Young bean plants (Phaseolus vulgaris L. var Saxa) were fed with three different types of inorganic nitrogen, after being grown on nitrogen-free nutrient solution for 8 days. The pattern of "CO2 fixation was investigated in photosynthesizing primary leaf discs of 11-day-old plants (3 days with nitrogen source) and in a pulse-chase experiment in 13-day-old plants (5 days with nitrogen source).Ammonium caused, in contrast to nitrate nutrition, a higher level of 14C incorporation into sugar phosphates but a lower incorporation of label into malate, glycolate, glycerate, aspartate, and alanine. The labeling kinetics of glycine and serine were little changed by the nitrogen source. Ammonium feeding also produced an increase in the ratio of extractable activities of ribulose-1,5-bisphosphate carboxylase to phosphoenolpyruvate carboxylase and an increase in dark respiration and the CO2 compensation concentration. Net photosynthesis was higher in plants assimilating nitrate.The results point to stimulated turnover of the photosynthetic carbon reduction cycle metabolites, reduced phosphoenolpyruvate carboxylation, and altered turnover rates within the photosynthetic carbon oxidation cycle in ammonium-fed plants. Mechanisms of the regulation of primary carbon metabolism are proposed and discussed.The effect of NH4' on photosynthetic carbon assimilation has been repeatedly reported (3, 8, 12, 13, 16, 17, 19-23, 27, 29) a decrease in the concentration of malate and an inhibition of malic enzyme activity (2). However, the supply of N03-to cells of Spinacia oleracea had no apparent effect on the distribution of photosynthetic products compared with N-free nutrition, whereas in NH4t-fed cells, fixation of CO2 into carbohydrates decreased but increased in carboxylic and amino acids (16).The effect of inorganic N sources on the composition of N compounds in the bleeding sap and leaves of the bushbean Phaseolus vulgaris has previously been reported (6, 27). Coupled with the above observations, this has led us to investigate the effect of different N sources on various carboxylating enzymes and on the pattern of "4CO2 fixation in primary leaves of nonnodulated bean plants grown in liquid culture. Our results show that different N sources, fed through the roots of Phaseolus vulgaris, cause changes in photosynthetic carbon metabolism. MATERIALS AND METHODSPlant Material. Seeds of Plaseolus vulgaris L. var Saxa (strain Vatter) were soaked overnight in tap water, rinsed, and placed on wet tissue paper in darkness for 3 d at 24 ± 2°C. Seedlings were transferred to coarse quartz sand, watered with N-free nutrient (27), and grown under an illumination of 7000 to 9000 lux. After a further 4 d (day 8), the plants were set into plastic tanks filled with 14 L nutrient solution containing a total N concentration of 3.5 mm either as NH4', or NH4NO3 (27). The pH of the nutrient solution was adjusted with either KOH or H3PO4. All subsequent investigations were carried out on primary leaves. "4C02/'2C02 Pulse-Chase ExpeAments. After...

