RESUMEN
BACKGROUND: The formation of infection threads in the symbiotic infection of rhizobacteria in legumes is a unique, fascinating, and poorly understood process. Infection threads are tubes of cell wall material that transport rhizobacteria from root hair cells to developing nodules in host roots. They form in a type of reverse tip-growth from an inversion of the root hair cell wall, but the mechanism driving this growth is unknown, and the composition of the thread wall remains unclear. High resolution, 3-dimensional imaging of infection threads, and cell wall component specific labelling, would greatly aid in our understanding of the nature and development of these structures. To date, such imaging has not been done, with infection threads typically imaged by GFP-tagged rhizobia within them, or histochemically in thin sections. RESULTS: We have developed new methods of imaging infection threads using novel and traditional cell wall fluorescent labels, and laser confocal scanning microscopy. We applied a new Periodic Acid Schiff (PAS) stain using rhodamine-123 to the labelling of whole cleared infected roots of Medicago truncatula; which allowed for imaging of infection threads in greater 3D detail than had previously been achieved. By the combination of the above method and a calcofluor-white counter-stain, we also succeeded in labelling infection threads and plant cell walls separately, and have potentially discovered a way in which the infection thread matrix can be visualized. CONCLUSIONS: Our methods have made the imaging and study of infection threads more effective and informative, and present exciting new opportunities for future research in the area.
RESUMEN
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.
