While there is an explosion in Correlative Electron and Light Microscopy (CLEM) protocols, many have been developed on different eukaryotic models, though reports on plant cell exploration by CLEM remain scarce (Bell et al., 2013). Indeed, plant cells represent a challenge for imaging in many ways. Plant specificities have to be taken into account when optimizing CLEM protocols. To name some, strong autofluorescence of cellular components specifically present in plant cells (chlorophyll, pigments, etc.) and interfering with fluorescent signals of reporter proteins, presence of a large central vacuole complicating the fixation and the dehydration processes needed for TEM processing, cell wall and cuticles acting as physical barriers and impairing resin embedding processes (Hawes et al., 2001).

Here we report two “in hand” robust and easily reproducible protocols to make such CLEM approaches on plant material. The proposed protocols had been worked out on the delicate question of autophagosomes identity in plant cells. Autophagy is a degradation process of intracellular components mainly implicated in response to environmental stresses. It consists in the delivery of cytosolic cargos entrapped by a compartment, named autophagosome, to lytic compartments for degradation and recycling. In plant cells as in any eukaryotic cells, autophagosomes have been identified as ring-shape or punctate structures, thanks to the light imaging of reporter proteins of markers for autophagosomes (Le Bars et al., 2014).

This study is using a Arabidopsis transgenic lines stably expressing GFP-ATG8, which is a common marker for autophagosomes. Two CLEM protocols are used:  firstly the high pressure freezing followed by acrylic resin embedding, and on the other hand the Tokuyasu method.  Here we compare the performance of the two protocol to preserve GFP fluorescence and enhanced ultrastructural features in plant cells.

References:

Hawes, C. and Satiat-Jeunemaitre, B. 2001 Plant Cell Biology: A Pratical Approach, 2nd edn. Oxford: Oxford University Press, 1–324.

Bell, K., Mitchell, S., Paultre, D., Posch, M., Oparka, K., 2013. Correlative Imaging of Fluorescent Proteins in Resin-Embedded Plant Material. Plant Physiol. 161, 1595–1603.

Le Bars, R., Marion, J., Le Borgne, R., Satiat-Jeunemaitre, B., Bianchi, M.W., 2014. ATG5 defines a phagophore domain connected to the endoplasmic reticulum during autophagosome formation in plants. Nat. Commun. 5.

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EMC Abstracts - https://emc-proceedings.com/abstract/optimizing-clem-protocols-for-plants-cells-a-good-preservation-of-gfp-fluorescence-and-ultrastructure-in-arabidopsis-roots/