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Intracellular localization of homoserine dehydrogenase in soybean (Glycine max) cell suspension cultures
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-
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
426
ABSTRACTS OF CONTRIBUTED MANUSCRIPTS
fractionated on" non-linear sucrose gradients. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact plastids. Fractions were electrophoresed on 7°'~ polyacrylamide gels and stained for homoserine dehydrogenase activity. Three enzyme forms, two of which were threonine sensitive, were electrophoretically detected in whole "ceff extracts. The two threonine-sensitive forms are localized within the plastid.
Steroids in cell and tissue culture of Solanum nigrum and S. dulcamara. PRASHANT BHATT and DACHA BX-mTT, United States Vegetable Laboratory, 2875 Savannah Highway, Charleston, SC 29407. The feasibility of using a somatic cell genetics approach for increasing the steroidal alkaloid content of certain solanaceous plants is being studied. Callus and cell suspension cultures were initiated from excised leaf explants on revised MS medium supplemented by 2 g/1 m-inositol and l mg/1 2,4-D in callus culture or 0.1 rag/1 2,4-D in cell suspension culture, lt~ For plant regeneration 2,4-D was replaced by various concentrations and combinations of IAA and BA. TM Steroids produced in tissue culture and of whole plants were analyzed by gas chromatography (GC), thin layer chromatography (TLC) and/or colorimetry. Plants of S. dulcamara were regenerated from single cell clones isolated from cell suspension cultures. The steroid concentration of S. nigrum callus culture is positively influenced by the concentration of sucrose and IAA added to the medium. Five sterols were detected in S. dulcamara by GC. Cholesterol and B sitosterol were found in both leaves and cell suspension cultures; stigmasterol was unique to the leaves and campesterol and an unidentified sterol were unique to cell suspensions. Two steroidal alkaloids with similar Rf value on T L C were detected from cell suspension, leaves, stem and roots of S. dulcamara. The biochemical basis of two selection procedures which are being used to isolate steroid overproducer-clones will be discussed. (PB is supported by a post-doctoral fellowship from Government of India.) 1. KHANNA P. and STABA E. J. (1968). Llyodia 31, 180.
2. BHA'Vr P. N., BI-mTT D. P. and SussEx I. M. (1979). Z. Pflanzenphysiol. 95, 355.
During embo~ogenesis, do cells exhibit polarity'prior to morpllological differentiation ? BEW-RLV B. SHELTON, GLEN F. EVANS-and WENDY F. Boss, Botany Department, N.C. State University, Raleigh, NC 27650. Chlorotetracycline ( C T C ) fluorescence has been used to visualize Ca 2+ gradients during pollen tube, fungal hyphae and root hair elongation. ~1~ C T C complexes with Ca 2 ÷ and will fluoresce in a hydrophobic environment such as a membrane. We report here the use of C T C to v i s u a l i z e C a 2+ gradients during somatic cell embryogenesis. Proembryonic masses (PEM's) cultured on wild carrot medium t2) were washed free of 2,4-D. Samples of the resulting embryogenic suspension with embryos at various stages of development were incubated for 10 min in 2 x 10 -4 M aqueous CTC (pH 5.0) at a cell to CTC volume ratio of 1:10. Fluorescence was observed using a Nikon epifluroescence microscope (395-425 nm excitation; 515nm emission). PEM's treated with C T C exhibited localized areas of fluorescence within individual clusters of cells. In some cases entire masses fluoresced and in others there was no fluorescence. Globular embryos produced localized areas of fluorescence often in a pattern suggesting the location of cotyledon and root formation. Localized fluorescence in heart and torpedo stage embryos was seen at the tips of the cotyledons and sometimes at the root tip. These results suggest that Ca 2 + may be a factor in the development of somatic embryo polarity. The use of C T C may enable us to visualize the development of a physiological polarity before polarity is morphologically evident. Experiments are currently in pro~ess to determine if there is any difference in the embryogenic capacity of fluorescing and nonfluorescing PEM's. 1. R~Iss H.-D. and HERTH W. (1979). Planta 146, 615--621. 2. WETg.ERELLD. F. (1969). Plant Physiol. 44, 17341737. Genetic and physiological factors effecting shoot formarion by tomato hypocotyl explants. R. D. Loev and R OvaE, Department of Horticultural
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
426
ABSTRACTS OF CONTRIBUTED MANUSCRIPTS
fractionated on" non-linear sucrose gradients. Homoserine dehydrogenase (EC 1.1.1.3) was identified in those fractions containing intact plastids. Fractions were electrophoresed on 7°'~ polyacrylamide gels and stained for homoserine dehydrogenase activity. Three enzyme forms, two of which were threonine sensitive, were electrophoretically detected in whole "ceff extracts. The two threonine-sensitive forms are localized within the plastid.
