Biochemical characterization of nitrate and nitrite reduction in the wild‐type and a nitrate reductase mutant of soybean

Abstract

Enzyme activities involved in nitrate assimilation were analyzed from crude leaf extracts of wild‐type (cv. Williams) and mutant (nr1) soybean [Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr1 soybean mutant (formerly LNR‐2), had decreased NADH‐NR, FMNH2‐NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH2‐NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH2 as a reductant source. Nitrite reductase activity was normal in the nr1 plant type in the presence of reduced methyl viologen. Assuming that constitutive NR is similar in structure to nitrate reductases from other plants, presence of xanthine dehydrogenase activity and loss of cytochrome c reductase activity indicated that the apoprotein and not the molybdenum cofactor had been affected in the constitutive enzyme of the mutant. Constitutive NR from urea‐grown wild‐type plants had 1) greater ability to use FMNH2 as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO3− and HCO3−, compared with inducible NR from NO3−‐grown nr1 plants. The presence in soybean leaves of a nitrate reductase with a pH optimum of 7.5 is contrary to previous reports and indicates that soybean is not an exception among higher plants for this activity.