Evaluation of methods to measure differential 15N labeling of soil and root N pools for studies of root exudation

To study patterns of root exudation, the effectiveness of different techniques for in situ N-15 labeling of Brassica napus, Centaurea jacea and Lolium perenne with ammonium nitrate was tested. Stem infiltration was found to effectively label plants with thicker stems, whereas, for grass species, cutting and immersing the leaf tips into N-15 solution proved to be most effective. A microdiffusion technique to isolate ammonium, combined with conventional cation-exchange chromatography to separate nitrate from amino-N compounds thereafter, was found suitable for separation of the N fractions of plant and soil extracts for N-15 determination. All three species were then cultivated in nutrient solution and labeled with (NH4NO3)-N-15-N-15 by stem feeding for 42 hours. Kinetics of N-15 labeling of bulk roots and shoots as well as hot water extractable material were assessed, and up to 1.1 at% N-15 excess (APE) was found in nutrient solutions. The main amino acids exuded by L. perenne were glycine, serine, alanine and aspartic acid. To assess the suitability of this set of methods to study root exudation in field settings, L. perenne was grown without fertiliser addition in pots containing low-nutrient soil. Plants were N-15 labeled via tip immersion and N-15 and N concentrations were analysed in shoots, roots and soils during a 48-h interval. Shoots reached 1.25 APE, roots and soil 0.10 and 0.005 APE, respectively. Between 4% (48 h) and 6% (24 h) of total plant N-15 was exuded by roots into the soil. In roots amino acids comprised the largest proportion of the soluble N-15 pool, whereas Soil N-15 levels were similar for amino acids and ammonium, exceeding those of nitrate. Mechanisms for the shift within N fractions from roots to soils are briefly discussed. Copyright (C) 2004 John Wiley Sons, Ltd.