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 ( nr 1 ) soybean [ Glycine max (L.) Merr.] plants lacking constitutive nitrate reductase (NR) activity. The nr 1 soybean mutant (formerly LNR‐2), had decreased NADH‐NR, FMNH 2 ‐NR and cytochrome c reductase activities, all of which were associated with the loss of constitutive NR activity. Measurement of FMNH 2 ‐NR activity, by nitrite determination, was accurate since nitrite reductase could not use FMNH 2 as a reductant source. Nitrite reductase activity was normal in the nr 1 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 FMNH 2 as an electron donor, 2) a lower pH optimum, and 3) decreased ability to distinguish between NO 3 − and HCO 3 − , compared with inducible NR from NO 3 − ‐grown nr 1 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.