Transmission Electron Microscopy equipped with Energy Dispersive Spectroscopy (TEM-EDS) is known to be a powerful tool to study metal speciation in biological and environmental samples. The characterisation of different metal pools present in organisms is indeed essential to understand optimal development under various environmental constraints. For instance in plant cells, iron (Fe) is known to be an essential co-factor for electron transfer in many biological reactions. Fe remobilization by specific membrane transporters in seeds is especially important for germination. In Arabidopsis thaliana seeds, the AtVIT1 transporter is involved in Fe influx into perivascular vacuoles whereas the AtNRAMP3 and AtNRAMP4 function in Fe retrieval. The vit1-1 and nramp3nramp4vit1-1 mutants display an altered Fe pattern.
To examine the subcellular localization of Fe and Mn in cotyledons from wild-type, vit1-1 and nramp3nramp4vit1-1 triple mutant dried seeds, we used Energy Dispersive Spectroscopy (EDS) combined with Transmission Electron Microscope (TEM). This technique collects the X-rays spectrum emitted by plant sample sections bombarded with a focused beam of electrons to obtain a localized chemical analysis (Figure 1). A cartography using STEM-EDX data complete the characterization of these mutants and open new questions of the iron localization and chemistry in the triple mutant.
Results (Figure 2) show that in vit1-1 mutant embryos, Fe and Mn were both concentrated in subepidermal cell globoids, whereas Fe is localized in perivascular globoids in wild-type. In the nramp3nramp4vit1-1 triple mutant Fe was detected at low level in globoids from subepidermal and perivascular cells although vit1-1 and nramp3nramp4vit1-1 have the same pattern revealed by Perl’s/DAB staining (1).
To further localize and quantify metal elements such as Fe or Ni in cells, we also developed a CEMOVIS approach which will allow to perform elemental chemical analysis from lyophilized cryosections observed by STEM-EDX.
(1) Mary V. et al. (2015). Bypassing Iron storage in endodermal vacuoles rescues the iron mobilization defect in the natural resistance associated- macrophage protein3natural resistance associated-macrophage protein4 double mutant. Plant Physiology, 169, 748-760
To cite this abstract:Cynthia GILLET, Viviane MARY, Vanesa SANCHEZ GARCIA DE LA TORRE, Sébastien THOMINE, Béatrice SATIAT-JEUNEMAÎTRE; Subcellular localization of metal pools determined by TEM-EDS in embryo Arabidopsis thaliana mutants. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/subcellular-localization-of-metal-pools-determined-by-tem-eds-in-embryo-arabidopsis-thaliana-mutants/. Accessed: December 5, 2022
EMC Abstracts - https://emc-proceedings.com/abstract/subcellular-localization-of-metal-pools-determined-by-tem-eds-in-embryo-arabidopsis-thaliana-mutants/