Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach ϩ Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO 2 assimilation by analysis of the reduced nitrogen derived from NO 2 (NO 2 -RN). Twelve independent transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between the total NiR mRNA and total NiR protein contents (r ϭ 0.74), between the total NiR protein and NiRA (r ϭ 0.71), and between NiRA and NO 2 -RN (r ϭ 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P Ͻ 0.01), and three had significantly higher NO 2 -RN (P Ͻ 0.01). Each of the latter three had one to two copies of spinach NiR cDNA per haploid genome. The NiR flux control coefficient for NO 2 assimilation was estimated to be about 0.4. A similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR is a controlling enzyme in NO 2 assimilation by plants.

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