Charles W. Parker

Regulators of cell division in plant tissues. XVI : Metabolism of zeatin by radish cotyledons and hypocotyls.

Summary[3H]Zeatin was supplied through the transpiration stream to radish (Raphanus sativus L.) seedlings with roots excised. Formation of dihydrozeatin was not detected but numerous other metabolites were formed, including adenine, adenosine, AMP, zeatin riboside and zeatin riboside-5′-monophosphate. However, in labelled seedlings which had been left in water for 15 h, an unknown compound (raphanatin) was the dominant metabolite and accounted for about 25% of the total radioactivity extracted. A procedure for the isolation of this metabolite was devised and yielded 70 μg from 1600 seedlings. Raphanatin was characterized by mass and ultraviolet spectra and has been identified as 7-glucosylzeatin. It is an active and very stable metabolite which was located mainly in the cotyledon laminae and may be a storage form of the hormone. In contrast, labelled nucleotides, the other major metabolites of zeatin, were largely confined to the hypocotyls and petioles. Zeatin riboside-5′-monophosphate was the dominant metabolite in hypocotyls of de-rooted seedlings supplied with zeatin for 0.5–2 h. The majority of the radioactivity in the xylem sap was due to zeatin, but about 10% was present as zeatin riboside; nucleotides accounted for less than 10% of the radioactivity and labelled raphanatin was not detected.

Regulators of cell division in plant tissues : XXV. Metabolism of zeatin by lupin seedlings.

Abstract[3H]zeatin was supplied through the transpiration stream to de-rooted lupin (Lupinus angustifolius L.) seedlings. The following previously known metabolites were identified chromatographically: 5′-phosphates of zeatin riboside and dihydrozeatin riboside, adenosine-5′-phosphate, zeatin riboside, zeatin-7-glucopyranoside, zeatin-9-glucopyranoside, adenine, adenosine and dihydrozeatin. Five new metabolites were purified; four of these contain an intact zeatin moiety. Two were identified unequivocally, one as l-β-[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]alanine, a metabolite now termed lupinic acid, and the second as O-β-d-glucopyranosylzeatin. These two compounds were the major metabolites formed when zeatin solution (100 μM) was supplied to the de-rooted seedlings. The radioactivity in the xylem sap of intact seedlings, supplied with [3H]zeatin via the roots, was largely due to zeatin, dihydrozeatin and zeatin riboside. When [3H]zeatin (5 μM) was supplied via the transpiration stream to de-rooted Lupinus luteus L. seedlings, the principal metabolite in the lamina was adenosine, while in the stem nucleotides of zeatin and adenine were the dominant metabolites. O-Glucosylzeatin and lupinic acid were also detected as metabolites. The level of the latter varied greatly in the tissues of the shoot, and was greatest in the lower region of the stem and in the expanding lamina. Minor metabolites also detected chromatographically were: (a) dihydrolupinic acid, (b) a partially characterized metabolite which appears to be a 9-substituted adenine (also formed in L. angustifolius), (c) glucosides of zeatin riboside and/or dihydrozeatin riboside, and (d) O-glucosyldihydrozeatin. While lupinic acid supplied exogenously to L. luteus leaves underwent little metabolism, chromatographic studies indicated that O-glucosylzeatin was converted to its riboside, the principal metabolite formed, and also to adenosine, zeatin and dihydrozeatin. A thinlayer chromatography procedure for separating zeatin, dihydrozeatin, zeatin riboside and dihydrozeatin riboside is described.

Regulators of cell division in plant tissues: XXIII. The identity of an unusual metabolite of 6-benzylaminopurine☆

When the cytokinin 6-benzylaminopurine was supplied to de-rooted radish seedings, the principal metabolites formed were the 7- and 9-glucosides. However the cytokinin activity of these glucosides was much less than that of a minor metabolite. This metabolite was purified (yield 550 mug from 40 600 seedings), identified as 6-benzylamino-3beta-D-glucopyranosylpurine and synthesized. It is the first compound with a glycosidic linkage at position 3 of a purine ring to be isolated from a plant tissue.

Inhibitors of two enzymes which metabolize cytokinins

Abstract Compounds which inhibit the natural metabolic inactivation of cytokinins are of considerable physiological significance. In this study, inhibitors have been found for two enzymes which form glucose and alanine conjugates of cytokinin bases, namely, cytokinin 7-glucosyltransferase and β-(9-cytokinin)alanine synthase. The most effective inhibitors found for the former enzyme were the cytokinin analogues 3-methyl-7-n-pentylaminopyrazolo[4,3-d]pyrimidine, which acted competitively (Ki, 22 μM), and the diaminopurine, 6-benzylamino-2-(2-hydroxyethylamino)-9-methylpurine (Ki, 3.3 μM). However these compounds were ineffective as inhibitors of the cytokinin-alanine synthase which was inhibited competitively by IAA (Ki 70 μM) and related compounds, especially 5,7-dichloro-IAA (Ki 0.4 μM). Certain urea derivatives were moderately effective inhibitors of the enzymes (Kica 100μM).

