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

Abstract

The cytokinin, N6-(A2-isopentenyl)adenine, is found to be at least 3.3 times as active as N6-(A2-isopentenyl)adenosine in promoting the growth of cytokinin-requiring tobacco (Nicotiana tabacum) callus. Absorption rates of N6-(A2-isopentenyl)adenine and N6-(A2-isopentenyl)adenosine by tobacco cells in liquid suspension do not differ significantly. In these cells, N6-(A2-isopentenyl)adenosine-5'-monophosphate, di-, and triphosphate are synthesized in both cases, but 7-glucosylation occurs significantly only with N6-(A2-isopentenyl)adenine, protecting thereby its N6-isopentenyl side chain from cleavage. Degradation by N6-side chain removal appears to be intense, leading to the formation of adenine, adenosine, and adenylic nucleotides. Thus, it is suggested that N6-(A2-isopentenyl)adenine-7-glucoside is a protected or storage form of the cytokinin which could account for the higher biological activity of N6-(A2-isopentenyl)adenine than of N6-(A2-_sopentenyl)adenosine.In the last years, metabolism of exogenously supplied cytokinins has been studied in various plant tissues and plant cells. Since the reports of the formation of the unusual 7-glucosylbenzyladenine in various plant tissues (1) and 7-glucosylzeatin in radish cotyledons (19) as major and highly active (4, 18) cytokinin metabolites of the supplied cytokinin, attention has been focussed on cytokinin glucosides (7,10,13,20,26).However, cytokinin glucosides are not the only cytokinin metabolites synthesized from exogenously supplied cytokinin that keep the intact cytokinin base moiety. In most of the studies cited above and in other studies (2, 24), cytokinin riboside and cytokinin riboside-5'-monophosphate were also found to be synthesized. In a previous communication (12), we established the formation of cytokinin riboside-5'-di and triphosphates in tobacco and Acer cells.So far, the only correlation that could be established between cytokinin metabolism and cytokinin activity were based on the cytokinin-requiring growth of plant tissues grown as callus and cytokinin metabolites that were present in this callus. Fox et al.(5) found that 7-glucosylbenzyladenine was the only cytokinin molecule that persisted during the active growth of cytokininrequiring soybean callus and tobacco KX4 callus, suggesting that 7-glucosylbenzyladenine could be the "physiologically active form of the cytokinin" (4). Horgan (7) showed that 7-glucosylzeatin was not formed in cytokinin-requiring soybean callus, 6(4-0-,8-D-glucosyl-3-methyltrans-2-butenylamino) purine being the major metabolite present 24 hr after the zeatin had been sup-

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

“…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. One of the major metabolites formed from a cytokinin ribonucleoside has been reported to be its corresponding base (9,11,13). In the de novo biosynthesis of cytokinins using a crude enzyme system prepared from plant cells, a cytokinin nucleotide, nucleoside, and base were formed from 5'-AMP and A2-isopentenylpyrophosphate (5).…”

Isolation and characterization of a new glucopyranosyl derivative of 6‐(3‐methyl‐2‐butenylamino)purine from sweet potato tubers

“…In [16,17] i6Ade was converted to the 7-glucosyl derivative in tobacco-cell suspension cultures; it was suggested that cleavage of the isopentenyl side chain was protected by the presence of the glycosidic linkage. The new 9-glucosyl derivative exhibited cytokinin activity in the Amaranths bioassay, although the activity was -1%0 of that of N6-benzyladenine on a molar basis.…”

“…It can be readily transported in plant tissue, as shown in [ 181 using synthetic 6-benzylamino-9glucofuranosyl purine. This glucosyl cytokinin can also serve as a storage form which can be activated when needed [ 17,19,20].…”

Metabolism of Cytokinin

“…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).…”

“…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).…”