Cytokinin Biosynthesis in Cultured Rootless Tobacco Plants

Abstract

Biosynthesis of cytokinin in shoots was examined by growling rootless tobacco (Nicodans tabacum) plants in vitro. The rootless plants were originated by culturing tobacco callus on a high cytokinin-low auxin medium to induce the formation of plantlets which were then grown on medium without exogenous cytokinin and auxin. The rootless plants supplied with I14Cladenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N6-(A2-isopentenyl)adenine, N"-(A2-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-@-D-ribofuranosylpurine. The total amount of these four major cytokinins was estimated to be present at a concentration of 14 to 23 nanomoles per kilogram of rootless plant. These data indicate that adenine serves as a precursor of the purine moiety of cytokinin molecules and that the cytokinin biosynthetic sites are also located in the shoot in addition to the presumed root sites.i6Ade,2 io6Ade, and closely related derivatives constitute a group of plant hormones, the cytokinins, which promote cell division and differentiation. Precursor of the purine moiety of cytokinins has been shown to be Ade (3, 7). Although there is evidence that cytokinin does exist in the root tip, either free (14) or as constituents of tRNA (1,9,14), and that cytokinins from xylem exudate may come from the roots (11, 15), the question of whether the intact root tips and/or other plant parts serve as a primary site of cytokinin biosynthesis remains to be answered.Information about the biosynthesis of cytokinins in roots comes from indirect evidence based on collection of materials by diffusion and extraction together with the bioassay of substances from root exudates. This evidence has been reviewed recently in some detail by Torrey (17). 2 Abbreviations: Ade: adenine; i6Ade: N6-(A2-isopentenyl)adenine; i6Ado: N6-(A&2-isopenten6yl)adenosine; io6Ade: 6-(4-hydroxy-3-methyl-2-butenylamino)purine; io Ado: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-/?-ribofuranosylpurine. Chromatograms were cut into 1-cm sections and placed in vials containing scintillation fluid (4). Radioactivity was measured in a Nuclear-Chicago Unilux II scintillation system. For liquid samples, an aliquot no more than 0.5 ml was added to 10 ml of Bray's solution (2). Counting efficiency of 14C samples was 74% for paper chromatogram sections and 90%7o for liquid samples.A Cary model 14 spectrophotometer was used to measure the quantity of purine bases and cytokinins.Histology. A modified procedure of Johansen (10) was used in histological examination of the rootless plants. The plants harvested from the culture media were rinsed gently in distilled H20 to remove adherent medium from the stem bases. Stem bases were cut into 1-cm lengths which included any development in the base area except for leaves. The stem bases were dropped immediately into a fixative containing 100 ml of 50%1o ethanol, 50 ml of formalin,...

Metabolism of Cytokinin: Deribosylation of Cytokinin Ribonucleoside by Adenosine Nucleosidase from Wheat Germ Cells

“…Km values for the cytokinin nucleoside and esine are 238 and 1.43 micromolar, respectively, in 50 milHolar Tris-citrate buffer (pH 4.7) at 30 C. The presence of adenosine and other cytokinn nucleosides inlhbited the hydrolysis of N64A'.isopentenyl)adenosine but this reaction wa insensitive to guanosine, uridine, or 3'deoxy oine. It is hypothesized that an adequate level of "active cytokinin" in plant cells may be provided through the deribosylation of cytokinin riboside in concert with other cytokinin metabolic enzymes.Cytokinin bases and cytokinin ribonucleosides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (3,4,9,11,16,18). The relative amount of a specific metabolite formed may differ not only from one plant to another, but also in one particular plant or tissue under different physiological conditions.…”

“…Cytokinin bases and cytokinin ribonucleosides have been found in various plant cells, and these cytokinins are metabolized in the plant cell to form different metabolites (3,4,9,11,16,18). The relative amount of a specific metabolite formed may differ not only from one plant to another, but also in one particular plant or tissue under different physiological conditions.…”

Metabolism of Cytokinin

“…The occurrence of cytokinin nrbonucleotide and cytokinin ribonucleoside in plant cells is well documented (2,4,5,10,11).…”

“…Phenylphosphate and 32-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5'-nucleotidases may play an important role in regulating levels of "active cytokinin" in plant cells.The occurrence of cytokinin nrbonucleotide and cytokinin ribonucleoside in plant cells is well documented (2,4,5,10,11).The cytokinin ribonucleotide can be formed from cytokinin base (3,11,18,20), the ribonucleoside (3,7,17,18) or turnover of cytokinin-containing tRNA (13,22). Alternatively, the ribonucleotide i6Ado-5'-P2 can be synthesized by a de novo pathway from the simple metabolites AMP and A2-isopentenylpyrophosphate (6, 24).…”

Competência organogênica in vitro das linhagens MT-Rg1 e MT-pro em tomateiro (Solanum lycopersicum L. cv Micro-Tom)

“…As citocininas são sintetizadas principalmente nas raízes, de onde são transportadas para o caule pelo xilema, porém, também são produzidas em embriões em desenvolvimento, folhas jovens, ápices caulinares e nos tecidos da galha da coroa (AKIYOSHI et al, 1984;CHEN;PETSCHOW, 1978;SCHMÜLLING, 2004;VAN STADEN;SMITH, 1978). A atuação deste hormônio pode ocorrer nas próprias células em que ele é produzido, nas células próximas ou a longas distâncias dos centros de produção (SCHMÜLLING, 2004 (AKIYOSHI et al, 1984;BARRY et al, 1984).…”