D. S. Letham

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34cdc2-like protein, recoverable in the p13suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.

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.

Cytokinins from Zea mays

Abstract A number of adenine derivatives with cytokinin activity were isolated from immature sweet corn (Zea mays) kernels. The following structures were assigned: 9-β- d -ribofuranosylzeatin, 9-β- d -ribofuranosylzeatin 5′-monophosphate, 6-(1-carboxy-2-hydroxypropylamino)-9-ribofuranosylpurine, 6-(2,3,4-trihydroxy-3-methylbutylamino)purine, 2-hydroxy-6-(4-hydroxy-3-methylbut- trans -2-enylamino)purine, 6-(3,4-dihydroxy-3-methylbutylamino)purine, a 9-glycoside of zeatin(identity of sugar moiety not established), and 6-(1,2-dicarboxyethylamino)-9-β- d -ribofuranosylpurine.

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene.

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.

Regulators of Cell Division in Plant Tissues XXIX.* The Activities of Cytokinin Glucosides and Alanine Conjugates in Cytokinin Bioassays

In a number of cytokinin bioassays, the activities of the following compounds were compared: 3-, 7-, and 9-glucosides of 6-benzylaminopurine (BAP); 7- and 9-glucosides of zeatin; O-glucosides of zeatin, dihydrozeatin, and their ribosides; 9-alanine conjugates of zeatin, and BAP. The bioassays included the radish cotyledon, theAmaranthus betacyanin, the oat leaf senescence, and the tobacco pith callus. Cytokinin activity was markedly reduced by 7- and 9-glucosylation in nearly all bioassays, but 3-glucosylation of BAP and O-glucosylation of the zeatin sidechain usually had little effect on activity. However, there were two notable exceptions to this generalization: the activity of O-glucosylzeatin markedly exceeded that of zeatin in the oat leaf senescence assay; 9-glucosyl-BAP and free BAP were similarly active in retarding the senescence of radish leaf discs. The 9-alanine conjugate of zeatin (lupinic acid) and of BAP were markedly less active than zeatin and BAP, respectively, in all bioassays, but the responses evoked by these conjugates at high concentrations in theAmaranthus bioassay approached those caused by the corresponding base. The activities of several new compounds related to the alanine conjugate of BAP were also assessed. To serve as a guide in the selection of the most suitable bioassay for detection of the above-mentioned cytokinin conjugates, the lowest detectable amounts in selected bioassays have been compared.

Detection of cytokinins in a seaweed extract

Abstract trans-Zeatin, trans-zeatin riboside, their dihydro derivatives, isopentenyladenine and isopentenyladenosine have been identified and quantified in Seasol, a commercial extract of Tasmanian Giant Bull kelp, Durvillea potatorum.

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).

Phytohormones,Rhizobium mutants, and nodulation in legumes. IV. Auxin metabolites in pea root nodules

High specific activity [3H]indole-3-acetic acid (IAA) was applied directly to root nodules of intact pea plants. After 24 h, radioactivity was detected in all plant tissues. In nodule and root tissue, only 2–3% of3H remained as IAA, and analysis by thin layer chromatography suggested that indole-3-acetyl-L-aspartic acid (IAAsp) was a major metabolite. The occurrence of IAAsp in pea root and nodule tissue was confirmed unequivocally by gas chromatography-mass spectrometry (GC-MS). The following endogenous indole compounds were also unequivocally identified in pea root nodules by GC-MS: IAA, indole-3-pyruvic acid, indole-3-lactic acid, indole-3-propionic acid, indole-3-butyric acid, and indole-3-carboxylic acid. Evidence of the occurrence of indole-3-methanol was also obtained. With the exception of IAA and indole-3-propionic acid, these compounds have not previously been unequivocally identified in a higher plant tissue.