Kinetin Inhibition of ³H-Uracil and ¹⁴C-Leucine Incorporation by Tobacco Cells in Suspension Culture

Abstract

The rate of "4C-leucine and 3H-uracil incorporation by tobacco cells (Nicotiana tabaccum var. Samsun N.N.) in suspension culture was simultaneously decreased by the addition of kinetin at concentrations above 2.5 X 10-5 M.Ribosomal RNA was the first RNA species affected by kinetin. The purine derivatives, adenine and N6-methylaminopurine, which exhibit low cytokinin activity overcame the inhibitory effects of kinetin. However, purine derivatives without cytokinin activity, guanine, N6. -dimethylaminopurine, and 2-aminopurine, did not relieve kinetin inhibition.Since the early studies of cytokinins, it has been shown that in addition to their stimulatory effect on the growth of plant tissues they exhibited some inhibitory effects. Skoog and Miller (20) have shown that tobacco stem segments which were plated on medium containing 0.5 mg/l of kinetin formed only few buds, and that these buds were retarded in their growth. The growth of the basal callus was also inhibited by kinetin. Other reports demonstrate the inhibitory effect of kinetin on buds, roots, and shoot formation in intact plant and plant tissues (4,5,8,23).In general, substances with cytokinin activity stimulated the growth of tobacco callus tissue, but high concentrations of them inhibited the growth of the same tissue (10,19). Cytokinins stimulate the synthesis of RNA and protein in plant tissue (16,21,22), in isolated nuclei (3, 17), and isolated mitochondria (1). In this paper we studied the kinetics of the inhibition by high concentration of kinetin, of 3H-uracil, and '4C-leucine incorporation by tobacco cells in suspension culture. A preliminary report has been published elsewhere (14 3H-Uracil and 14C-Leucine Incorporation. The rate of incorporation of 3H-uracil and '4C-leucine was determined in suspensions of single cells and very small cell clusters. These suspensions were obtained by precipitating the large clusters and collecting the supernatant of the stock cultures. Unless otherwise mentioned in the text, radioactive precursors, hormones, or drugs were added to the suspensions at zero time. After varying periods of time, aliquots of the suspensions were transferred into trichloroacetic acid (10% final concentration) for 20 min. The cells were precipitated on Whatman fiberglass filters and washed with 5% trichloroacetic acid. Radioactivity was measured in a Packard Tricarb liquid scintillation spectrometer. Most of the 3H-and '4C radioactivity could be removed from the filters by treatment with ribonuclease A (Worthington) and pronase (Calbiochem), respectively.Labeling ofRNA with 32P, Extraction and Separation on Methylated Albumin Kieselguhr Columns. Four days prior to the labeling experiments, the cultures were transferred into a phosphate-poor medium (one-eighth of its concentration in the growth medium). Following the incubation of the cultures with 1 mc of carrier-free 32PO43-(Amersham) the tissue (10 g fresh wt) was collected on a precooled Buchner funnel and washed with an excess of ice cold water. The tissue was then froze...

Generation and Suppression of Microsomal Ribonuclease Activity after Treatments with Auxin and Cytokinin

“…plant tissues, but effects are often opposite in different species or growth situations and they do not bear any consistent relationship to total RNA levels. It has been suggested (19,27) that the ability of auxins and cytokinins to stimulate or to inhibit RNA metabolism depends in part on relative hormone concentrations. Another source of confusion may be the fact that, in most studies to date where RNase activity has been studied during plant development, only total levels are measured or, in cases where tissue is fractionated, only one fraction is assayed.…”

Cytokinin Effects on Growth of Quiescent Tobacco Pith Cells

“…When tested against the kinetin-requiring Wisconsin 38, adenine showed no effect at inhibitory kinetin concentrations. Thus, the earlier observations on adenine reversal (15) appear to relate solely to precursor incorporation, and not to the kinetin effects on cell growth.…”

“…Adenine. Nudel and Bamberger (15) showed that addition of growth-inhibitory levels of kinetin to kinetin-requiring tobacco cells (see Fig. la) is accompanied by a large decrease in the incorporation of radioactive precursors into protein and RNA and that the decreased incorporation could be reversed by adenine.…”

Respiratory Deficient Mutants Induced in Baker's Yeast by the Action of Acridine Orange and some Plant Growth Hormones طفرات نقص التنفس المستحدثة فی خمیرة الخباز کنتیجة للمعاملة بالأکریدین وهرمونات النمو النباتیة

“…These results agreed with Murai et al (1977), who found a close relationship between DNA and RNA biosynthesis and the ability of cells to continue growing. In addition, Nudel and Bamberger (1971) reported that kinetin inhibits incorporation of uracil by tobacco cells suspension culture. Furthermore, Barciszewski et al (1992) showed that radioactive kinetin was incorporated sitespecifically into prokaryotic tRNAs.…”