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.

No abstract

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.

The effects of N source (6 mm nitrogen as NOs-or urea) and tungstate (0, 100, 200, 300, and 400 FM Na2WO4) on nitrate metabolism, nodulation, and growth of soybean (Glycine max IL.1 Menf.) plants were evaluated.Nitrate reductase activty and, to a lesser extent, N03-content of leaf tissue decreased with the addition of tungtate to the nutrient growth medium. Concomitantly, nodule mass and acetylene reduction activity of NO3-grown plants Increased with addition of tungstate to the nutrient solution. In contrast, nodule mass and acetylene reduction activity of ureagrown plants decreased with increased nutrient tungstate levels. The acetylene reductin actvity of nodulated roots of NOs--grown plants was less than 10% of the activity of nodulated roots of urea-grown plants when no tungstate was added. At 300 and 400 pM tungstate levels, acetylene reduction actity of nodulated roots of NO3-grown plants exceeded the activity of comprable urea-grown plants.Addition of tungstate to the nutrient solution decreased plant growth, regardless of the N source, although the effect was more pronounced with NOs-nutrition. The Increased nodulation and decreased nitrate reductase activity noted with plants grown In the presence of tungstate and a high (6 mM) external supply of NO. suggests that N03-does not directly inhibit nodulatin but rather affects nodulation indirectly through subsequent metabolism of NO5.The inhibitory effect of NO3 on nodulation of soybean is well documented (2). Munns (6) and Trifolium subterraneum L., respectively) any better than did parent strains with nitrate reductase activity. They concluded that it was unlikely that reduction of N03-to N02 by the rhizobia played a significant role in the inhibition of nodulation by NO3-. The more marked inhibitory effect of NO3 than of urea on soybean nodulation (4) also suggested a more direct effect of N03-on nodulation. However, urea was taken up more slowly than N03 (12), and the more marked inhibition by N03 may still involve a less favorable C:N ratio. Therefore, the mechanism of inhibition of nodulation by combined N is not clear.The known inhibitory effect of tungstate on nitrate reductase activity in higher plants (5,8,10) suggested that the addition of tungstate may be a means of separating the direct and indirect effects of N03 on nodulation. The inhibition of nitrate reductase by tungstate results from the formation of a tungstate protein analog lacking nitrate reductase activity (8). Rao and Rains (10) showed that tungstate had no effect on N03 uptake in short term (3 hr) studies with barley, although it completely inhibited nitrate reductase activity.Data concerning effects of tungstate on nodulated legumes are limited. Quin and Hoglund (9) reported that 5 ,ug/g tungstate in nutrient solution slightly depressed growth and N accumulation of white clover (Trifolium repens L.) which was solely dependent on atmospheric N2 as the N source. In a field study with white clover, Davies and Stockdili (1) showed that addition of tungstate without molybdat...

An in vivo assay was used to determine if differences existed in nitrate reductase activity among 16 soybean (Glycine max L. Merr.) varieties within four maturity groups. Canopy and seasonal profiles of nitrate reductase activity were determined. All varieties exhibited highest nitrate reductase activity per leaf ✕ nr in upper leaves of the plant canopy during growth stages up to mid pod fill. Activity levels were similar throughout the plant leal canopy following mid pod fill. Nitrate reductase activity per leaf × hr at a given node was generally highest the initial date at which the leaf was sampled and activity declined with leaf age. Mean nitrate reductase activity (µmoles NO‐2 formed per g fresh wt × hr) for the entire leaf canopy was highest in the seedling stage for all maturity groups and declined as the plants matured. Activity of the total plant (µmoles NO‐2 formed per plant × hr) was maximal at approximately the full bloom stage with all maturity groups, irrespective of the calendar date at which the maturity groups attained full bloom. Thus, enzyme activity appeared to be closely associated with physiological growth stage. Genetic variation (up to 1.8 fold) in enzyme activity (seasonal means of µmoles NO‐2 formed per plant × hr) was found within maturity groups II, III, and IV. However, enzyme activity did not correlate with either seed yield or seed protein content.

No abstract

Experiments were conducted to determine if nitrate ("5N-labeled) was taken up and assimilated by intact soybean (Glyciae max [L.] Merr. cv Williams) plants during extended periods of dark. Nitrate was taken up by soybean roots throughout a 12-hour dark period. The '5N-labeled nitrogen was also translocated to the plant shoots, but at a slower rate than the rate of accumulation in the roots. Much of the nitrogen ("5N-labeled) was present in a nonreduced form, although considerable solublereduced nitrogen ('5N-labeled) accumulated throughout the dark period. The '5N-labeled, soluble-reduced nitrogen fraction accounted for nearly 30% of the total '5N found in plant roots and more than 63% of the total '5N found in plant tops after 12 hours of dark. This provided evidence that intact soybean plants take up and metabolize significant quantities of nitrate to reduced N forms in the dark.In addition to nitrate influx during the dark, it was shown that there was a concomitant loss of '5N-labeled nitrogen compounds from previously '5N-labeled plants to a natural abundance '5N nutrient solution. Thus, evidence was obtained which indicated that light was not directly essential for flux and reduction of nitrate by intact soybean plants.Initial investigations concerning diurnal NRA' in higher plants indicated that assimilation of NO3-and NO2-to reduced N compounds (amino acids) was highly dependent on light and was negligible in darkness (2). More recent studies (1,6,7,8,10,15,19) have reported substantial rates of "5NO3 and '5NO2 assimilation in the dark in a wide variety of plant species. Evidence for dark assimilation of N into reduced N compounds has been derived from NR assay methods (5,6,16,19), from xylem exudate analysis (10), and from analysis of modified plant parts such as excised leaves (8), or excised roots (10). Evidence that soybeans are capable ofNO3-metabolism in the dark comes from in vivo assays on leaf sections where '5NO3 was shown to be fully reduced to amino N (15). However, no data was found in the literature to indicate that intact soybean plants were capable of reducing NO3-to the amino level in the dark.In the present study, hydroponically grown soybean plants were subjected to changes in '5N enrichment immediately prior to a dark cycle. Whole plants, plant parts, and nutrient solutions were analyzed for '5N content to determine flux and translocation of NO3-in the dark, and to establish that NO3-was fully reduced by intact soybeans in the dark.