COMPREHENSIVE ANALYSIS OF ORGANIC-LIGANDS IN WHOLE ROOT EXUDATES USING NUCLEAR-MAGNETIC-RESONANCE AND GAS-CHROMATOGRAPHY MASS-SPECTROMETRY

Root exudates in the rhizosphere are vital to the normal life cycle of plants. A key factor is phytometallophores, which function in the nut ritional acquisition of iron and zinc and are likely to be important i n the uptake of pollutant metals by plants, Unraveling the biochemistr y of these compounds is tedious using traditional analyses, which also fall short in providing the overall chemical composition or in detect ing unknown or unexpected organic ligands in the exudates. Here, we de monstrate a comprehensive analysis of the exudate composition directly by H-1 and C-13 multidimensional NMR and silylation GC-MS. The advant ages are (a) minimal sample preparation, with no loss of unknown compo unds, and reduced met analysis time; (b) structure-based analysis for universal detection and identification; and (c) simultaneous analysis of a large number of constituents in a complex mixture. Using barley r oot exudates, a large number of common organic and amino acids were id entified. Three derivatives of mugineic acid phytosiderophores were al so determined, the major one being 3-epihydroxymugineic acid, for whic h complete H-1 and C-13 NMR assignments were obtained, Quantification of all major components using these methods revealed a sevenfold incre ase in total exudation under moderate iron deficiency, with 3-epihydro xymugineic acid comprising approximately 22% of the exudate mixture. A s iron deficiency increased, total quantities of exudate per gram of r oot remained unchanged, but the relative quantity of carbon allocated to phytosiderophore increased to approximately 50% of the total exudat e in response to severe iron deficiency. (C) 1997 Academic Press.