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.