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 x 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.
ABSTRACT
NADP+-isocitrate dehydrogenase (NADP+-IDH; EC 1.1.1.42) is involved in the supply of 2-oxoglutarate for ammonia assimilation and glutamate synthesis in higher plants through the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Only one NADP+-IDH form of cytosolic localization was detected in green cotyledons of pine (Pinus spp.) seedlings. The pine enzyme was purified and exhibited molecular and kinetic properties similar to those described for NADP+-IDH from angiosperm, with a higher catalytic efficiency (10(5) M-1 s-1) than the deduced efficiencies for GS and GOGAT in higher plants. A polyclonal antiserum was raised against pine NADP+-IDH and used to assess protein expression in the seedlings. Steady-state levels of NADP+-IDH were coordinated with GS during seed germination and were associated with GS/GOGAT enzymes during chloroplast biogenesis, suggesting that NADP+-IDH is involved in the provision of carbon skeletons for the synthesis of nitrogen-containing molecules. However, a noncoordinated pattern of NADP+-IDH and GS/GOGAT was observed in advanced stages of cotyledon development and in the hypocotyl. A detailed analysis in hypocotyl sections revealed that NADP+-IDH abundance was inversely correlated with the presence of GS, GOGAT, and ribulose-1,5-bisphosphate carboxylase/oxygenase but was associated with the differentiation of the organ. These results cannot be explained by the accepted role of the enzyme in nitrogen assimilation and strongly suggest that NADP+-IDH may have other, as-yet-unknown, biological functions.