Steroids in cell and tissue culture of Solanum nigrum and S. dulcamara. PRASHANT BHATT and DACHA BX-mTT, United States Vegetable Laboratory, 2875 Savannah Highway, Charleston, SC 29407. The feasibility of using a somatic cell genetics approach for increasing the steroidal alkaloid content of certain solanaceous plants is being studied. Callus and cell suspension cultures were initiated from excised leaf explants on revised MS medium supplemented by 2 g/1 m-inositol and l mg/1 2,4-D in callus culture or 0.1 rag/1 2,4-D in cell suspension culture, lt~ For plant regeneration 2,4-D was replaced by various concentrations and combinations of IAA and BA. TM Steroids produced in tissue culture and of whole plants were analyzed by gas chromatography (GC), thin layer chromatography (TLC) and/or colorimetry. Plants of S. dulcamara were regenerated from single cell clones isolated from cell suspension cultures. The steroid concentration of S. nigrum callus culture is positively influenced by the concentration of sucrose and IAA added to the medium. Five sterols were detected in S. dulcamara by GC. Cholesterol and B sitosterol were found in both leaves and cell suspension cultures; stigmasterol was unique to the leaves and campesterol and an unidentified sterol were unique to cell suspensions. Two steroidal alkaloids with similar Rf value on T L C were detected from cell suspension, leaves, stem and roots of S. dulcamara. The biochemical basis of two selection procedures which are being used to isolate steroid overproducer-clones will be discussed. (PB is supported by a post-doctoral fellowship from Government of India.) 1. KHANNA P. and STABA E. J. (1968). Llyodia 31, 180.
2. BHA'Vr P. N., BI-mTT D. P. and SussEx I. M. (1979). Z. Pflanzenphysiol. 95, 355.
During embo~ogenesis, do cells exhibit polarity'prior to morpllological differentiation ? BEW-RLV B. SHELTON, GLEN F. EVANS-and WENDY F. Boss, Botany Department, N.C. State University, Raleigh, NC 27650. Chlorotetracycline ( C T C ) fluorescence has been used to visualize Ca 2+ gradients during pollen tube, fungal hyphae and root hair elongation. ~1~ C T C complexes with Ca 2 ÷ and will fluoresce in a hydrophobic environment such as a membrane. We report here the use of C T C to v i s u a l i z e C a 2+ gradients during somatic cell embryogenesis. Proembryonic masses (PEM's) cultured on wild carrot medium t2) were washed free of 2,4-D. Samples of the resulting embryogenic suspension with embryos at various stages of development were incubated for 10 min in 2 x 10 -4 M aqueous CTC (pH 5.0) at a cell to CTC volume ratio of 1:10. Fluorescence was observed using a Nikon epifluroescence microscope (395-425 nm excitation; 515nm emission). PEM's treated with C T C exhibited localized areas of fluorescence within individual clusters of cells. In some cases entire masses fluoresced and in others there was no fluorescence. Globular embryos produced localized areas of fluorescence often in a pattern suggesting the location of cotyledon and root formation. Localized fluorescence in heart and torpedo stage embryos was seen at the tips of the cotyledons and sometimes at the root tip. These results suggest that Ca 2 + may be a factor in the development of somatic embryo polarity. The use of C T C may enable us to visualize the development of a physiological polarity before polarity is morphologically evident. Experiments are currently in pro~ess to determine if there is any difference in the embryogenic capacity of fluorescing and nonfluorescing PEM's. 1. R~Iss H.-D. and HERTH W. (1979). Planta 146, 615--621. 2. WETg.ERELLD. F. (1969). Plant Physiol. 44, 17341737. Genetic and physiological factors effecting shoot formarion by tomato hypocotyl explants. R. D. Loev and R OvaE, Department of Horticultural