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Reactive nitrogen gases progressively contribute to the global warming. Development of gas-gas denitrifying plants that can efficiently reduce reactive nitrogen gases to dinitrogen (N2) could help to mitigate the effect of these gases. Taking the advances in gene manipulation technology, tobacco BY-2 cells were transformed with the fungus Cylindrocarpon tonkinense cytochrome P-450nor2 (Cnor2) gene. The product of this gene acts as nitric oxide reductase (nor). Transgenic BY-2 cell clones cultured in 15N-labelled nitrate (15NO3-) actively evolved 15N2O gas up to 35-folds compared to the wild-type cells. In 15N-labelled ammonium (15NH4+), the transgenic and wild-type cells produced comparable amounts of 15N2O. This indicates that ammonium is not a direct substrate for nor and the small amount of N2O observed may be due to the nitrification of ammonium to nitrite. Addition of tungstate (a nitrate reductase inhibitor) and cyanide to the transgenic cell cultures strongly inhibited 15N2O production. Activity of nor enzyme was also confirmed by in vitro activity assay. These observations together suggest that Cnor2 is actively expressed and enhanced the reduction of nitrate to N2O in plant cells. This finding indicates that plant cells are capable to tackle the denitrification pathway.
The biological function of Reactive Nitrogen Species (RNS) is not well understood, however, they actively contribute to the effect of green house gases. Development of plants that could efficiently denitrify intermediates of the RNS to the dinitrogen (N2) is a rationale that could help amelioration the effect of these gases. Fusarium oxysporum cytochrome P-450 nor gene (Fnor) was constitutively expressed in tobacco BY-2 cells. The gene product functions as nitric oxide reductase (nor), which catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N2O) in the fungal denitrification pathway. Intact transgenic BY-2 cells cultured in 15N-labeled nitrate (15NO3¯) actively produced 15N2O gas up to 59 folds higher than the wild-type cells. Activity of the enzyme was also confirmed by an in vitro nor activity assay. Tungstate (a nitrate reductase inhibitor) and cyanide (an inhibitor of the last protein complex of electron transport chain) strongly inhibited 15N2O production. These observations together suggest that Fnor enhanced the reduction of nitrate to N2O in plant cells. This finding indicates that plant cells are capable to tackle the denitrification pathway.
