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. Secondary Ion Mass Spectrometry microscopy offers a new possibility to study the locations of steps of nitrogen metabolism in tissues of higher plants. After a critical choice of preparation techniques we present a physiological application of SIMS microscopy to plants. Using localized measurements of nitrogen isotopic ratios, we localized the pathway of nitrogen newly imported into the leaf through the petiole, traced with N-15 stable isotope enrichment. [N-15]nitrate was supplied by the transpiration stream to excised soybean (Glycine max.) leaves as a short pulse, followed by a [N-14]nitrate chase 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 elements, namely carbon and the natural isotopic ratio of nitrogen in the embedding resin. Empirical quantification was carried out with reference to a calibration curve. The biological standards used for drawing this curve were obtained from leaves of plants grown since germination in media containing different N-15/N-14 ratios. The calibration curve matched a curve obtained by classical mass spectrometry using the same leaves. The nitrogen isotopes were imaged in the leaf tissues and the isotopic ratios N-15/N-14 were measured in numerous spots. Various localized N-15 enrichments were quantified. The distribution of the N-15 newly imported into the leaf was not uniform. Large differences of N isotopic ratios were observed between the epidermis, the bundle sheath and the mesophyll of the leaf. Heterogeneous distributions were observed even among cells of the same tissue. The largest N-15 accumulation occurred in cells in the epidermis, although they were the most remote from the site of solute importation into the leaf. The results indicate that the nitrate delivered by the xylem was transported firstly to the epidermis, then to inner tissues. From the observed pattern of N-15 distribution it may be inferred that these two successive steps correspond to distinct routes, respectively through the apoplast and the symplast of the leaf.