Cellular Imaging of Cadmium in Resin Sections of Arbuscular Mycorrhizas Using Synchrotron Micro X-ray Fluorescence

Arbuscular mycorrhizal (AM) fungi function as extended roots and take an active part in plant acquisition of nutrients and also soil pollutants, such as heavy metals. The objective of this study was to establish a method to observe the localization of cadmium (Cd) K alpha at subcellular levels using X-ray fluorescence (XRF) imaging with a synchrotron irradiation microbeam in resin-embedded sections of mycorrhizas. To evaluate the methodology, distributions of Cd in high-pressure-frozen Lotus japonicus-Rhizophagus irregularis mycorrhizal roots were compared between two treatments; Cd was exposed either to the roots or to the extraradical hyphae. Results showed that, in the latter treatment, Cd was restricted to fungal structures, whereas in the former, Cd was detected in cell walls of the two organisms. Plunge-frozen extraradical mycelium of Gigaspora margarita exposed to Cd showed high signals of Cd in the cell walls and vacuoles, and low in the cytoplasm. With selective staining and elemental mapping by electron-dispersive X-ray spectrometry (EDS), a positive correlation between distributions of Cd and P was revealed in the vacuole, which suggested polyP as a counter ion of Cd. These results indicated that there was no Cd relocation in rapidly frozen resin-embedded materials, therefore supporting the usefulness of this methodology.