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Comparative glutamine metabolism in tobacco callus and leaf tissues
ABSTRACTS OF CONTRIBUTED MANUSCRIPTS leaves produced the highest activity among the combinations tested. A decrease in photosynthetic activity to 0.835/~mol COz/mg chlorophyll/h by changing the kinetin concentration to 1.0mgfl was observed. Reducing the cytokinin level reduces the photosynthetic activity for most hormone concentrations tested. Plastid ultrastructure was examined for the various hormone combinations.
Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of illumination and auxin. ARTHUR L. LAWYER,KAREN L. GRhDY and JAMES A. BASSrtAM, Laboratory of Chemical Biodynamics, LaWrence Berkeley Lab, University of California, Berkeley, CA 94720. Callus cultures derived from pith of dVicotiana tabacura (cv. Wisconsin 38) were grown on two media under either continuous illumination or in complete darkness. The first medium limited greening ability when grown in light (3.0 mg/1 naphthalene acetic acid, NAA; 0.3 mg/1 isopentenylaminopurine, 2iP; M and S salts and 2% sucrose) and the second encouraged chlorophyll synthesis (greening) but not shoot formation (0.3 mg/1 NAA; 0.3 mg/1 2iP). To measure intracellular concentrations callus were put on standard media containing [U-X4C]-sucrose for 15 days. Glutamine (from 4 to 2 6 m M ) accounted for over 95% of the free amino acids found, possibly due to the high NH 3 content of the media. Proline concentration increased 20fold in light-grown callus on low auxin media (green cells), possibly a stress response to the high osmotic potentials observed in those cultures. Citrate concentrations were lower in light-grown cells suggesting that citrate synthetase was light activated. To analyze sucrose metabolism, callus were allowed to take up 0.2°0 (w/v) [U-14C]sucrose for up to 90rain at 30°C. In callus tissues and in pith sections from which the callus were derived, the observed sucrose metabolism was through invertase activity, producing glucose and fructose (1 : 1 ). The hexoses were then phosphorylated and metabolized through glycolysis. The apparent primary sources-of 14CO2 release from c a l l u s were tricarboxyfic acid .. cycle intermediates: Photorespiration activity, as indicated by re-
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lative labeling in glycine and serine, was negligible under all growth conditions. (Supported .by the Division of Biological Energy Conversion and Conservation, Office of Basic Energy Sciences, Department of Energy under contract No. W-7405-ENG-48, and by a Rockefeller Foundation Postdoctoral Fellowship to A.L.L. )
Comparative glutamine metabolism in tobacco callus and leaf tissues. M. D. LAZAR and G. B. COLLINS, University of Kentucky, Lexington, Kentucky 40546. We have observed excessive accumulation of free glutamine (40-50-fold) in callus of several burley tobacco (Nicotiana tabacum L.) genotypes when compared to leaf tissue of the same genotypes. Other free amino acid pools were generally larger in callus than in leaf, though the differences did not compare to those observed for glutamine. Amino acid profiles differed among genotypes, although such differences were small compared to those between tissues, within genotypes. The difference in free glutamine pool size between leaf and callus of genotype Burley 21 was largely eliminated when ammonia was supplied to leaf in concentrations equal to those usually provided to callus cultures. In vivo labeling studies have shown that label from Ca4-sucrose accumulated in glutamine more rapidly than it disappeared from glutamate, wlfich probably results from the activity of glutamate synthase (glutamate-2oxoglutai'ate amino transferase, EC 2.6.1.53). In vitro studies established that in both leaf and callus, apparent peak activity of glutamine synthetase (EC 6.3.1.2) existed at exogenous ammonia levels of 0.3-1.0mM while apparent peak activity of the alternative enzyme, glutamate dehydrogenase (EC 1.4.1.2), existed at ca 10 mM exogenous ammonia. Intracellular localization of homoserine dehydrogenase in soybean (Glycine max) cell suspension cultures. DAVID F. EIERMAN and KENNETH G. WILSON, Botany Department, Miami University, Oxford, o H 45056. Protoplasts isolated from two-day-old, lightgrown soybean cell suspension cultures were
