Expression of a conifer glutamine synthetase gene in transgenic poplar

Abstract. The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887–898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tremula × P. alba) via Agrobacterium tumefaciens. Transformed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The presence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.

[1]  P. Lea.  7 – Primary Nitrogen Metabolism , 1997 .

[2]  F. Cánovas,et al.  Glutamine synthetase activity during the ripening of tomato , 1988 .

[3]  M. Vincentz,et al.  Adaptations of Photosynthetic Electron Transport, Carbon Assimilation, and Carbon Partitioning in Transgenic Nicotiana plumbaginifolia Plants to Changes in Nitrate Reductase Activity , 1994, Plant physiology.

[4]  M. Bevan,et al.  Binary Agrobacterium vectors for plant transformation. , 1984, Nucleic acids research.

[5]  N. Kawakami,et al.  Senescence-specific increase in cytosolic glutamine synthetase and its mRNA in radish cotyledons. , 1988, Plant physiology.

[6]  F. Cánovas,et al.  High‐level expression of Pinus sylvestris glutamine synthetase in Escherichia coli , 1996, FEBS letters.

[7]  S. Chaillou,et al.  Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development , 1997, Planta.

[8]  S. Chaillou,et al.  Manipulating the pathway of ammonia assimilation in transgenic non-legumes and legumes , 1997 .

[9]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[10]  B. Miflin,et al.  4 – Ammonia Assimilation , 1980 .

[11]  C. Foyer,et al.  Modulation of carbon and nitrogen metabolism in transgenic plants with a view to improved biomass production. , 1994, Biochemical Society transactions.

[12]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[13]  F. Cánovas,et al.  Two different modes of early development and nitrogen assimilation in gymnosperm seedlings , 1998 .

[14]  M. R. Ahuja.  In Vitro Propagation of Poplar and Aspen , 1987 .

[15]  F. Cánovas,et al.  Differential expression of glutamine synthetase isoforms in tomato detached leaflets infected with Pseudomonas syringae pv. tomato , 1995 .

[16]  A. Migge,et al.  A role for cytosolic glutamine synthetase in the remobilization of leaf nitrogen during water stress in tomato , 1997 .

[17]  U. K. Laemmli,et al.  Cleavage of structural proteins during , 1970 .

[18]  F. Cánovas,et al.  The occurrence of glutamine synthetase isoenzymes in tomato plants is correlated to plastid differentiation , 1990 .

[19]  G. Edwards,et al.  Expression of maize phosphoenolpyruvate carboxylase in transgenic tobacco : effects on biochemistry and physiology. , 1992, Plant physiology.

[20]  G. Coruzzi,et al.  Use of Arabidopsis mutants and genes to study amide amino acid biosynthesis. , 1995, The Plant cell.

[21]  D. Cole,et al.  Elemental cycling in forest ecosystems , 1981 .

[22]  G. Coruzzi,et al.  Ectopic Overexpression of Asparagine Synthetase in Transgenic Tobacco , 1993, Plant physiology.

[23]  V. Walbot,et al.  Stable transformation of maize after gene transfer by electroporation , 1986, Nature.

[24]  G. Coruzzi,et al.  THE MOLECULAR-GENETICS OF NITROGEN ASSIMILATION INTO AMINO ACIDS IN HIGHER PLANTS. , 1996, Annual review of plant physiology and plant molecular biology.

[25]  R. Wetmore,et al.  FERN CALLUS TISSUE CULTURE , 1951 .

[26]  D. Ort,et al.  Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts. , 1984, The Journal of biological chemistry.