Plant Physiol, June 2001, Vol. 126, pp. 731-741
Nitrite Reductase Gene Enrichment Improves Assimilation of
NO2 in Arabidopsis1
Misa
Takahashi,
Yukari
Sasaki,
Shoji
Ida, and
Hiromichi
Morikawa*
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.
1
This work was supported in part by the program
Research for the Future from the Japanese Society for the Promotion of
Science (no. JSPS-RFTF96L00604) and by a Grant-in-Aid for Scientific
Research on Priority Areas (no. 05266213) from the Ministry of
Education, Science, Culture and Sports, Japan. We also gratefully
acknowledge financial support from the Naito Foundation and Electric
Technology Research Foundation of Chugoku.
*
Corresponding author; e-mail hmorikaw{at}sci.hiroshima-u.ac.jp;
fax 81-824-24-0749.
© 2001 American Society of Plant Physiologists
