Synthesis and cytokinin activity of (R)-(+)- and (S)-(−)-dihydrozeatins and their ribosides

(R)-(+)- and (S)-(?)-dihydrozeatins [(R)-(+)- and (S)-(?)-6-(4-hydroxy-3-methylbutylamino)purines, 1a and 1b] and their ribosides {(?)-6-[(R)-4-hydroxy-3-methylbutylamino]- and (?)-6-[(S)-4-hydroxy-3-methyl-butylamino]-9-β-D-ribofuranosylpurines, 3a and 3b} were synthesized and tested for their cytokinin activity by four bioassay systems, the growth of tobacco callus, the seed germination of lettuce, the fr. wt increase of excised radish cotyledons and the retardation of chlorophyll degradation in radish cotyledons. In tobacco callus bioassay, 1a was more active than 1b. The ribosides 3a and 3b were not less active than their corresponding aglycones 1a and 1b. In other bioassays used the activity followed the order: 1a >3a >1b >3b. In tobacco callus bioassay and lettuce seed germination, trans-zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] showed stronger cytokinin activity than 1a.     

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[³H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate     

Loading and translocation of various cytokinins in phloem and xylem of the seedlings of Ricinus communis L.

The phloem sap of Ricinus seedlings was analyzed for cytokinins and the concentration was compared with that in cotyledons and xylem sap. The dominant cytokinin in the phloem sap was isopentenyladenine (70 nM) when the endosperm was attached to the cotyledons; zeatin, dihydrozeatin and cytokinin-ribosides were present at relatively low concentrations (1–2 nM). Removal of the endosperm and incubation of the cotyledons in buffer led to a sharp decrease in the level of isopentenyladenine in the phloem sap, down to the value for zeatin, namely 1–2 nM. Similar low cytokinin concentrations were found in the xylem sap, too, whereas in the cotyledons the cytokinin content was at least 10-fold higher. Incubation of the cotyledons with various cytokinins (isopentenyladenine, zeatin and their ribosides) led to an increase of each of the applied cytokinins in the phloem sap, including also the metabolically closely related cytokinins. Zeatin was especially well loaded. It is concluded that the phloem translocates most free bases and ribosides of the various cytokinin species, if they are offered to the phloem. The data also show that the cytokinin levels in the phloem, which may be far higher than in the xylem, are subject to strong fluctuations depending on the physiological situation.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 137). The experimental assistance by P. Geigenberger and the help in cytokinin analysis by Dr. A. Fußeder, Dr. B. Wagner, W. Peters (all Bayreuth) and by Prof. E. Weiler (Bochum) is gratefully acknowledged. Also the constructive discussions with Profs. E. Weiler (Bochum) and E. Beck (Bayreuth) are much appreciated.     

The role of cytokinins in root development of pea seedlings

Endogenous cytokinin-like activity was detected in pea seedlings usingthe soybean callus bioassay. Higher levels of activity were found in two-day-oldseedling roots and in the root free zone of four-day-old seedlings compared tothe lateral root zone of four-day-old seedlings. By day six, the levels ofendogenous cytokinin-like activity was greatly reduced in both the lateral rootzone and root free zone. Decapitation of the root tip disrupted the spatialorganization of the root. Lateral roots were subsequently found along the entirelength of the root rather than in a discreet lateral root zone. Application of10^–3 M dihydrozeatin to decapitated root tipsovercame the effect of root tip removal and restored the situation to what isnormally found in intact roots. There was little mobility of dihydrozeatin inthe root, with applied ³H-DHZ not moving from the root free zone,even 24 h after application.     

The Involvement of Cytokinin Oxidase/Dehydrogenase and Zeatin Reductase in Regulation of Cytokinin Levels in Pea (Pisum sativum L.) Leaves

Cytokinin metabolism in plants is very complex. More than 20 cytokinins bearing isoprenoid and aromatic side chains were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS) in pea (Pisum sativum L. cv. Gotik) leaves, indicating diverse metabolic conversions of primary products of cytokinin biosynthesis. To determine the potential involvement of two enzymes metabolizing cytokinins, cytokinin oxidase/dehydrogenase (CKX, EC 1.5.99.12) and zeatin reductase (ZRED, EC 1.3.1.69), in the control of endogenous cytokinin levels, their in vitro activities were investigated in relation to the uptake and metabolism of [2−³H]trans-zeatin ([2−³H]Z) in shoot explants of pea. Trans-zeatin 9-riboside, trans-zeatin 9-riboside-5′-monophosphate and cytokinin degradation products adenine and adenosine were detected as predominant [2−³H]Z metabolites during 2, 5, 8, and 24 h incubation. Increasing formation of adenine and adenosine indicated extensive degradation of [2−³H]Z by CKX. High CKX activity was confirmed in protein preparations from pea leaves, stems, and roots by in vitro assays. Inhibition of CKX by dithiothreitol (15 mM) in the enzyme assays revealed relatively high activity of ZRED catalyzing conversion of Z to dihydrozeatin (DHZ) and evidently competing for the same substrate cytokinin (Z) in protein preparations from pea leaves, but not from pea roots and stems. The conversion of Z to DHZ by pea leaf enzyme was NADPH dependent and was significantly inhibited or completely suppressed in vitro by diethyldithiocarbamic acid (DIECA; 10 mM). Relations of CKX and ZRED in the control of cytokinin levels in pea leaves with respect to their potential role in establishment and maintenance of cytokinin homeostasis in plants are discussed.