Plant Physiol, June 2001, Vol. 126, pp. 731-741

Nitrite Reductase Gene Enrichment Improves Assimilation of NO₂ in Arabidopsis¹

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, 739-8526 Japan (M.T., Y.S., H.M.); and Research Institute for Food Science, Kyoto University, Uji, Kyoto, 611-0011 Japan (S.I.)

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 + Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO₂ assimilation by analysis of the reduced nitrogen derived from NO₂ (NO₂-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 = 0.74), between the total NiR protein and NiRA (r = 0.71), and between NiRA and NO₂-RN (r = 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P < 0.01), and three had significantly higher NO₂-RN (P < 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 NO₂ 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 NO₂ assimilation by plants.