Nitrate assay by multiple wavelength spectroscopy and analysis of kinetic parameters of net nitrate uptake by barley seedlings

Abstract

A method for determination of nitrate concentration and estimation of kinetic parameters of nitrate uptake by spectroscopy based on absorbances at multiple wavelengths has been developed to estimate nitrate uptake by barley (Hordeum vulgare L. cv. Steptoe) seedlings. Nitrate concentration in the nutrient solution was determined from the slope of the linear regression line of the absorbances to nitrate absorption coefficients at 12 wavelengths. Interference by root exudates was only due to an absorption component changing with wavelength in correlation with nitrate absorption. The standard error of the determination decreased in reverse proportion to the square root of the number of the wavelengths. A linear form of the net uptake equation, NUR = I_maxC/K_m+C‐E, could be expressed as NUR = (I_max ‐ E) ‐ K_m NUR/C ‐ K_mE I/C where NUR is net uptake rate, Imax is maximum influx, C is concentration, Km is the Michaelis constant, and E is an efflux constant. The method described here was used to determine the time course of nitrate depletion by barley seedlings from their nutrient solution. The isotherm of net nitrate uptake rates derived from the time course was analyzed after modifications based on the linear form of the net uptake equation. The analysis yielded highly significant results (P<0.0001).