Ribosyl-cis-zeatin in a Leucyl Transfer RNA Species from Peas

Abstract

tRNAI-U in Pisum sativum seed has been purified. This tRNA species contains a cytokinin-active nucleoside and accounts for approximately 7% of the total cytokinin activity in acid hydrolysates of pea tRNA. Tle cytokinin has been identified as ribosyl-cis-zeatin, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-f3-D-ribofuranosylpurine.The modified nucleoside c-io6A3 was first tentatively identified by Hall et al. (7) in tRNA preparations from peas, corn, and spinach and has since been isolated from tRNAs of several other plants (6,12). It has also been reported, on the basis of bioassays, to occur in tRNA preparations of certain bacterial species;i.e. Corynebacterium fascians (8), a plant pathogen, and in species of Rhizobium (10), the bacterium associated with nitrogen-fixing nodules in legumes. The 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9-,8-D-ribofuranosylpurine, t-io6A, the isomer generally found in free form in plants, has also been isolated from Pisum tRNA (14).Gas chromatographic evidence for the presence of c-io6A in tRNA'U from peas has been reported (2). This paper reports the isolation of this tRNA species and the definitive identification of c-io6A as its cytokinin-active nucleoside constituent.MATERIALS AND METHODS Purification of Leucyl tRNA. Pea seeds (Pisum sativum cv. Alaska) that had been surface-sterilized and soaked for 48 hr in aerated, sterile distilled H20 were homogenized with 1 ml/g fresh weight of 0.1 M tris-HCl (pH 7.8), 0.1 mm EDTA, 0.2% SDS, and then this homogenate was extracted twice with equal volumes of phenol saturated with the same buffer. The final aqueous phase was adjusted to 0.1 M NaCl and the RNA was precipitated by addition of 2 volumes of cold 95% (v/v) ethanol. The RNA was then suspended in 0.02 M sodium acetate (pH 4.5), 1 mm EDTA, and applied to a column of DEAE-cellulose previously equilibrated with the same buffer. After washing the column with 5 bed volumes of buffer containing 0.3 M NaCl, the tRNA was eluted with 2.5 bed volumes of the same buffer containing 1 M NaCl.The tRNA was applied to a column of BD-cellulose (5) Bioassay of Cytokinin Activity. The fractionation of plant tRNA species containing c-io6A was monitored with the tobacco callus bioassay, as this compound is active as a cytokinin in the free form (11). The tRNA fractions were dissolved in 10 ml of distilled H20, acid-hydrolyzed, and incorporated in serial dilutions into nutrient medium (12). Cytokinin content is expressed either as kinetin equivalents (KE, where 1 KE is the growth response obtained on medium containing 1 ,ug/1 kinetin) or as ,umol of c-io6A calculated by comparison of the growth response with the response given by acid-hydrolyzed samples of this nucleoside tested in serial dilutions.Determination of Leucine Acceptor Activity. The leucyl tRNA synthetase was prepared from soaked pea seeds by the procedure of Scott and Morris (10) except that the chromatography step on Bio-Gel P-150 was omitted. Reaction mixtures for aminoacylation were incubated at 30 C and contained tR...

Distribution of Cytokinin-active Ribonucleosides in Wheat Germ tRNA Species

“…The latter is probably equivalent to tRNAsLu from peas, which has been shown to contain ribosylzeatin (8). The identification of these wheat germ tRNA species as cytokinin-containing species is tentative in the sense that complete purification was not achieved.…”

“…Information concerning the distribution of cytokinins with respect to particular plant tRNA species is much more limited. Ribosyl-cis-zeatin has been identified as a constituent of a minor tRNALu species from peas (8), but the distribution of cytokinin moieties in other tRNA species from this source was not examined.…”

Cytokinin-Active Ribonucleosides in Phaseolus RNA

“…The most detailed investigations of the distribution of cytokinin moieties with respect to individual tRNA species have been conducted with tRNA from microbial sources, but all available evidence concerning cytokinin distribution in tRNA from higher plant and animal tissues is consistent with this concept (2,6,12,(19)(20)(21). Not all tRNA species within the U group necessarily contain a cytokinin moiety.…”

“…tRNALu species from peas (2,6) and soybeans (12), but the other U-group tRNA species have not been examined in these plant materials. In Euglena (4,11) and Phaseolus vulgaris (8,9), the cytokinin 2-methylthio-N6-(A2-isopentenyl)adenosine has been identified as a constituent of the corresponding chloroplast tRNAPhe species.…”