Use of Protein in Extraction and Stabilization of Nitrate Reductase

The study presented here was an extension of a preceding field project concerned with changes in N metabolism of four maize hybrids during grain development. The objectives were to relate uptake, flux, and reduction of nitrate to accumulation of reduced N in growth-chamber-grown seedlings of the same four hybrids and to compare these results with those obtained in the field study.Hybrid D took up more nitrate than the other three hybrids, primarily because of a larger root system. The correlations between total N (nitrate plus reduced N plant-) accumulated by harvest and root dry weight or shoot to root ratios were r = +0.97 and -0. In wheat and corn, significant correlations between the estimated amount of reduced N supplied to the plant (by the in vitro or in vivo NR3 assay) and the actual amount accumulated by the plant (2,5,7,8) support the concept that nitrate reduction is the rate-limiting step in the assimilation of nitrate to reduced N. Significant correlations were found between integrated seasonal leaf NRA and grain yield, plant reduced N, and grain reduced N for maize. In these and other experiments, the correlation values were low, indicating that factors other than the level of leaf NRA affect these relationships.Dalling et al. (5) showed that transport of vegetative N to the grain of wheat was one of these factors. They also found that wheat cultivars with comparable levels of NRA could accumulate different amounts of reduced N. Deckard et al. (6) identified one maize genotype with a relatively low NRA but a high capacity to accumulate reduced N. The fact that this genotype had a high leaf nitrate concentration suggests that the availability of nitrate to NR in the leaf, as well as the level of enzyme, may affect the rate of nitrate reduction (3). This possibility is supported by (a) the stimulation of the in vivo NR assay with nitrate (10), and (b) the correlation found between nitrate uptake and accumulation of reduced N in several maize genotypes (4).Although nitrate flux to the leaves of maize regulates the level of NR (21), it has not been shown that nitrate flux and NR are directly related among genotypes. Since the nitrate flux provides both substrate for and inducer of NR, genotypic differences in the partitioning of nitrate and/or responsiveness of the induction mechanism to nitrate could explain the discrepancy observed for the one genotype in the study by Deckard et al. (7).The study presented here was an extension of a preceding field project that utilized the same four maize hybrids (19). The field study was concerned with changes in N metabolism (enzymes and N components) in various plant parts during grain development.The objectives of the current study were: (a) to relate nitrate uptake, nitrate flux, shoot to root partitioning of nitrate reduction, and NRA to the accumulation of reduced N by the plants during early vegetative growth; and (b) to compare these results with seedlings to those obtained in the preceding field study.

Section: Resultsmentioning

confidence: 80%

mentioning

confidence: 99%

Relationship between Nitrate Uptake, Flux, and Reduction and the Accumulation of Reduced Nitrogen in Maize (Zea mays L.)

Reed

Hageman²

1980

“…The overestimation would be anticipated as the optimum conditions used for the in vitro assay would seldom be obtained in situ. The underestimation which is most common with mature leaf tissue could result from exposure to NR to inhibitors or proteases during extraction as indicated by the work of Schrader et al (15) and Wallace (17).…”

mentioning

confidence: 99%

Comparison of in Vivo and in Vitro Assays of Nitrate Reductase in Wheat (Triticum aestivum L.) Seedlings

