The metabolism of 6-(2,3,4-trihydroxy-3-methylbutylamino) purine by soybean callus

6-(2,3,4-trihydroxy-3-methylbutylamino) purine (trihydroxyzeatin) applied to soybean callus is metabolised slowly. After 48 h only one peak of biological activity which co-eluted with the applied cytokinin was detected. When the callus was incubated on a medium which contained 10^–5 M trihydroxyzeatin, spiked with 8 {¹⁴C} trihydroxyzeatin, for 28 days, three peaks of biological activity and three peaks of radioactivity were detected. One of the biologically active and radioactive peaks co-eluted with zeatin. Another of the radioactive peaks co-eluted with N-(purin-6-yl) glycine. From the data obtained it apears that trihydroxyzeatin can be both oxidized and reduced by soybean callus. The potential to be converted to zeatin may explain why trihydroxyzeatin and its parent compound, which is usually rapidly metabolised by living material, are equally active in the soybean callus bioassay. From the radioactive data obtained it appears that trihydroxyzeatin is susceptible to oxidation to form N-(purin-6-yl) glycine.     

The Effect of Adenine and Mevalonic acid on the Endogenous Cytokinins of a Cytokinin-dependent Strain of Soya Bean Callus

The combined application of 10^–6 M adenine and 10^–6M mevalonic acid to soya bean callus accelerated its growth.Two biologically active compounds that co-chromatographed withzeatin and isopentenyl adenine were extracted from this callus.Studies with labelled adenine and mevalonic acid indicated thatthe cytokinin-dependent soya bean callus incorporated only avery small amount of the radioactive precursors into the biologically-activecompounds, making it extremely difficult to determine whetherthese compounds were synthesized de novo or whether they aroseas by-products of tRNA turnover. As cytokinins do not accumulatein rapidly-growing cytokinin-dependent soya bean callus culturedon kinetin as a source of cytokinin it seems as if biosynthesisde novo occurs when the callus is supplied with adenine andmevalonic acid. Glycine max (L.) Merrill, soya bean, callus culture, adenine, mevalonic acid, endogenous cytokinins     

Cytokinin biosynthesis in crown-gall tissue ofVinca rosea

When care was taken to minimise the effects of phosphatase activity during extraction ofVinca rosea crown-gall tumour tissue, a large proportion of extractable cytolinin activity was present in the nucleotide fraction. Analysis using ion-exchange chromatography followed by enzymic or chemical degradation and subsequent identification of the biologically active material indicated that this activity was due to zeatin riboside 5′-monophosphate. This was also the major radiolabelled cytokinin formed when this tissue was supplied with [¹⁴C]adenine. The incorporation of radioactivity from [¹⁴C]adenosine into free cytokinins was also shown, but no incorporation of radioactivity was found when [³H]mevalonic acid lactone was supplied to this tissue under the same conditions. In parallel experiments using normal stem callus tissue ofV. rosea, no incorporation of [¹⁴C]adenine into free cytokinins was observed. The significance of these results is discussed in relation to a possible transfer-RNA-independent pathway of cytokinin biosynthesis, operating primarily at the mononucleotide level.     

Electron microscopic autoradiographic study of RNA isolated from apple-tree callus tissue labelled with 6-Benzylaminopurine-8-C14

In the present paper RNA from apple-tree callus tissue labelled with 6-benzyl-minopurine¹⁴) was studied. The RNA isolated from this tissue was prepared as sample for electron microscopic studies and was also used as biochemical control. The electron microscopic autoradiograms obtained showed the labelled structure of RNA, sRNA and rRNA. The incorporation of labelled purine rings was confirmed in all three RNA types by the radioactivity, which was also proved in nucleotides after hydrolysation of RNA fractions. The results were compared with RNA isolated from tissue cultivated on a non-radiactive medium.     

