Electron Tomography to Study the Three-dimensional Structure of Plasmodesmata in Plant Tissues-from High Pressure Freezing Preparation to Ultrathin Section Collection.

Plasmodesmata (PD) are nanometric (~20 nm wide) membrane lined pores encased in the cell walls of the adjacent plant cells. They allow the cells to exchange all types of molecules ranging from nutrients like sugar, hormones, to RNAs and various proteins. Unfortunately, they are also hijacked by phyto-viruses, enabling them to spread from cell-to-cell and then systematically throughout the whole plant. Their central position in plant biology makes it crucial to understand their physiology and especially link their function to their structure. Over the past 50 years, electron microscopists have observed them and attempted to ultrastructurally characterize them. They laid the foundation of what is known about these pores (Tilney et al., 1991; Ding et al., 1992; Oparka and Roberts, 2001; Nicolas et al., 2017a). Despite the explosion of three-dimensional electron microscopy (3D-EM), PD ultrastructure remained recalcitrant to such technique. The first technical difficulty is to process them in such a way where they are as close to their native state as possible. Secondly, plant samples reveal themselves as being difficult to process due to the poor staining/fixating reagents penetration rates, their increased size, their high water content and the presence of an acidic vacuole. On top of this, their very unique position in the cell wall and their nanometric size make them difficult to conveniently stain in order to see the inner-workings of these pores. Here we describe in detail the protocol used in Nicolas et al. (2017b) to image PD in fine detail and produce high-resolution tomograms.