Super-Resolution Fluorescence Imaging of Arabidopsis thaliana Transfer Cell Wall Ingrowths using Pseudo-Schiff Labelling Adapted for the Use of Different Dyes.

To understand plant growth and development, it is often necessary to investigate the organization of plant cells and plant cell walls. Plant cell walls are often fluorescently labeled for confocal imaging with the dye propidium iodide using a pseudo-Schiff reaction. This reaction binds free amine groups on dye molecules to aldehyde groups on cellulose that result from oxidation with periodic acid. We tested a range of fluorescent dyes carrying free amine groups for their ability to act as pseudo-Schiff reagents. Using the low-pH solution historically used for the Schiff reaction, these alternative dyes failed to label cell walls of Arabidopsis cotyledon vascular tissue as strongly as propidium iodide but replacing the acidic solution with water greatly improved fluorescence labeling. Under these conditions, rhodamine-123 provided improved staining of plant cell walls compared to propidium iodide. We also developed protocols for pseudo-Schiff labeling with ATTO 647N-amine, a dye compatible for super-resolution Stimulated Emission Depletion (STED) imaging. ATTO 647N-amine was used for super-resolution imaging of cell wall ingrowths that occur in phloem parenchyma transfer cells of Arabidopsis, structures whose small size is only slightly larger than the resolution limit of conventional confocal microscopy. Application of surface-rendering software demonstrated the increase in plasma membrane surface area as a consequence of wall ingrowth deposition and suggests that STED-based approaches will be useful for more detailed morphological analysis of wall ingrowth formation. These improvements in pseudo-Schiff labeling for conventional confocal microscopy and STED imaging will be broadly applicable for high-resolution imaging of plant cell walls.