On the biogenesis of cytokinins in roots of Phaseolus vulgaris

Roots of intact bean plants were supplied with [¹⁴C]adenine by pulse-chase experiments. The rate of incorporation of radioactivity into tRNA and oligonucleotides of roots as well as the content of radioactive labeled cytokinin nucleotides in these RNA fractions were determined. On the average, 1/70 of the radioactivity incorporated into tRNA was localized in N⁶(²isopentenyl)adenosine. The half life of tRNA was estimated to be 65–70 h. Shortly after the pulse period, oligonucleotides contained zeatin riboside at a ratio of 1:800, on the basis of radioactivity. The half life of these oligonucleotides was determined to be about 8 h. The main free radioactive cytokinin of roots and leaves was zeatin. Comparing the rate of degradation of ¹⁴C-labeled tRNA and the oligonucleotides of roots and the rate of appearance of radioactive cytokinins in roots and leaves, we found strong indications for their dependency. The results contradict the hypothesis of de novo synthesis of cytokinins in roots of intact bean plants.Abbreviations AMP adenosine monophosphate - IPA N⁶(²isopentenyl)adenosine - IPAde N⁶(²isopentenyl)adenosine - Z zeatin - ZR zeatinriboside - TLC thin-layer chromatography - HPLC high performance liquid chromatography Part of the doctoral thesis, Bonn 1980     

Purification and identification of a cytokinin from moss callus cells

A cytokinin was isolated from the culture medium of callus cells of the moss hybridFunaria hygrometrica (L.) Sibth xPhyscomitrium piriforme Brid. The purification procedure included ethyl-acetate extraction, silver-salt precipitation, crystallization as picrate, and ion exchange chromatography. The structure of the cytokinin was confirmed as N⁶–(²-isopentenyl)adenine by means of gas chromatography and mass spectrometry. The concentration of the compound in the culture medium was determined at ca. 10^-6 M.Abbreviation 2iP N⁶–(²-isopentenyl) adenine     

Influence of phytohormones,carbohydrates, aminoacids,growth supplements and antibiotics on somatic embryogenesis and plant differentiation in finger millet

Cultured caryopses of finger millet (Eleusine coracana GAERTN) produced callus from shoot apices or mesocotyls depending upon the concentration of picloram and combination of cytokinins in MS basal medium. On subsequent subcultures, numerous somatic embryos differentiated from the callus on MS medium supplemented with picloram and kinetin. The embryos germinated into complete plants on medium devoid of phytohormones. When different carbohydrates were tested, basal medium containing glucose and sucrose produced the highest frequency of germinating somatic embryos. Supplementation of MS basal medium with a variety of aminoacids, osmotic agents and growth supplements had an adverse effect on the germination of embryos. Incorporation of different antibiotics such as carbenicillin, cefotaxime and streptomycin sulfate enhanced plant differentiation from somatic embryos. Cytological analysis of regenerated plants showed normal diploid chromosome number in their root tips.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - BA benzyl adenine - 2,iP 6---dimethylallylamino purine - Kn kinetin - Z zeatin     

Transport,localization and physiological effect of 6-benzyladenme-8-14C in apple shoots

The transport of radioactivity of 6-benzyladenine-8-¹⁴C applied to one-year old apple shoots was studied. Simultaneously, labelled metabolites of this cytokinin were studied separately in the xylem and phloem above and below the place of application. According to the results obtained, it can be assumed that 6-B and its metabolites are transported in one-year old apple shoots acropetally through the xylem and basipetally through the phloem, while penetrating from the xylem to the phloem. Besides 6-B-8-¹⁴C, a complex of cytokinin with sugar, also adenosine and adenine were found in the phloem.     

