DIVERSITY OF NITRITE REDUCTASE GENES AND THEIR EXPRESSION IN TOBACCO

A ferredoxin-nitrite reductase (NiR), which catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway, is localized in chloroplasts in leaves and plastids in roots. Its gene is nuclear-encoded. The number of NiR genes varies among plant species. We therefore first cloned a partial cDNA of this gene, and analyzed expression of the "leaf-(nii1 and nii3)" and " root-(nii2 and nii4)" types of NiR genes in tobacco by the competitive RT-PCR. The ratio of mRNA level of "leaf-" to "root-" type appeared to be 3：1 in leaves before induction by nitrate, and this value was not changed after nitrate induction, suggesting that "root-type" NiR plays an important role in leaves as well. On the other hand, this ratio in roots was 3：100 both before and after nitrate induction, indicating that NiR in roots is almost exclusively "root-type". We studied reduction of 15N-labeled nitrate in the antisense transgenic tobacco (clone 271) (Vaucheret et al. , 1992) in which root-type NiR (nii1) cDNA antisense expressed and 98% of NiR activity is lost. The transgenic tobacco had the same ability to assimilate nitrate as that of wild-type tobacco. This suggests that 98% of NiR activity is not used to assimilate nitrate in wild type tobacco. In the transgenic tobacco the expression of leaf-type of NiR genes was reduced, but the expression of root-type of NiR expression was same as that of wild-type tobacco. This suggests that nitrate reduction in the transgenic tobacco is done by the root-type NiR genes (nii2 and nii4).