Cytokinin Translocation and Metabolism in Lupin Species. I. Zeatin Riboside Introduced Into the Xylem at the Base of Lupinus angustifolius Stems

The principal cytokinins in xylem exudate of blue lupin (Lupinus angustifolius L.) identified by radioimmunoassay and mass spectrometry were zeatin riboside (ZR), dihydrozeatin riboside and zeatin, but cytokinin O-glucosides and nucleotides were also present. Radioactive [3H]ZR was supplied to the transpiration stream of both derooted and intact blue lupin plants. The distribution of methanol-extracted radioactivity throughout the shoot and the identities of 3H-labelled metabolites were determined; for the latter work, a new bilayer thin-layer chromatography system was developed. In derooted plants supplied with ZR for about 20 h, radioactivity per unit fresh weight of tissue was greatest in the peduncle, stem and axillary shoots and least in the seed. Differences in metabolism were more marked between tissue types than within tissues of different maturity. The principal metabolites in derooted plants (ZR supplied for up to 24 h) were: in laminae, O-glucosyldihydrozeatin, lupinic acid, dihydrozeatin riboside and adenosine; in petioles, dihydrozeatin riboside and adenosine; in pod walls, cytokinin nucleotides, dihydrozeatin riboside, adenine and adenosine; in developing lateral shoots, cytokinin nucleotides; in stem, dihydrozeatin riboside, dihydrozeatin and cytokinin nucleotides. Apart from adenosine, no metabolites could be identified with certainty in seed. In experiments with intact plants conducted over a longer period (plants extracted 2 days after 30 h uptake), metabolism was more extensive but, in all tissues except seed, low percentages of radioactivity were still due to metabolites with an intact zeatin moiety (most frequently O-glucosyldihydrozeatin and lupinic acid). Free zeatin and dihydrozeatin were not found in any tissue and their ribosides were detected with certainty only in pod walls (<2% of extracted 3H). Extraction with 0.5 N KOH after extraction with methanol yielded, on average, an additional equal amount of radioactivity due to nucleotides of adenine and guanine. Features of the translocation studies include: (1) the direct lateral movement of ZR and/or related cytokinins from xylem to bark which was established by bark ringing experiments; (2) the relatively high level of radioactivity per unit tissue fresh weight in developing lateral shoots; (3) the lack of appreciable movement to the seed although the cytokinin reached the pod walls and was partially conserved there; (4) the disproportionately high retention of radioactivity by the petioles which increased with leaf age; (5) changes in ZR metabolites in laminae associated with age; (6) the detection of a new nucleotide metabolite of ZR in pod walls, bark and lateral shoots; this appears to be a transient metabolite with an intact ZR moiety and may be involved in ZR uptake and/or transport. The studies of ZR translocation and metabolism are discussed in relation to sequential leaf senescence, the proposal that developing seeds accumulate xylem cytokinins, and the origin of phloem cytokinins.

Gas Chromatographic/Thin-Layer Chromatographic Analysis of Acetylated Codeine and Morphine in Urine

This procedure positively identifies codeine and morphine in urine. Urine samples are hydrolyzed and extracted with organic solvent, and the extracts are evaporated and acetylated. The presence of codeine and morphine is ascertained by gas chromatography (3% OV-25 and 3% Poly-A 103 columns) and confirmed by thin-layer chromatography (system: ethyl acetate/acetone/concd ammonium hydroxide, 100/10/4.5 by vol; reagent: iodoplatinate). As little as 0.5 mg each of codeine and morphine per liter, in free and conjugated forms, is detectable by this method.

SYNTHESIS OF THE 2,4-DI-O-METHYL TETROSES

The four isomeric 2,4-di-O-methyl tetroses were prepared by periodate oxidation of known methylated sugars. 2,4-Di-O-methyl-D- and L-erythroses were prepared from 4,6-di-O-methyl-D-glucose and 3,5-di-O-methyl-L-arabinose respectively. 2,4-Di-O-methyl-D- and L-threoses were prepared from 3,5-di-O-methyl-D-xylose and 1,4,6-tri-O-methyl-L-sorbose.The tetroses were characterized by their crystalline 2,4-dinitrophenylhydrazones. The RF and RG values of the free sugars were recorded in a variety of solvents including a silica gel thin-layer chromatography system.