Properties of purified L-tyrosine decarboxylase (EC 4.1.1.25) from elicitor-induced cell suspension cultures of Eschscholtzia californica Cham. and Thalictrum rugosum Ait. are described. L-Tyrosine decarboxylase is a dimeric enzyme with a molecular weight of 112,600 ± 600 daltons. The isoelectric point was estimated to be at pH 5.2 and pH 5.4for the enzyme from E. californica and T. rugosum, respectively. The purified enzymes were stabilized in the presence of pyridoxal-5-phosphate. Optimum pH for the enzyme from both plants was found to be 8.4. Enzyme activity was dependent on exogeneously supplied pyridoxal-5-phosphate. The enzyme decarboxylated L-tyrosine and L-,a-3,4-dihydroxyphenylalanine but was inactive toward L-phenylalanine and Ltryptophan. Apparent Km values of Eschscholtzia-and Thalictrum-decarboxylase for L-tyrosine were 0.25 ± 0.03 and 0.27 ± 0.04 millimolar, respectively. Similar affinities were found for L-3,4-dihydroxyphenylalanine. Eschscholtzia L-tyrosine decarboxylase was strongly inhibited by the phenylalanine analogue L-a-aminooxy-#-phenylpropionate and largely unaffected by D,L-a-monofluoromethyl-3,4-dihydroxyphenylalanine and a-difluoromethyltyrosine.An enhanced biosynthesis of berberine in cell suspension cultures of Thalictrum rugosum Ait. was achieved by treating the cells with a yeast carbohydrate elicitor (6). This increased product formation followed an increase of TDC2 (EC 4.1.1.25) activity (8). These findings, coupled with results from feeding experiments with radioactive labeled tyrosine (8), indicate that TDC might control the branching point between primary and secondary metabolisms. Furthermore, the synthesis ofdopamine (one of the direct precursors of norlaudanosoline) from tyrosine during the initial steps of alkaloid biosynthesis is not clear. Two possible routes could be considered: first, hydroxylation of tyrosine to DOPA followed by a decarboxylation of DOPA to dopamine; second, decarboxylation of tyrosine to tyramine and subsequent hydroxylation of tyramine to dopamine.Data about the catalytic and molecular properties of plant DOPA and tyrosine decarboxylase would be valuable in establishing the function of these enzymes in alkaloid biosynthesis. However, only very limited information about these enzymes has been published (2,7,9,14,15 A sample (0.5 ml) ofpurified TDC was desalted by centrifugation through a 5 ml Sephadex G-25 column equilibrated in 25 mM bis-Tris-HCI (pH 6.7) and applied to the Mono P column, which had been equilibrated with the same buffer. Elution was performed with a decreasing pH gradient (30 ml) using a 10-fold diluted Polybuffer 74-HCI (pH 4.9). The pI values were also estimated by isoelectric focusing. IEF was carried out on tubes as described (5) using ampholine pH 4 to 6. 52 www.plantphysiol.org on May 11, 2018 -Published by Downloaded from

No abstract

No abstract

Photosynthetic C02-fixation in isolated pea (Pisum sativum L., cv Little Marvel) chloroplasts during induction is markedly inhibited by 0.4 millimolar sulfite. Sulfate at the same concentration has almost no effect.The "C02-fixation pattern indicates that the primary effect of sulfite is inhibition of the reaction catalyzed by ribulose bisphosphate carboxylase and a stimulation of export of intermediates out of the chloroplasts. Inhibition of light modulation of stromal enzyme activity does not appear to account for the toxicity of SO2 in this Pisum variety. Arsenite at 0.2 millimolar concentrations inhibits light activation and inhibits photosynthetic CO2 fixation. The '4C02-fixation pattern indicates that the primary effect of arsenite is inhibition of light activation of reductive pentose phosphate pathway enzyme activity.Several sites of action have been proposed to explain the effect of SO2, one of the major air pollutants, on plants. In chloroplasts sulfite (at pH 8.2: 9.2% HSO3-and 90.8% S032-) and effectors induced by SO2 fumigation can attack reactions of photosynthetic electron transport (7, 26), photophosphorylation (5, 27), and the reductive pentose phosphate pathway (10,14,17,24,29,(31)(32)(33). Light modulation of stromal enzyme activity in the chloroplast is known to be sulfite sensitive (3,20,21).In previous experiments involving '4C02 fixation in intact chloroplasts the RPP3 pathway intermediates were only partially resolved (16). To verify the reported SO2 effects and to determine their significance in situ, we have examined the effect of sulfite on the "4CO2 fixation pattern and the distribution of '4C-labeled metabolites between chloroplasts and the incubation medium during photosynthetic induction.S02-fumigated plants can accumulate sulfate (9, 30), which has been reported to be both relatively harmless (8,27) and to quite effectively inhibit photosynthesis in isolated chloroplasts (4,27). We treated isolated pea chloroplasts with sulfate in order to compare effects with those of sulfite.The major causes of the sulfite-induced reduction in the pho-'Supported