Preparation and characterization, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins.

Cytokinins can occur naturally as glycosides with beta-D-glucose as the sugar substituent. From radish (Raphanus sativus) cotyledons, an enzyme has been partly purified which synthesizes the 7-glucopyranoside of zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine], a compound known to occur in this species. High-performance reverse-phase liquid chromatography was uniquely useful as the analytical procedure for quantitative study of the minute amounts of enzyme available. The enzyme uses UDPglucose as the source of the sugar residue. A large number of derivatives of purine are glucosylated, but adenine derivatives with an alkyl side chain at least three carbon atoms in length at position N6 are preferentially glucosylated. This corresponds to the structural features required for high cytokinin activity. The 7-glucoside of zeatin is known to be very weakly active in cytokinin bioassays. Hence, this enzyme, and others catalyzing the same reaction, have a role in the regulation of cytokinin activity.

The complex of O-glucosylzeatin derivatives formed in Populus species☆

Abstract When zeatin was supplied to excised leaves of Populus alba , the principal metabolites formed were adenosine, O -β- d -glucopyranosyl- cis -zeatin (derived from cis -zeatin in the commercial zeatin used), O -β- d -glucopyranosylzeatin, and two new metabolites, namely, O -β- d -glucopyranosyldihydrozeatin and O -β- d -glycopyranosyl-9-β- d -ribofuranosyldihydrozeatin, the structures of which were confirmed by unambiguous synthesis. Chromatographic studies indicated that adenosine 5′-phosphate, zeatin 7-glucopyranoside, zeatin 9-glucopyranoside, dihydrozeatin and zeatin 9-riboside were minor metabolites. The principal metabolites of zeatin 9-riboside in P. nigra leaves were the new metabolites O -β- d -glucopyranosyl-9-β- d -ribofuranosylzeatin (synthesized chemically) and O -β- d -glucopyranosl-9-β- d -ribofuranosyldihydrozeatin.

The glucosylation of cytokinins

Summary When [3H]zeatin was supplied to the roots of Zea mays seedlings, the principal metabolite formed was identified as 9-glucosylzeatin. Other metabolites produced were adenosine 5′-phosphate, zeatin riboside 5′-phosphate, adenine, adenosine, and zeatin riboside. Chromatographic evidence indicated that 7-glucosylzeatin formed as a minor metabolite in Zea mays roots and in de-rooted Zea mays seedlings. [3H]6-Benzylaminopurine was converted by de-rooted radish seedlings to both the 7-glucoside and the 9-glucoside. These two glucosides accounted for about 90% of the radioactivity in the form of extractable metabolites.

Raphanatin, an unusual purine derivative and a metabolite of zeatin

Abstract When zeatin was supplied to radish seedlings with roots excised, a number of metabolites were formed. These included adenine, adenosine, adenosine 5′-phosphate, zeatin riboside and zeatin riboside 5′-phosphate. However the major metabolite was a glucoside of zeatin which differed from 9-β-D-glucopyranosylzeatin. This metabolite termed raphanatin, which is active as a cytokinin, occurred mainly in the cotyledons of the seedlings.

The occurrence of raphanatin as an endogenous cytokinin in radish seed Identification and quantitation by gas chromatographic—mass spectrometric analysis using deuterium-labelled standards

When the phytohormone zeatin IIa [6-(4-hydroxy3-methylbut-trans-2-enylamino)purine] is supplied exogenously to plant tissues, it is converted to a diversity of metabolites including a number of glucosides, namely, 7Q-D-glucopyranosylzeatin (Ia, termed raphanatin), 9

Glucosylation of cytokinin analogues

-D-glucopyranosylzeatin IVa, 0/3-D-glucopyranosylzeatin and its riboside, and 0(3-D-glucopyranosyldihydrozeatin and its riboside [l-3] . The 7-glucoside Ia is of particular interest because of its very unusual structure. The only known endogenous purines in which a sugar (ribose) is linked to the 7 position are analogs of vitamin Blz [4] . In this communication the occurrence of raphanatin as an endogenous cytokinin is established and quantitated using [‘H2]raphanatin Ib as an internal standard.