Brunetti¹,

Hageman²

1976

110

The effectiveness of the in vivo and in vitro assays for nitrate reductase (NR) in estimating the amounts of reduced N made available to plants was tested against the daily increases in reduced N (Nesslerization) actually accumulated by the plant. With growth-chamber-grown wheat seedlings, the average ratio values (input of reduced N as estimated by the in vitro assay to actual accumulation of N by the plant) were 3.9 for shoots, 3.7 for the roots, and 4.1 for the entire plant, over a 10-day period. With the in vivo assay, the average ratio values were 0.7 for the shoot, 1.8 for the root, and 0.9 for the entire plant. Although the linear regressions between the accumulated N in the plant and the estimated N input (by both in vitro and in vivo assays) were significant and positive, the in vivo assay provided the closest approximation of the actual amount of N accumulated.The in vivo NR assay effectively distinguished between two wheat varieties. The variety known to have the higher percentage of seed protein also had the higher amounts of NR activity.With seedling wheat leaves, the addition of NADH plus a surfactant increased in vivo NR activity approximately 2-fold over comparable controls. Because the tissue contained high levels of nitrate and enzyme, we concluded that reducing potential was the rate-limiting factor in nitrate reduction in situ in these growth-chamber-grown plants.Because NR3 is the rate-limiting step of NO3-reduction to NH4+ in cereals, the assimilation of NO3-can be used to estimate the amount of reduced nitrogen made available to the plant. This is supported by various studies with wheat (2, 4) and corn (3) that show a highly significant correlation between the amount of N supplied to the plant as estimated by the in vitro NR assay and the actual amount of N accumulated by the plant. Although, in most cases, the ratio of estimated N exceeds the actual accumulated N by a factor of 2 to 8, values as low as 0.6 were found during the reproductive phase of wheat (4). The overestimation would be anticipated as the optimum conditions used for the in vitro assay would seldom be obtained in situ. The underestimation which is most common with mature leaf tissue could result from exposure to NR to inhibitors or proteases during extraction as indicated by the work of Schrader et al. (15) and Wallace (17).

“…Nitrate reductase was extracted from nitrate-treated pea leaves or roots with a Tekmar homogenizer using 7 ml of grinding medium/g fresh weight of materials as described by Hageman and Hucklesby (5) except that 3% (w/v) casein was added to replace the 10 mm cysteine (11), and the solution was adjusted to pH 8.8. Root extracts were passed through Sephadex G-25 to Plant Physiol.…”

mentioning

confidence: 99%

Induction of Root Nodule Senescence by Combined Nitrogen in Pisum sativum L

Chen

Phillips²

1977

Root nodule senescence induced by nitrate and ammonium in Pisum sativum L. was defined by determining nitrogenase activity and leghemoglobin content with the acetylene reduction and pyridine hemochrome assays. Root systems supplied with 100 mM KNO3 or 100 mM NH4CI exhibited a decrease in nitrogenase activity foUlowed by a decline in leghemoglobin content. Increasing the CO2 concentration from 0.000320 atm to 0.00120 atm had no effect on the time course of root nodule senescence when 20 mM KNO3 was supplied to the roots; in vitro nitrate reductase activity was detected in leaves and roots, but not bacteroids. Nitrate appeared in leaves, roots, and the nodule cytosol fraction but not bacteroids when 20 mM KNO3 was supplied to roots. When nitrate entered through the shoots, however, no root nodule senescence was observed, and no nitrate was detected in root or nodule cytosol fractions although nitrate and nitrate reductase were found in leaves. The results suggest that nitrate does not induce root nodule senescence through competition between nitrate reductase and nitrogenase for products of photosynthesis.Field-grown soybeans reportedly obtain only 25% of their nitrogen from N2 (6). The remaining nitrogen is drawn from soil reserves of this element. Understanding the manner in which nitrate and ammonium decrease biological N2 reduction in legume root nodules is important for devising strategies for maximizing N2 fixation.One method for studying interactions between combined nitrogen and N2 fixation is to supply excess nitrate or ammonium to legumes and examine the effect on root nodule functioning. Morphological effects of supplying NH4NO3 to root nodules have been described (2), but the physiological basis of this response has not been elucidated. Small and Leonard (12) Nitrogen treatments were started 28 days after planting the peas. Each 15-cm diameter pot containing five plants was treated with 500 ml of the appropriate nitrogen-containing solution in experiments utilizing root-supplied nitrogen. When nitrate was supplied to the shoot, the apical meristem was removed, and the entire shoot was dipped into a 1-liter solution containing 3 g KNO and 0.5 ml Triton X-100 for 4 hr. Control plants were treated with a solution containing only Triton X-100.In vivo nitrogenase activity of intact pea plants was measured with the acetylene reduction assay (3). Pea nodules were extracted with 0