In Vivo Nitrite Reduction in Leaf Tissue of Phaseolus vulgaris L.

Abstract

Experiments were performed to establish a procedure for in vivo measurement of nitrite utilization by leaf tissue of bean (Phaseolus vulgaris L. cv. Top Crop).To measure light-dependent nitrite disappearance, a single disc of leaf tissue was exposed to light for 1 hour at 30 C while immersed in incubation medium (approximately 0.11 milliliter per square centimeter of leaf area) in the bottom of a tall-form glass beaker. The incubation medium was 100 millimolar phosphate buffer (pH 7.5) with added wetting agent and nitrite. The wetting agent combination of 1% 1-propanol plus 0.05% Neutronyx-600 was used in some experiments for compatibility with established in vivo nitrate reductase (NR) assays; however, 0.05% Neutronyx-600 alone was found to be a suitable substitute. Parallel assays run in the dark on related tissue are recommended as a means to determine the amount of nitrite synthesized within the tissue by the NR system. Adding the results of the two assays gives an estimate of total nitrite utilization by the leaf tissue. It was found that 20 millimolar nitrite in the incubation medium was the most suitable level of external nitrite for promoting light-dependent nitrite disappearance. This was also found to reduce, sometimes to zero, the rate of synthesis of nitrite by NR. NR activity declined steadily with advancing age. Except for very young tissue, the rate of nitrite disappearance was independent of age. Nitrite disappearance was completely blocked by diuron.