Rapid detection of plasmodesmata in purified cell walls

Abstract

Plasmodesmata are complex channels within the plant cell wall, which create plasma membrane and symplastic continuity between neighbouring cells. To detect plasmodesmata in cell wall preparations fromNicotiana cle elandii, we have used 3,3′-dihexyl-oxacarbocyanine iodide (DiOC6), a cationic amphiphilic fluorescent probe, widely employed for general studies of membrane structure and dynamics. Punctate fluorescent staining was readily seen in pit fields, small depressions within the cell wall known to be rich in plasmodesmata. Scanning electron microscopy was used to demonstrate that the punctate staining corresponded to plasmodesmata. Treatment of cell wall fragments with chloroform-methanol to remove lipids did not alter the staining of plasmodesmata. In contrast, pronase E-sodium dodecyl sulfate treatment completely abolished staining, indicating that the DiOC6 labelling of plasmodesmata may be protein rather than lipid specific. Although not membrane mediated, DiOC6 staining of plasmodesmata is a simple, rapid, and specific tool for the detection of plasmodesmata in isolated cell walls and will prove useful for studies of plasmodesmal location, structure, and composition.