Subcellular Localization of Iron in Rhizophora mangle Leaves Revealed by Integrated Perls Reaction, TEM, STEM-HAADF,...

Mangrove ecosystems are frequently exposed to high concentrations of iron (Fe) in sediments, resulting in Fe accumulation in plant tissues. Although Fe is an essential micronutrient involved in several metabolic processes, its excess requires efficient mechanisms of compartmentalization and storage to maintain cellular homeostasis. Histochemical detection using the Perls reaction has usually been applied to identify ferric iron (Fe³⁺) in biological tissues; however, the combination of this technique with ultrastructural and elemental analyses remains relatively unexplored in plant cells. In this study, we investigated Fe localization in leaf tissues of Rhizophora mangle L. (Rhizophoraceae), a dominant mangrove species, by combining complementary approaches, including Perls cytochemical reaction, transmission electron microscopy (TEM), scanning transmission electron microscopy coupled with high-angle annular dark-field imaging (STEM-HAADF), and energy-dispersive X-ray spectroscopy (EDS). Perls-positive electron-dense deposits were visualized at the ultrastructural level, and their elemental composition was further characterized by EDS analyses. Fe-containing deposits were detected in the epidermis, mesophyll parenchyma, mucilage cells, and vascular tissues, as well as in multiple cellular compartments, including plastids, mitochondria, vacuoles, cell walls, intercellular spaces, and plasmodesmata, whereas sclerenchyma cells showed no detectable Fe-containing deposits. The combination of Perls reaction with TEM, STEM-HAADF, and EDS provides a complementary approach for high-resolution visualization and elemental characterization of Fe-containing deposits at the subcellular level. This integrated methodology may facilitate the investigation of Fe distribution and compartmentalization in plant tissues under contrasting conditions of Fe availability.