Strategies to Prepare Frozen-Hydrated Duckweed for Cryogenic Electron Tomography of Thylakoid Membranes

Le résumé fourni par la source

Plant chloroplasts have dynamic thylakoid membrane organization to orchestrate photosynthesis. High resolution inelastic neutron scattering spectroscopy has recently been shown to measure bulk thylakoid membrane dynamics in living plant leaves [1]. Changes in membrane thickness, bilayer-bilayer interactions and embedded protein decoration and protrusion across the membrane can contribute to this membrane flexibility. Transmission electron microscopy (TEM) can be used as a complementary technique to directly visualize flexible membranes and embedded proteins in appressed and non-appressed thylakoid membranes. Plant material has routinely been chemically fixed and metal stained for high contrast TEM imaging of thylakoid membranes for ultrastructural architecture. However, this technique may lower the detail of embedded proteins that mediate membrane curvature [2-4]. Chemical fixation may cause membrane-embedded proteins to adopt a non-native state due to fixative-induced rigidity, and metal staining loses protein atomic detail. Therefore, maintaining the protein native state with frozen-hydrated plant cells gives us a closer representation of thylakoid membrane dynamics in live plants [5]. It is currently a challenge to find workflows focused on preparing frozen-hydrated plant tissue. In this work, we seek to design a preparation technique for frozen-hydrated duckweed to visualize native thylakoid membranes and embedded proteins using cryogenic electron tomography (cryoET). Once the tissue is prepared, frozen hydrated duckweed will be thinned using the cryogenic ultramicrotome and the cryogenic focused ion beam (cryoFIB), then imaged with cryoET. These instruments will be key to process the plant tissue in a consistent cryogenic state to maintain frozen-hydrated cell integrity [6].

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