Summary
The main nitrogen source for most higher plants is soil nitrate. Prior to its incorporation into amino acids, plants reduce nitrate to ammonia in two enzymatic steps. Nitrate is reduced by nitrate reductase to nitrite, which is further reduced to ammonia by nitrite reductase. In this paper, the complete primary sequence of the precursor protein for spinach nitrite reductase has been deduced from cloned cDNAs. The cDNA clones were isolated from a nitrate-induced cDNA library in two ways: through the use of oligonucleotide probes based on partial amino acid sequences of nitrite reductase and through the use of antibodies raised against purified nitrite reductase. The precursor protein for nitrite reductase is 594 amino acids long and has a 32 amino acid extension at the N-terminal end of the mature protein. These 32 amino acids most likely serve as a transit peptide involved in directing this nuclearencoded protein into the chloroplast. The cDNA hybridizes to a 2.3 kb RNA whose steady-state level is markedly increased upon induction with nitrate.
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Allen G (1981) Sequencing of proteins and peptides. In: Work TS, Burdon RH (eds) Laboratory Techniques in Biochemistry and Molecular Biology, vol 9. Elsevier/North-Holland, Amsterdam
Aparicio PJ, Knaff DB, Malkin R (1975) The role of an iron-sulfur center and siroheme in spinach nitrite reductase. Arch Biochem Biophys 169:102–107
Beevers L, Hageman RH (1969) Nitrate reduction in higher plants. Annu Rev Plant Physiol 20:495–522
Broglie R, Coruzzi G, Fraley RT, Rogers SG, Horsch RB, Niedermeyer JG, Fink CL, Flick JS, Chua NH (1984) Light regulation expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science 224:838–843
Cammack R, Hucklesby DP, Hewitt EJ (1978) Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase: Signals from the iron-sulfur centre and haem under turnover conditions. Biochem J 171:519–526
Cheng CL, Dewdney J, Kleinhofs A, Goodman HM (1986) Cloning and nitrate induction of nitrate reductase mRNA. Proc Natl Acad Sci USA 83:6825–6828
Chua NH, Schmidt GW (1978) Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci USA 75:6110–6114
Coruzzi G, Broglie R, Cashmore A, Chua NH (1983) Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptid. J Biol Chem 258:1399–1402
Crawford NM, Cambell WH, Davis RW (1986) Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proc Natl Acad Sci USA 83:8073–8076
Dalling MJ, Hucklesby DP, Hageman RH (1973) A comparison of nitrite reductase from green leaves, scutella and roots of corn. Plant Physiol 51:481–484
Ferrario S, Hirel B, Gadal P (1983) Immunochemical characterization of nitrate reductase from spinach Spinacia oleracea leaves and roots. Biochem Biophys Res Comm 113:733–737
Guerrero MG, Vega JM, Losada M (1981) The assimilatory nitrate-reducing system and its regulation. Annu Rev Plant Physiol 32:169–204
Gupta SC, Beevers L (1984) Synthesis and degradation of nitrite reductase in pea leaves. Plant Physiol 74:251–252
Haynes RJ (1986) Uptake and assimilation of mineral nitrogen by plants. In: Haynes RJ (ed) Mineral Nitrogen in the plant-soil system. Academic Press, Orlando, pp 303–358
Heidecker G, Messing J (1986) Structural analysis of plant genes. Annu Rev Plant Physiol 37:439–466
Hirasawa M, Fukushima K, Tamura G, Knaff DB (1984) Immunochemical characterization of nitrite reductases from spinach leaves, spinach roots and other higher plants. Biochim Biophys Acta 791:145–154
Ho CH, Tamura G (1973) Purification and properties of nitrite reductase from spinach leaves. Agric Biol Chem 37:37–44
Hucklesby DP, Dalling MJ, Hageman RH (1972) Some properties of two forms of nitrite reductase from corn scutellum. Planta 104:220–233
Ishiyama Y, Tamura G (1985) Isolation and partial characterization of homogeneous nitrite reductase from etiolated bean shoots. Plant Sci Lett 37:251–256
Ishiyama Y, Shinoda I, Fukushima K, Tamura G (1985) Some properties of ferredoxin-nitrite reductase from green shoots of bean and an immunological comparison with nitrite reductase from roots and etiolated shoots. Plant Sci 39:89–95
