Abstract
Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) γ polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia β-F1 ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS γ polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the β-F1 ATPase has successfully targeted the GS γ polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the γ polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the γ polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.
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References
Baulcombe DC, Saunders GR, Bevan MW, Mayo MA, Harrison BD: Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants. Nature: 321: 446–449 (1986).
Bennett MJ, Cullimore JV: Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules. Planta 179: 433–440 (1989).
Bennett MJ, Cullimore JV: Expression of three plant glutamine synthetase cDNAs in Escherichia coli: Formation of catalytically active isoenzymes and complementation of a glnA mutant. Eur J Biochem: in press (1990).
Bennett MJ, Lightfoot DA, Cullimore JV: cDNA sequence and differential expression of the gene encoding the glutamine synthetase γ polypeptide of Phaseolus vulgaris L. Plant Mol Biol 12: 553–565 (1989).
Bevan MW: Binary Agrobacterium vectors for plant transformation. Nucl Acids Res 12: 8711–8721 (1984).
Boutry M, Chua N-H: A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia. EMBO J 4: 2159–2165 (1985).
Boutry M, Faber A-M, Charbonnier M, Briquet M: Microanalysis of plant mitochondrial protein synthesis products: detection of various polypeptides associated with cytoplasmic male sterility. Plant Mol Biol 3: 445–452 (1984).
Boutry M, Nagy F, Poulsen C, Aoyagi K, Chua N-H: Targeting of bacterial chloramphenicol acetyl transferase to mitochondria in transgenic plants. Nature 328: 340–342 (1987).
Chen F-L, Cullimore JV: Two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L. Plant Physiol 88: 1411–1417 (1988).
Dellaporta S, Wood J, Hicks J: A rapid method for DNA extraction from plant tissue. Plant Mol Biol Rep 1: 19–21 (1983).
Dewey RE, Timothy DH, LevingsIII CS: A mitochondrial protein associated with cytoplasmic male sterility in the T cytoplasm of maize. Proc Natl Acad Sci USA 84: 5374–5378 (1987).
Eckes P, Schmitt P, Daub W, Wengenmayer F: Overproduction of alfalfa glutamine synthetase in transgenic tobacco plants. Mol Gen Genet 217: 263–268 (1989).
Ellis RJ, Robinson C: Protein targeting. Adv Bot Res 14: 1–24 (1987).
Feinberg A, Vogelstein B: A technique for labelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13 (1983).
Forde BG, Cullimore JV: The molecular biology of glutamine synthetase in higher plants. In: Miflin BJ (ed) Oxford Surveys of Plant Molecular and Cell Biology, Oxford University Press, Oxford, pp 247–296 (1989).
Givan CV, Joy KW, Kleczkowski LA: A decade of photorespiratory nitrogen cycling. TIBS 13: 433–437 (1988).
Horsch RB, Fry JE, Hoffman NL, Eichholtz D, Rogers SG, Fraley RT: A simple and general method for transferring genes into plants. Science 227: 1229–1231 (1985).
Keys AJ, Bird IF, Cornelius MJ, Lea PJ, Wallsgrove RM, Miflin BJ: Photorespiratory nitrogen cycle. Nature 275: 741–743 (1978).
Kobayashi K, Iwasaki Y, Sasaki T, Nakamura K, Asahi T: Putative amino-terminal presequence for β-subunit of plant mitochondrial F1 ATPase deduced from the aminoterminal sequence of the mature subunit. FEBS Lett 203: 144–148 (1986).
Maniatis T, Fritsch E, Sambrook J: Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982).
McNally SF, Hirel B, Gadal P, Mann AF, Stewart GR: Glutamine synthetases of higher plants. Plant Physiol 72: 22–25 (1983).
Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue culture. Plant Physiol 15: 473–497 (1962).
Robinson C: Targeting of proteins to chloroplasts and mitochondria. In: Grierson D (ed) Plant Genetic Engineering. Blackie Press, Glasgow, in press (1990).
Schmitz UK, Lonsdale DM: A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo. Plant Cell 1: 783–791 (1989).
Tingey SV, Coruzzi GM: Glutamine synthetase of Nicotiana plumbaginifolia: cloning and in vivo expression. Plant Physiol 84: 366–373 (1987).
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Hemon, P., Robbins, M.P. & Cullimore, J.V. Targeting of glutamine synthetase to the mitochondria of transgenic tabacco. Plant Mol Biol 15, 895–904 (1990). https://doi.org/10.1007/BF00039428
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DOI: https://doi.org/10.1007/BF00039428