SIMS localization of nitrogen in the leaf of soybean: basis of quantitative procedures by localized measurements of isotopic ratios

Localizing and quantifying the compartmentation of mineral nutrients in the leaf is essential to understand plant mineral nutrition. Stable nitrogen N -15 is commonly used as a marker of natural nitrogen fluxes in plants. Seco ndary Ion Mass Spectrometry microscopy offers a new possibility to study th e locations of steps of nitrogen metabolism in tissues of higher plants. Af ter a critical choice of preparation techniques we present a physiological application of SIMS microscopy to plants. Using localized measurements of n itrogen isotopic ratios, we localized the pathway of nitrogen newly importe d into the leaf through the petiole, traced with N-15 stable isotope enrich ment. [N-15]nitrate was supplied by the transpiration stream to excised soy bean (Glycine max.) leaves as a short pulse, followed by a [N-14]nitrate ch ase for 15 or 60 min. Isotopic ratios in leaf samples were analyzed by SIMS and compared to data obtained by bulk classical gaseous mass spectrometry. To standardize the measurements we used internal references of constant el ements, namely carbon and the natural isotopic ratio of nitrogen in the emb edding resin. Empirical quantification was carried out with reference to a calibration curve. The biological standards used for drawing this curve wer e obtained from leaves of plants grown since germination in media containin g different N-15/N-14 ratios. The calibration curve matched a curve obtaine d by classical mass spectrometry using the same leaves. The nitrogen isotop es were imaged in the leaf tissues and the isotopic ratios N-15/N-14 were m easured in numerous spots. Various localized N-15 enrichments were quantifi ed. The distribution of the N-15 newly imported into the leaf was not unifo rm. Large differences of N isotopic ratios were observed between the epider mis, the bundle sheath and the mesophyll of the leaf. Heterogeneous distrib utions were observed even among cells of the same tissue. The largest N-15 accumulation occurred in cells in the epidermis, although they were the mos t remote from the site of solute importation into the leaf. The results ind icate that the nitrate delivered by the xylem was transported firstly to th e epidermis, then to inner tissues. From the observed pattern of N-15 distr ibution it may be inferred that these two successive steps correspond to di stinct routes, respectively through the apoplast and the symplast of the le af.