Cytokinins in the leaves of Ginkgo biloba. II. Metabolism and transport of 8 (14C) zeatin applied to shoot explants

Irrespective of their age, leaves of Ginkgo biloba metabolised applied 8 (¹⁴C) zeatin to compounds of similar chromatographic properties. Glucosylation is apparently not a normal feature of cytokinin metabolism in immature leaves. However, the application of zeatin to these leaves did result in the formation of metabolites which co-chromatographed with glucosylated cytokinins. As far as cytokinin metabolism is concerned therefore, this application of excess zeatin allowed immature leaves to behave as mature or senescing leaves. Overall metabolism was fastest in immature leaves. From the metabolites formed it would appear as if oxidation, which resulted in the formation of a metabolite which co-eluted with N-(purin-6-yl)glycine, was also important in immature leaves. In senescing leaves glucosylation was the major form of metabolism. Extraction and re-application of the polar metabolites (formed from zeatin) to mature leaves resulted in the formation of compounds which co-chromatographed with zeatin. This suggests that these compounds could serve as precursors for zeatin or could be hydrolysed to form zeatin.Very little of the applied radioactivity was exported from the leaves irrespective of their physiological age. When the metabolites, obtained after zeatin application to mature leaves, were extracted and reapplied to the leaves, export of radioactive material was much improved. The results suggest that should cytokinins such as zeatin be translocated to mature leaves of this deciduous gymnosperm their export from the leaves would be unlikely unless first metabolised. In all probability the metabolites concerned are cytokinin glucosides.The financial support of the C.S.I.R., Pretoria, is gratefully acknowledged.     

The senescence retarding ability and metabolism of trihydroxy-zeatin in Avena leaf segments

6-(2,3,4-Trihydroxy-3-methylbutylamino) purine, trivial name trihydroxy-zeatin (THZ), an oxidation product of zeatin was applied via the epidermis or cut end of Avena leaf segments. THZ did not retard senescence of the segments in either case. With epidermal application THZ remained unmetabolised while the cut end method of application resulted in its metabolism to more polar THZ metabolites of unknown identity. The Avena leaf tissue did not apparently cleave the isoprenoid side chain of the THZ molecule.     

Increases in cytokinin levels in Scots pine in relation to chilling and bud burst

Levels of isopentenyladenosine and zeatin riboside were monitored in buds and needles of Scots pine ( Pinus sylvestris L.) seedlings grown under controlled climatic conditions and in field-grown trees. Extracts were purified by immunoaffinity chromatography and high-performance liquid chromatography. Cytokinin levels were quantified with an enzyme-linked immunosorbent assay. The cytokinin content was low in buds and needles of dormant seedlings but increased during dormancy release, reaching peak values in buds just before resumption of shoot growth. Samples collected in the field also showed a marked increase in the levels of cytokinins just prior to bud burst. Further, the biological activity of applied cytokinins in activating the dormant buds of Scots pine is shown. The results indicate a probable role of cytokinins in seedlings during dormancy release.     

Effect of zeatin on the growth and indolyl-3-acetic acid and abscisic acid levels in maize roots

Elongation, indolyl-3-acetic acid (IAA) and abscisic acid (ABA) levels, – gas chromatography-mass spectrometry quantification –, in the elongating zone were analysed for maize ( Zea mays L., Cv. LG11) roots immersed in buffer solution with or without zeatin (Z). The effect of Z depends on the initial extension rate of roots. The slower growing roots are more strongly inhibited by Z (10⁻⁷−10₋₅ M ) and they show a greater increase in IAA and ABA content. When compared to the rapidly growing roots, the larger reactivity of the 'slow'ones cannot be attributed to a higher Z uptake as shown when using [¹⁴C]-Z. It is suggested that Z could regulate root elongation by acting on the IAA and/or ABA level. The comparative action of these two hormones is discussed.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit ( Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O - and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O - and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g⁻¹ fresh weight at anthesis to 41 pmol g⁻¹ fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g⁻¹ fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g⁻¹ fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.     

Involvement of abscisic acid and cytokinins in the senescence and remobilization of carbon reserves in wheat subjected to water stress during grain filling

This study investigated the possibility that abscisic acid (ABA) and cytokinins may mediate the effect of water deficit that enhances plant senescence and remobilization of pre‐stored carbon reserves. Two high lodging‐resistant wheat (Triticum aestivum L.) cultivars were field grown and treated with either a normal or high amount of nitrogen at heading. Well‐watered (WW) and water‐stressed (WS) treatments were imposed from 9 d post‐anthesis until maturity. Chlorophyll (Chl) and photosynthetic rate (Pr) of the flag leaves declined faster in WS plants than in WW plants, indicating that the water deficit enhanced senescence. Water stress facilitated the reduction of non‐structural carbohydrate in the stems and promoted the re‐allocation of prefixed ¹⁴C from the stems to grains, shortened the grain filling period and increased the grain filling rate. Water stress substantially increased ABA but reduced zeatin (Z) + zeatin riboside (ZR) concentrations in the stems and leaves. ABA correlated significantly and negatively, whereas Z + ZR correlated positively, with Pr and Chl of the flag leaves. ABA but not Z + ZR, was positively and significantly correlated with remobilization of pre‐stored carbon and grain filling rate. Exogenous ABA reduced Chl in the flag leaves, enhanced the remobilization, and increased grain filling rate. Spraying with kinetin had the opposite effect. The results suggest that both ABA and cytokinins are involved in controlling plant senescence, and an enhanced carbon remobilization and accelerated grain filling rate are attributed to an elevated ABA level in wheat plants when subjected to water stress.     

Dynamics of endogenous cytokinin pools in tobacco seedlings: a modelling approach

Recent advances in cytokinin analysis have made it possible to measure the content of 22 cytokinin metabolites in the tissue of developing tobacco seedlings. Individual types of cytokinins in plants are interconverted to their respective forms by several enzymatic activities (5'-AMP-isopentenyltransferase, adenosine nucleosidase, 5'-nucleotidase, adenosine phosphorylase, adenosine kinase, trans-hydroxylase, zeatin reductase, beta-glucosidase, O-glucosyl transferase, N-glucosyl transferase, cytokinin oxidase). This paper reports modelling and measuring of the dynamics of endogenous cytokinins in tobacco plants grown on media supplemented with isopentenyl adenine (IP), zeatin (Z) and dihydrozeatin riboside (DHZR). Differences in phenotypes generated by the three cytokinins are shown and discussed, and the assumption that substrate concentration drives enzyme kinetics underpinned the construction of a simple mathematical model of cytokinin metabolism in developing seedlings. The model was tested on data obtained from liquid chromatography/tandem mass spectrometry cytokinin measurements on tobacco seedlings grown on Murashige and Skoog agar nutrient medium, and on plants grown in the presence of IP, Z and DHZR. A close match was found between measured and simulated data, especially after a series of iterative parameter searches, in which the parameters were set to obtain the best fit with one of the data sets.     

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.