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

Abstract

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).

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

“…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.…”

“…In Vivo. The leaf in vivo NR assay was the same as previously described (2), except 0.04% (v/v) Neutronyx 600 (nonionic surfactant, Onyx Chemicals, Jersey City, N. J.)…”

“…Plant samples were dried for 3 days in a forced-air oven at 60 C. Total reduced N of the plant parts was determined in the powdered samples as described (2). Nitrate was measured as described (I 1).…”

Molecular and physiological responses of diatoms to variable levels of irradiance and nitrogen availability: Growth of Skeletonema costatum in simulated upwelling conditions

“…Nitrate reductase activity-Nitrate reductase activity (NRA) was measured with an in vivo assay that measured NO,-production by alcohol-permeabilized cells (Brunetti and Hageman 1976). For S. costatum, 4% (vol/vol) 1 -propanol yielded the highest NRA, and the rate of NO*-' production was linear over 2 h. Cells were collected by centrifugation (1,000 x g, 5 min), resuspended in incubation medium (4% 1-propanol in sterile seawater containing 20 mM NaNO,), and incubated in the dark at 15°C on a rotary shaker table.…”

Distribution of Nitrate Reductase Activity in Six Legumes: The Importance of the Stem

“…The + NO3 in vivo assay is considered to be an indicator of nitrate reduction in the tissue when nitrate is not limiting (Nicholas, Harper & Hageman, \91()a\ Radin, 1978;Breteler, Ten Cate & Nissen, 1979) and is often directly related to NRA measured in vitro (Streeter & Bosler, 1972;Brunetti & Hageman, 1976;Timpo & Neyra, 1983). This latter assay is an indicator of NRA when neither reductant (NADH) nor nitrate are limiting (Nicholas et al, 1976a;Radin, 1978;Breteler et al, 1979).…”