Karlin-Neumann GA, Tobin EM (1986) Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework. EMBO J 5:9–13
Lancaster JR, Vega JM, Kamin H, Orme-Johnson NR, Orme-Johnson WH, Krueger RJ, Siegel LM (1979) Identification of iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR Studies of mechanism. J Biol Chem 254:1268–1272
Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differ in plants and animals. EMBO J 6:43–48
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York
McRee DE, Richardson DC, Richardson JS, Siegel LM (1986) The heme and Fe4S4 cluster in the crystallographic structure of Escherichia coli sulfite reductase. J Biol Chem 261:10277–10281
Mishkind ML, Wessler SR, Schmidt GW (1985) An intermediate in the maturation of the ribulose-1,5-bisphosphate carboxylase small subunit precursor. J Cell Biol 100:226–234
Murphy MJ, Siegel LM, Tove SR, Kamin H (1974) Siroheme: A new prosthetic group participating in six-electron reduction reaction catalyzed by both sulfite and nitrite reductases. Proc Natl Acad Sci USA 71:612–616
Okayama H, Berg P (1982) High efficiency cloning of full length complementary DNA. Mol Cell Biol 2:161–170
Ostrowski J, Back EW, Madden J, Siegel LM, Kredich NM (1987) Comparison of deduced amino acid sequences α and β subunits of S. typhimurium NADPH sulfite reductase with those of spinach nitrite reductase and cytochrome P450 oxidoreductase. Fed Proc 46:2231 (abstr 1780)
Robinson C, Ellis RJ (1984) Transport of proteins into chlorophasts: the precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps. Eur J Biochem 142:343–346
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schreier PH, Seftor EA, Schell J, Bohnert HJ (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J 4:25–32
Serra JL, Ibarlucea JM, Arizmendi JM, Llama MJ (1982) Purification and properties of the assimilatory nitrite reductase from barley (Hordeum vulgare) leaves. Biochem J 201:167–170
Shah DM, Hightower RC, Meagher RB (1982) Complete nucleotide sequence of a soybean actin gene. Proc Natl Acad Sci USA 79:1022–1026
Siegel LM, Wilkerson JO, Janick PA (1987) Structural studies on the siroheme-[4Fe−4S] cluster active centers of spinach ferredoxin-nitrite reductase and Escherichia coli sulfite reductase. In: Uldrich WR, Aparicio PJ, Syrett PJ, Castillo F (eds) Inorganic Nitrogen Metabolism. Springer-Verlag, Heidelberg. In press
Small IS, Gray JC (1984) Synthesis of wheat leaf nitrite reductase de novo following induction with nitrate and light. Eur J Biochem 145:291–297
Smeekens S, De Groot M, Van Binsbergen J, Weisbeek P (1985) Sequence of the precursor of the chloroplast thylakoid lumen protein plastcyanin. Nature 317:456–458
Smeekens S, Bauerle C, Hageman J, Keegstra K, Weisbeek P (1986) The role of the transit peptide in the routing of precursors toward different chloroplast compartments. Cell 46:365–375
Van den Broeck G, Timko MP, Kausch AP, Cashmore AR, Van Montagu M, Herrera-Esterella L (1985) Targeting of foreign proteins to chloroplasts by fusion of the transit peptide from the small subunit of ribulose-1,5-bisphosphate carboxylase. Nature 313:358–363
Vega JM, Kamin H (1977) Spinach nitrite reductase: Purification and properties of a siroheme-containing iron-sulfur enzyme. J Biol Chem 252:896–909
Wega JM, Cardenas J, Losada M (1980) Ferredoxin-nitrite reductase. Methods Enzymol 69:255–270
Wood WI, Gitschier J, Lasky LA, Lawn RM (1985) Base composition-independent hybridization in tetramethylammonium chloride: A method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci USA 82:1585–1588
Young RA, Davis RW (1983) Efficient isolation of genes using antibody probes. Proc Natl Acad Sci USA 80:1194–1198
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Back, E., Burkhart, W., Moyer, M. et al. Isolation of cDNA clones coding for spinach nitrite reductase: Complete sequence and nitrate induction. Molec. Gen. Genet. 212, 20–26 (1988). https://doi.org/10.1007/BF00322440
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DOI: https://doi.org/10.1007/BF00322440