patterns and some quantitative aspects of the utilization of the naturally occurring cytokinin N6-(A2-isopentenyl)adenosine (3) and of N6-(A2-isopentenyl)adenine in cytokinin-requiring and nonrequiring tobacco cells grown as cell suspensions in liquid medium. This study seemed relevant for three reasons.…”

Habituation of Tobacco Pith Cells for Factors Promoting Cell Division Is Heritable and Potentially Reversible

“…Habituated tissues maintained in culture produce significant amounts of the growth factor for which they are habituated (10)(11)(12)(13)(14), indicating that habituation results from persistent biosynthesis of specific growth factors. The fact that intact plants produce auxin and cell-division factors suggests that habituation is due to the heritable expression of genes normally inactive in cultured cells.…”