Co-regulation of nitrate reductase and nitrite reductase in cultured spinach cells

The analysis of nitrate reductase(NR)-deficient mutants provides an efficie nt approach to the study of the regulatory mechanisms of nitrate assimilati on. We previously isolated two cell lines, 12F and l(-1), and suggested tha t the mutation in the 12F cell line related to translation of NR mRNA which is expressed in the presence of nitrate, and that the mutation in the l(-1 ) cell line may be in a regulatory gene controlling both genes encoding NR and nitrite reductase (NiR). We investigated transformants of the two cell lines using particle bombardment with tobacco NR cDNA and analyzed the expr ession of NR and NiR in the transformants. In the 12F cell line transforman ts, NR activity and protein was rescued completely and the transformants co uld grow on NO3- medium. This result indicates that the 12F cell line has a mutation that prevents the synthesis of NR protein from NR mRNA. In the l( -1) cell line, the activities of NR and NiR were detected in cells grown on NO3- medium, but at low levels. In the transformants however, activities o f NR and NiR and the NR mRNA levels attained levels of observed in the wild -type cells. In addition. the transformants could also grow on NO3- medium. These results indicate that the l(-1) cell line has a mutation related to the signal transfer cascade from the NO3- ion to the NR gene. This suggests that NR and NiR genes are co-regulated, and that the presence of NR mRNA m ay be post-transcriptionally essential for the synthesis of NiR protein.