Ammonia Regulation of Carbon Metabolism in Photosynthesizing Leaf Discs '

Alfalfa (Medicago sadiva L., var. El Unico) leaf discs, floating on buffer contining NH4CI and photosynthesizing with "4CO2, produced more labeled amino acid d les scrose th did control discs (no added NH4CI). The level of pyruvate increased and that of phosphoenlpyruvate decreased. Tlese and other changes in levels of labeled compouds led us to condude that pyrvate kiae was activated by ammoml, resuing In inemased transfer of photosynthetcaly incorporated carbon to synthesis of amino acid skeletons at the expense of ucre synthesis. Carbon flow throngh enzymes catalyzing the anaplerotic reactions was apparently stimulated. The over-all process of nitrate reduction and ammonia incorporation into amino acids in higher plant leaves (along with the role of light in that process) has been a subject of recent research (3, 10, 12). Given the production of amino acids as well as sucrose during photosynthesis (16), it is of interest to determine whether leaf regulatory mechanisms involve ammonia. The intracellular NH4+ level could influence leaf carbon metabolism during photosynthesis with respect to sucrose synthesis versus the amino acid synthesis necessary for protein production and leaf growth. Kinetic studies of "4C-labeled compounds formed in the unicellular green alga Chlorella pyrenoidosa during photosynthesis with "4CO2 have indicated that ammonia brings about increased amino acid synthesis in part due to stimulation of pyruvate kinase (8). We have now investigated whether a similar regulatory mechanism is active in the leaves of higher plants. The specific plant chosen for investigation was alfalfa because of its potential as a source of leaf protein for direct human consumption (5). In our experiments we have used the techniques of kinetic tracer analysis of steady-state photosynthesis (2, 19). Labeled 1This work was carried out at the Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley, California 94720. It was supported in part by the Division of Biomedical and Environmental Research of the U.S. Energy Research and Development Administration, and in part by the U.S. Department of Agriculture. 2To whom requests for reprints should be addressed. Address for correspondence: Western Regional Research Center, A.R. S. , U.S.D.A. , Berkeley, California 94710. 3Present address: Agricultural Research Organization, Bet-Dagan, Israel. CO2 fixation by alfalfa leaf discs in the presence and absence of NH4Cl was examined. Analysis of the labeled products formed as a function of time indicates that ammonia can function as a regulatory agent in alfalfa leaves. MATERIALS AND METHODS Plant Material. Alfalfa (Medicago sativa L., var. El Unico) seeds were planted in 12 cm of vermiculite. The plants were grown at 3,000 ft-c and 15 C with a 9-hr light period and a 15hr dark period. Plants were fertilized with modified Hoagland solution. Second through fourth unfolded leaves (numbered from the plant apex; not fully expanded) were excised after 1 hr of light from 6-week-old plants. The leaves were placed on a rubber sheet and 1-cm-diameter leaf discs were cut out with a cork borer. Photosynthetic Carbon Fixation. The closed steady-state gas circulation system described previously was employed (19). Leaf discs were exposed to CO2 in 4.5-cm-diameter flasks made from ground-glass joints and having transparent upper and lower surfaces. Each flask was fitted with gas inlet and outlet tubes, a serum stopper (allowing for addition of reagents during experiments), and five glass leaf disc holders (serving to prevent leaf overlap). Immediately after being cut, the leaf discs were floated on 4 ml of pH 7.4 buffer (0.05 M K2HPO4, pH adjusted with HCl) in the disc exposure flasks. Twelve flasks were utilized; each contained five discs comprising a single sample. The flasks were placed in a temperature-regulated shaking device (previously described for chloroplast experiments) (7) and attached to the closed gas circulation system through manifolds. Gas flow was commenced with 0.04% CO2 in air (20% 02); flask temperature was 27 C. After 12 min of preincubation in the dark, the lights were turned on and ammonium chloride in the pH 7.4 buffer was injected into six of the flasks to give a final concentration of 5 mm NH4Cl. Photosynthesis with 12CO2 was observed for 17 min. The unlabeled CO2 in air was then replaced by 0.038% "4CO2 (30.2 ,uCi/Amol) in air (20% 02). Samples of control and ammoniumtreated leaf discs were removed at the time intervals shown under "Results. " As each sample was removed, the leaf discs were immediately frozen in liquid N2Analysis of 14C-labeled Products. Each sample was ground and successively extracted with 80% ethanol (v/v), 20% ethanol, and then water essentially as described previously (20). The extracts of each sample were combined. The residue from each sample was collected on filter paper, washed with 3 drops of formic acid, and dried in a vacuum desiccator over silica gel and KOH. The residue was then combusted (Packard automatic 739 www.plantphysiol.org on September 6, 2017 Published by Downloaded from Copyright © 1977 American Society of Plant Biologists. All rights reserved. PLATT, PLAUT, AND BASSHAM combustion apparatus) to give data on 14CO2 fixation into insoluble materials. Sample extracts were analyzed by liquid scintillation for fixation into total soluble metabolites. Fixation of '4CO2 into most soluble metabolites was determined from chromatograms developed for 24 hr and also 48 hr in each direction using, with one modification, the solvent system of Pedersen et al. (17). The pH of the phenol-wateracetic acid-EDTA solvent used in the 24-hr development was adjusted to pH 4.2 (18). Glycolate, glycine, and serine were isolated as described earlier (1 8). Radioactive areas were located and major labeled metabolites identified as described earlier (17-19). Radioactivity in each compound was determined by elution with water, followed by liquid scintillation counting. Samples were analyzed for a-ketoacids by first preparing and purifying the 2,4-dinitrophenylhydrazone derivatives (1). Unlabeled sodium pyruvate was added to aliquots of each sample prior to reaction with 2,4-dinitrophenylhydrazine. The mixture of hydrazones was analyzed on Whatman 1 paper; chromatograms were first developed for 24 hr with 1-butanol-ethanol-1 N NH40H (13:2:5) (11) and then in the second dimension for 19 hr with butanol-propionic acid-water (17). Labeled materials were located by autoradiography (17). The pyruvic and aketoglutaric acid derivatives (yellow in color) were identified by coincidence with added unlabeled carrier. The OAA4 derivative was identified by its mobility in butanol-ethanol-1 N NH40H (11). The a-KG and OAA derivative spots were excised from the paper prior to development in the second dimension. The locations of the derivatives of hydroxypyruvate and glyoxylate were determined by chromatography of standards and were found to be separated well from the pyruvate, a-KG, and OAA derivative spots. Several of the derivatives gave double spots on the chromatograms as has been long noted (11) apparently due to formation of syn-anti isomers. Radioactivity was determined by automatic Geiger counter (17). All fixation results were expressed on the basis of microgram atoms 14C/dm2 of leaf area. The microgram atoms of 14C were calculated by dividing the radioactivity of the product, in,uCi, by the specific radioactivity of the entering 14CO2.