Regeneration of Ensete ventricosum through somatic embryogenesis and adventitious buds

In southern and south-western Ethiopia, Ensete ventricosum is grown as an important starchy, staple food crop, supporting the diet of a quarter of the Ethiopian population. Due to difficulty in germinating seeds and the long vegetative period, breeding enset is extremely difficult. Adventitious buds and somatic embryos have been induced from callus derived from corm tissues and cultured on Murashige and Skoog's (MS) basal medium supplemented with benzylaminopurine (BAP) or 6 --dimethylallylamino purine 2iP. Elongation of somatic embryos was achieved on the same medium and rooting was induced on half-strength MS basal medium supplemented with IBA. No phenotypic variation was observed among more than 200 potted regenerants. The possible implications for mutation breeding in this crop are discussed.Abbreviations IAA Indole-acetic acid - IBA Indole-butyric acid - BAP Benzylaminopurine - 2iP 6 --dimethylallylamino purine     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Interactions of γ-L-glutamyltaurine with excitatory aminoacidergic neurotransmission

The in vitro effects of -L-glutamyltaurine on different stages of excitatory aminoacidergic neurotransmission were tested with -D-glutamyltaurine as reference. -L-Glutamyltaurine enhanced the K⁺-stimulated release of [³H]glutamate from cerebral cortical slices (25% at 0.1 mM) and slightly inhibited the uptake by crude brain synaptosomal preparations (about 10% at 1 mM). -L-Glutamyltaurine was also a weak displacer of glutamate and its agonists from their binding sites in brain synaptic membrane preparations, being, however, less selective to quisqualate (QA) sites than -D-glutamyltaurine. The basal influx of Ca²⁺ into cultured cerebellar granular cells was not affected by 1 mM -L-glutamyltaurine, but the glutamate- and its agonist-activated influx was significantly inhibited in low-Mg²⁺ (0.1 mM) and Mg²⁺-free media. The glutamate-evoked increase in free intracellular Ca²⁺ and the kainate-activated formation of cGMP in cerebellar slices were both markedly inhibited by 0.1 mM -L-giutamyltaurine. We propose that -L-glutamyltaurine may act as endogenous modulator in excitatory aminoacidergic neurotransmission.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

On the Significance of Cytokinin Incorporation into RNA

The clarification of the following 2 questions was attempted: (a) are cytokinins precursors in the formation of sRNA, (b) is the observed incorporation of cytokinins into sRNA related to the action of the hormone? Although Escherichia coli contains cytokinins in its sRNA, no cytokinin auxotroph mutants of E. coli could be found and the statistical probability for the existence of such mutants is extremely low. This suggests that cytokinins are not precursors in the synthesis of sRNA. A radioactive cytokinin, 6-benzylamino-9-methyl-purine was synthesized and it was tested whether or not it is incorporated into sRNA of soybean callus tissue. Masking the 9-position of the purine inhibited the incorporation of this cytokinin into RNA while not affecting its biological activity. This is taken as an indication that the observed incorporation of cytokinins such as benzyladenine into sRNA is not related to the action of this hormone.     

Effect of aminooxyacetic acid on the release of preloaded [3H]GABA and radioactive metabolites from slices of developing mouse brain

The release of [³H]-aminobutyric acid (GABA) and its radioactive metabolites from slices of the cerebral cortex, cerebellum, striatum and brain stem of developing and adult mice was studied. The slices were incubated and superfused in the absence and presence of the GABA aminotransferase (GABA-T) inhibitor aminooxyacetic acid (AOAA). Exposure to 100 M AOAA totally inhibited GABA-T and all radioactivity released from slices was in authentic GABA. In studies on developing brain the 10-M concentration was also effective enough, except in cerebellar slices. In the absence of AOAA the major part of radioactivity spontaneously released from slices of adult cerebral cortex and cerebellum was tritiated water and still about one third part in the presence of 10 M AOAA. Potassium stimulation induced only the release of radioactive GABA but not labeled metabolites in both presence and absence of AOAA. AOAA reduced the stimulation-induced release of GABA. It is recommended that the use of GABA-T inhibitors should be discontinued in release experiments. Then labeled GABA must be separated in the effluents from its radioactive breakdown products.     

