Naomasa Shiraishi

Isolation and Characterization of Nitrate Reductase-Deficient Mutants of Cultured Spinach Cells: Biochemical, Immunological and mRNA Analysis

Summary Nitrate reductase (NR)-deficient mutants of cultured spinach cells were selected by chlorate resistance. Two mutants (12F and I-1 cell lines) were isolated and analyzed for activities, protein levels, and the expression of mRNAs of NR and nitrite reductase (NiR). When mutant cells were transferred to a medium containing 20 mM KNO 3 and 20 mM NH 4 NO 3 as a nitrogen source, the 12F cell line had no detectable NR activity or NR protein, but the NR mRNA was expressed clearly in contrast with the data of activity and protein levels. The NiR activity and protein were still induced and the NiR mRNA was also expressed clearly. In the I-1 cell line, however, the NR activity and protein were still present at low levels, and the NiR activity and protein were no longer inducible. Extremely low levels of the NR and NiR mRNAs were found in the I-1 cell line. These results suggest that the 12F and I-1 cell lines are structural gene mutant and regulatory gene mutant, respectively.

Nitrogen fixation and soil N level during maturation affect the contents of storage compounds of soybean seeds

Abstract We compared the concentrations and contents of protein and oil in mature seeds from nodulated and non-nodulated soybean plants grown on soils with four different N levels during maturation. We observed a positive correlation between the contents of protein and oil in seeds from nodulated plants. Seeds from nodulated plants grown on urea-treated soil showed higher protein and lower oil contents than those from plants grown on soil treated with coated slow release N fertilizer (LP-100). Contents of these compounds in seeds from nodulated plants grown on LP-100 soil were almost the same as those from non-nodulated plants on the same soil. These observations indicated that N economy in roots during seed maturation affects the contents of storage compounds. We suggested that the control of the N2 fixation activity of soybean plants and management of soil N level during seed maturation are important to determine the contents of protein and oil in seeds.

Effects of Glutamine on the Induction of Nitrate Reductase and Nitrite Reductase in Cultured Spinach Cells

Summary Regulation by glutamine of the expression of nitrate reductase (NR) and nitrite reductase (NiR) was investigated in cultured spinach cells. After addition of glutamine to nitrate-containing medium, NR activity was repressed but NiR activity was not affected. Low levels of NiR protein and activity were observed in cells cultured in medium that contained glutamine as the only source of nitrogen. Changes in levels of mRNA for NR and NiR showed patterns similar to those of changes in levels of activities and proteins in glutamine-containing medium. The results suggest that genes for NR and NiR might be co-regulated by nitrate but not by glutamine and, moreover, that expression of genes for NR and NiR might be regulated by different mechanisms in cultured spinach cells.

Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach.

Chemical modifications of spinach leaf nitrate reductase, and its 28,000 M(r) fragment with phenylglyoxal, 2,3-butanedione and pyridoxal phosphate reduce the catalytic activity of the enzyme. The kinetics of the modification indicate a rapid inactivation followed by a slower rate of inactivation. NADH-nitrate reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase activities of the nitrate reductase complex are inactivated at a faster rate when compared to the loss of FMNH2-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28,000 M(r) fragment of nitrate reductase. These data suggest that nitrate reductase contains active sites of arginine and lysine residues that are involved in the NADH binding site of the enzyme.