Intracellular concentrations and metabolism of carbon compounds in tobacco callus cultures: effects of illumination and auxin. ARTHUR L. LAWYER,KAREN L. GRhDY and JAMES A. BASSrtAM, Laboratory of Chemical Biodynamics, LaWrence Berkeley Lab, University of California, Berkeley, CA 94720. Callus cultures derived from pith of dVicotiana tabacura (cv. Wisconsin 38) were grown on two media under either continuous illumination or in complete darkness. The first medium limited greening ability when grown in light (3.0 mg/1 naphthalene acetic acid, NAA; 0.3 mg/1 isopentenylaminopurine, 2iP; M and S salts and 2% sucrose) and the second encouraged chlorophyll synthesis (greening) but not shoot formation (0.3 mg/1 NAA; 0.3 mg/1 2iP). To measure intracellular concentrations callus were put on standard media containing [U-X4C]-sucrose for 15 days. Glutamine (from 4 to 2 6 m M ) accounted for over 95% of the free amino acids found, possibly due to the high NH 3 content of the media. Proline concentration increased 20fold in light-grown callus on low auxin media (green cells), possibly a stress response to the high osmotic potentials observed in those cultures. Citrate concentrations were lower in light-grown cells suggesting that citrate synthetase was light activated. To analyze sucrose metabolism, callus were allowed to take up 0.2°0 (w/v) [U-14C]sucrose for up to 90rain at 30°C. In callus tissues and in pith sections from which the callus were derived, the observed sucrose metabolism was through invertase activity, producing glucose and fructose (1 : 1 ). The hexoses were then phosphorylated and metabolized through glycolysis. The apparent primary sources-of 14CO2 release from c a l l u s were tricarboxyfic acid .. cycle intermediates: Photorespiration activity, as indicated by re-
425
lative labeling in glycine and serine, was negligible under all growth conditions. (Supported .by the Division of Biological Energy Conversion and Conservation, Office of Basic Energy Sciences, Department of Energy under contract No. W-7405-ENG-48, and by a Rockefeller Foundation Postdoctoral Fellowship to A.L.L. )
Comparative glutamine metabolism in tobacco callus and leaf tissues. M. D. LAZAR and G. B. COLLINS, University of Kentucky, Lexington, Kentucky 40546. We have observed excessive accumulation of free glutamine (40-50-fold) in callus of several burley tobacco (Nicotiana tabacum L.) genotypes when compared to leaf tissue of the same genotypes. Other free amino acid pools were generally larger in callus than in leaf, though the differences did not compare to those observed for glutamine. Amino acid profiles differed among genotypes, although such differences were small compared to those between tissues, within genotypes. The difference in free glutamine pool size between leaf and callus of genotype Burley 21 was largely eliminated when ammonia was supplied to leaf in concentrations equal to those usually provided to callus cultures. In vivo labeling studies have shown that label from Ca4-sucrose accumulated in glutamine more rapidly than it disappeared from glutamate, wlfich probably results from the activity of glutamate synthase (glutamate-2oxoglutai'ate amino transferase, EC 2.6.1.53). In vitro studies established that in both leaf and callus, apparent peak activity of glutamine synthetase (EC 6.3.1.2) existed at exogenous ammonia levels of 0.3-1.0mM while apparent peak activity of the alternative enzyme, glutamate dehydrogenase (EC 1.4.1.2), existed at ca 10 mM exogenous ammonia. Intracellular localization of homoserine dehydrogenase in soybean (Glycine max) cell suspension cultures. DAVID F. EIERMAN and KENNETH G. WILSON, Botany Department, Miami University, Oxford, o H 45056. Protoplasts isolated from two-day-old, lightgrown soybean cell suspension cultures were