Abstract
Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C16) from a tobacco λgt11 expression library. A longer Fd-GOGAT cDNA clone (C35) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco λgt10 cDNA library using C16 as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C35 was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.
Similar content being viewed by others
Abbreviations
- bp:
-
base pairs
- Fd-GOGAT:
-
ferredoxin-dependent glutamate synthase
- GS:
-
glutamine synthetase
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulfate
References
Avila, C., Botella, J.R., Canovas, F.M., Nuniez de Castro, I.N., Valpuesta, V. (1987) Different characteristics of the two glutamate synthases in the green leaves of Lycopersicon esculentum. Plant Physiol. 85, 1036–1039
Becker, T.W., Caboche, M., Carrayol, E., Hirel, B. (1992) Nucleotide sequence of a tobacco cDNA encoding plastidic glutamine synthetase and light inducibility, organ specificity and diurnal rhythmicity in the expression of the corresponding genes of tobacco and tomato. Plant Mol. Biol. (in press)
Berger, M.G., Fock, H.P. (1983) Effects of methionine sulfoximine and glycine on nitrogen metabolism of maize in the light. Aust. J. Plant Physiol. 10, 187–194
Calza, H., Huttner, E., Vincentz, M., Rouzé, P., Galangau, F. Vaucheret, H., Chérel, I., Meyer, C., Kronenberger, J., Caboche, M. (1987) Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductase from higher plants. Mol. Gen. Genet. 209, 552–562
Coïc, Y., Lesaint, C. (1975) Comment assurer une bonne nutrition en eau et ions minéraux en horticulture. Hortic. Franç. 8, 11–14
Dellaporta, S., Wood, J., Hicks, J. (1983) A plant DNA mini-preparation: version II. Plant Mol. Biol. Rep. 1, 19–21
Elmlinger, M.W., Mohr, H. (1991) Coaction of blue/ultraviolet-A light and light absorbed by phytochrome in controlling the appearance of ferredoxin-dependent glutamate synthase in the scots pine (Pinus sylvestris L.) seedling. Planta 183, 374–380
Goodspeed, G.H. (1954) The genus Nicotiana. Chron. Bot. 16, 372–375
Hirel, B., Vidal, J., Gadal, P. (1982) Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves. Planta 155, 17–23
Kendall, A.C., Wallsgrove, R.M., Hall, N.P., Turner, J.C., Lea, P.J. (1986) Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase. Planta 168, 316–323
Keys, A.J., Bird, I.F., Cornelius, M.J., Lea, P.J., Wallsgrove, R.M., Miflin, B.J. (1978) Photorespiratory nitrogen cycle. Nature 275, 741–743
Laemmli, U.K., (1974) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lea, P.J., Miflin, B.J. (1974) An alternative route for nitrogen assimilation in higher plants. Nature 251, 614–616
Lederer, F., Cortial, S., Becam, A.M., Haumont, P.Y., Perez L. (1985) Complete amino acid sequence of flavocytochrome b2 from baker's yeast. Eur. J. Biochem. 152, 419–428
Maniatis, T., Fritsh, E.F., Sambrock, J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor laboratory press, New York
Martin, F., Suzuki, A., Hirel, B. (1982) A new high-performance liquid chromatography assay for glutamine synthetase and glutamate synthase in plant tissue. Anal. Biochem. 125, 24–29
Matoh, T., Suzuki, F., Ida. (1979) Corn leaf glutamate synthase: purification and properties of the enzyme. Plant Cell Physiol. 20, 1329–1340
Mayhew, S.G. (1971) Non-denaturing procedure for rapid preparation of ferredoxin from Clostridium pasteurianum. Anal. Biochem. 42, 191–194
Miflin, B.J., Lea, P.J. (1982) Ammonia assimilation and amino acid metabolism. In: Encyclopedia of plant physiology, N.S. pp, 5–54, Boulter, D., Parthier, B., eds. Springer Verlag, Berlin
Olivier, G., Gosset, G., Sanchez-Pescador, R., Lozoya, E., Ku, L.M., Flores, N., Becerill, B., Valle, F., Bolivar, F. (1987) Determination of the nucleotide sequence for the glutamate synthase structural gene of Escherichia coli K-12. Gene 60, 1–11
Sakakibara, H., Watanabe, M., Hase, T., Sugiyama, T. (1991) Molecular cloning and characterization of complementary DNA encoding for ferredoxin-dependent glutamate synthase in maize leaf. J. Biol. Chem. 266, 2028–2035
Sanger, F., Nicklen, S., Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467
Scopes, R.K. (1974) Measurement of proteins by spectrometry at 205 nm. Anal. Biochem. 59, 277–282
Somerville, C.R., Ogren, W.L. (1980) Inhibition of photosynthesis in Arabidopsis mutants lacking leaf glutamate synthase activity. Nature 286, 257–259
Southern, E. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517
Stewart, G.R., Mann, A.F., Fentem, P.A. (1980) Enzymes of glutamate formation: glutamate dehydrogenase, glutamine synthetase and glutamate synthase. In: The biochemistry of plants, pp. 271–327, Miflin, B.J., ed. Academic Press, London New York
Suzuki, A., Gadal, P. (1984) Glutamate synthase: physicochemical and functional properties of different forms in higher plants and other organisms. Physiol. Vég. 22, 471–461
Suzuki, A., Vidal, J., Gadal, P. (1982) Glutamate synthase from rice leaves. Plant Physiol. 69, 848–852
Suzuki, A., Oaks, A., Jacquot, J.P., Vidal, J., Gadal, P. (1985) An electron transport system in maize for reactions of glutamate synthase and nitrite reductase. Plant Physiol. 78, 374–378
Suzuki, A., Audet, C., Oaks, A. (1987) Influence of light on the ferredoxin-dependent glutamate synthase in maize leaves. Plant Physiol. 84, 578–581
Suzuki, A., Carrayol, E., Zehnacker, C., Deroche, M.E. (1988) Glutamate synthase in Medicago saliva L. Occurrence and properties of the Fd-dependent enzyme in plant cell fraction during root nodule development. Biochem. Biophys. Res. Commun. 156, 1130–1138
Tamura, G., Kanki, M., Hirasawa, M., Oto, M. (1980) The purification and properties of glutamate synthase from spinach leaves, and its dependence on ferredoxin. Agric. Biol. Chem. 44, 925–927
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354
Trotta, P.P., Platzer, K.E.B., Haschmeyer, R.H., Meister, A. (1974) Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proc. Natl. Acad. Sci. USA 71, 4607–4611
Verwoerd, T.C., Dekker, B.M., Hoekema, A. (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucl. Acid. Res. 17, 2362
Wallsgrove, R.M., Harel, E., Lea, P.J., Miflin, B.J. (1977) Studies on glutamate synthase from the leaves of higher plants. J. Exp. Bot. 28, 588–596
Wallsgrove, R.M., Lea, P.J., Miflin, B.J. (1982) The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves. Planta 154, 473–476
Young, R.A., Davis, R.W. (1983) Efficient isolation of genes by using antibody probes. Proc. Natl. Acad. Sci. USA 80, 1194–1198
Author information
Authors and Affiliations
Additional information
The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.
Rights and permissions
About this article
Cite this article
Zehnacker, C., Becker, T.W., Suzuki, A. et al. Purification and properties of tobacco ferredoxin-dependent glutamate synthase, and isolation of corresponding cDNA clones. Planta 187, 266–274 (1992). https://doi.org/10.1007/BF00201950
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00201950