The labeling patterns in malic acid from dark (13)CO2 fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and (13)C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12-14 h dark (13)CO2 fixation the ratio of [4-(13)C] to [1-(13)C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The (13)C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87-89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following (13)CO2 fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [(13)C] malic acid ((13)CO2 or [1-(13)C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to (13)CO2 in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284-291). In the shortest times, only singly-labeled [4-(13)C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.

The principal biologically active cytokinins in xylem exudate of young Phaseolus valgaris L. plants were identified by bioassay, high-performance liquid chromatography, enzymic degradation and combined gas chromatography-mass spectrometry (selected ion monitoring) a zeatin riboside, zeatin nucleotide, dihydrozeatin riboside, dihydrozeatin nucleotide, O-glucosyl zeatin, O-glycosyl dihydrozeatin, O-glucosyl dihydrozeatin riboside, and O-glucosyl dihydrozeatin nucleotide. Trace amounts of O-glucosyl zeatin riboside and O-glucosyl zeatin nucleotide were also detected.The major cytokinins in leaves, stems and roots of Phaseolus vulgaris L. have been rigorously identified by physicochemical methods (10,14,21,22), and there exists a wealth of information on cytokinin metabolism in this species as a result of studies using quantitative MS (9), bioassay (e.g. 1, 4, 7, 19), and radiolabeled cytokinins (e.g. 10, 11, 15). The nature and distribution of cytokinin-active ribonucleosides in P. vulgaris tRNA has been determined (2, 3) and this species has also been used as a model system to study cytokinin biosynthesis (8). Although the design and interpretation of these and other physiological experiments would benefit greatly from knowing the identity of translocated bean cytokinins, to date such analyses have provided only limited bioassay data (4,7,19). The present study seeks to identify the biologically active cytokinins in bean xylem exudate through high resolution chromatography and mass spectroscopic analysis. MATERIALS AND METHODSBean plants (P. vulgaris cv Hawkesbury Wonder) were grown in 2-L plastic pots containing sterilized garden soil, in a glasshouse, under full sunlight, 16 h days, and a temperature range of 18 to 32°C. Every 2 d, starting I week after germination, each plant received 100 ml of Hewitt's complete nutrient solution containing 12 mm nitrate (6). Plants were harvested 20 d after germination, at a stage ofdevelopment which has been correlated with high root system conductance (5), by decapitatingjust above the cotyledonary node. Exudate was removed continually for 1 h from the cut surfaces using a microsyringe, and transferred to a beaker containing 50 ml extraction solvent (methanol/chloroform/formic acid/water, 12:5:1:2), chilled to -5°C. A total of 24.6 ml exudate was collected from 120 plants.Exudate was stored in extraction solvent overnight at -5°C, then purified as described previously (10), by cation-exchange on cellulose phosphate, followed by alkaline phosphatase treatment of the acidic/neutral fraction, and partitioning of both the basic fraction (containing cytokinin bases and glycosides) and phosphatased fraction (containing nucleotide-derived cytokinins) against butan-1-ol at pH 8.0. The two butanol-soluble fractions were then purified by reversed-phase HPLC, as described previously (17). Initially, a Waters uBondapak C-18 column (7.8 mm x 30 cm) was used to effect a preparative separation ofcytokinin bases, ribosides, and glucosides (Fig.

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