The optimum In vivo nitrate reductase (NR) asay medium for soybean (Glycine mar [L.] Merr.) leaves was 50 mm KNO3, 1% (v/v) 1-propanol, and 100 mm potasim phosphate buffer (pH 7.5).Loss of in vivo NR activity from leaves of soybean exposed to dark was fastest at 40 C and slowest at 20 C. However, by the end of a 16-hr dark period, even those plants exposed to the lowest (20 C) temperature had lost 95% of the initial activity. Upon re-exposure to lght, following a 16 hr-30 C dark period, in vivo NR activity increased rapidly to maxmum levels after 4 hr lIght. The rate of increase was proportional to gbht intensity (6, 16, and 45 Iklux) and independent of temperature (20, 30, and 40 C).Studies with field-grown soybeaso indicated that nighttime temperature (16-27 C) had no effect on the subsequent in vivo NR activity in sunlight at ambient temperature. There was a marked decrease in in vivo NR activity in late afternoon with the field-grown plants. This decrease continued throughout the night with elevated temperature (27 C) while NR activity increased when a cooler (16 C) night temperature was imposed.The changes in in vivo NR activity in response to lght and dark treatments were quite rapid and thought to be related to energy limittions as well as enzyme level.

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Growth chamber studies with soybeans (Glycine max [L.] Mef.) were designed to determine the relative limitations of NO3-, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO3-in vivo, -NO3-in vivo, and in vitro) were compared. NR activity decreased with al assays when plants were exposed to dark. Addition of NO3-to the in vivo NR assay medium increased activity (over that of the -NO3-in vivo assay) at aUl sampUng periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO3-was rate-limiting. The stimulation of In vivo NR activity by NO3-was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO3-was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 ,umol NO2-[g fresh weight, hr]-1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting.The addition of NADH to the in vivo NR assay medium did not stimulate NR activity, although it was not established that NADH entered the tissue. The addition of glucose, fructose 1,6-diphosphate, pyruvate, citrate, succinate, or malate to the in vivo assay medium significantly increased measurable NR activity of leaf tissue from plants pretreated to extended dark periods at elevated temperature. Glucose additions were most effective, usually stimulating increases 2-to 3-fold greater than the other metabolites. Increased NR activities from the various additives were attributed to production of NADH. The loss of in vivo NR activity in soybeans during darkness appeared to be due to the combination of a net loss of enzyme per se and energy depletion. The subsequent light stimulation of NR activity was likely due to increased availability of reductant energy as well as a net synthesis of the NR enzyme.Since the original characterization of reductant energy requirements of NR2 (3), many investigators have reported on the capacity of various plant species to utilize NADH as the preferred electron donor for NR (1, 2, 5). Klepper et al. (5) concluded that sugars which migrated from the chloroplasts were the primary source of energy, and that the oxidation of glyceraldehyde 3-P was the in situ source of NADH for nitrate reduction in corn. Malate has also been implicated as an energy source for NADH generation in corn (7). Tingey (10) reported that addition of 48 mm glucose to the incubation medium significantly I Cooperative investigation of the North Central Region, Agricultural

Growth and nodulation response of soybean {Glycine max (L.) Merr.) to various single nitrogen sources in solution culture is confounded by unequal shifts in solution pH. A recirculating ion exchange system was designed in which a cation exchange resin (Amberlite IRC 50) was used to control the pH of solutions in which soybeans were grown. Nutrient solution pH levels were established at range extremes of 9.0 to 3.7 with 100% Ca^+ or H+ forms of resin, respectively. Intermediate pH levels were established by varying the ratio of Ca^+ to H"*" forms of resin. The system is capable of maintaining pH within 0.5 to 0.9 units of the initial pH over a two-week growth period of soybeans with either nitrate-or urea-N sources. In the absence of the resin column, pH of the urea nutrient solution rapidly declined to less than pH 4 which resulted in depressed plant nodule development. The optimum pH range for nodule mass and Nj fixation (measured by acetylene reduction) was between 5.2 and 7.0 with urea nutrition. Both nitrate-and ammonium-N sources were inhibitory to acetylene reduction in comparison with urea which allowed extensive nodule development and activity.