Cytokinin biosynthesis in cultured rootless tobacco plants

Biosynthesis of cytokinin in shoots was examined by growing rootless tobacco (Nicotiana 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 [(14)C]adenine synthesized ethanol-ethyl acetate-water-soluble radioactive components, portions of which had the same chromatographic and electrophoretic mobilities as N(6)-(Delta(2)-isopentenyl)adenine, N(6)-(Delta(2)-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)purine and 6-(4-hydroxy-3-methyl-2-butenylamino)-9-beta-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.     

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures

Summary Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.Abbreviations BM basal medium - BA 6-benzyladenine - CIM callus induction medium - CPM callus proliferation medium - (diH)Z dihydrozeatin - DW dry weight - ELISA enzyme linked immunoassay - FW fresh weight - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - iP N⁶-(²-isopentenyl)adenine - NAA 1-naphthaleneacetic acid - PBS phosphate buffer - PGR plant growth regulator - (diH)[9R]Z 9--D-ribofuranosyl-(diH) - [9R]iP 9--Dribofuranosyl-iP - [9R]Z 9--D-ribofuranosylzeatin - TBS trishydroxymethyl-aminomethane buffer - Z zeatin     

Furoquinoline alkaloid accumulation in Fagara zanthoxyloides cell cultures is highly dependent on the presence of exogenous benzylaminopurine

Culturing a non-habituated cell line of Fagara zanthoxyloides Lam. (Rutaceae) in either auxin-free medium or cytokinin-free medium led to opposite effects on furoquinoline accumulation. It appeared that production of skimmianine and -fagarine in the cells was strongly correlated with the presence of exogenous BAP: the levels of both alkaloids were 9 times lower when cells were cultured without cytokinin than in the control culture. NAA removal induced a slight stimulation of skimmianine and -fagarine accumulations, 1.2 and 1.9 times respectively. Culturing the cells in a PGR-free medium generated skimmianine and -fagarine levels that were 3.5 and 2.1 times lower, respectively, confirming the opposite effects of BAP and NAA on furoquinoline accumulation. Growth was only slightly inhibited when cells were cultured for one passage in the PGR-modified media.Abbreviations BAP 6-benzylaminopurine - MS Murashige-Skoog - NAA -naphtaleneacetic acid - PGR plant growth regulator     

Incorporation of [32P]- and [14C]-ATP intoEscherichia coli isocitrate lyase

InEscherichia coli, isocitrate lyase has been shown to be phosphorylated in vitro by [-³²P]-ATP on histidine residues. This phosphorylation is believed to be necessary for activity of this enzyme. Previous work has shown that treatment of isocitrate lyase with acid phosphatase leads to a decrease in activity as well as a loss of incorporated [³²P]-phosphate in a time-dependent manner. In addition to phosphorylation by [-³²P]ATP, isocitrate lyase has been found to incorporate radioactive label from [-³²P]ATP and from [¹⁴C]ATP. This finding may indicate that more than one type of covalent modification occurs on this enzyme. Isocitrate lyase activity, inE. coli, may be regulated by posttranslational modification in several ways.     

The metabolism of N6(Δ2-isosopentenyl) [3H]adenine by different stem sections of Pisum sativum

Serial segments of internodal stem tissue were isolated from Pisum sativum L. shoots and incubated in a medium containing N⁶(²-isosopentenyl) [³H]adenine. The recovery of radioactive derivatives separated using HPLC indicated a gradient of cytokinin metabolic activity in the stem. This gradient of activity was found to be greatest at the basal node in young seedlings but was high both at upper and lower nodes in older plants. An attempt to correlate this phenomenon with the basipetally decreasing concentration of indole-3-acetic acid in the stem led to an experiment in which stem segments were pretreated in indole-3-acetic acid solutions before incubating in a medium containing N⁶(²-isosopentenyl) [³H]adenine. Indole-3-acetic acid was found to have a marked effect on cytokinin metabolism in isolated stem segments. These results are discussed in relation to apical dominance in the shoot.     

Regulators of cell division in plant tissues. XVI

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 g 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.For Part XV, see Letham (1973).