Rapid detection of plasmodesmata in purified cell walls

Plasmodesmata are complex channels within the plant cell wall, which create plasma membrane and symplastic continuity between neighbouring cells. To d etect plasmodesmata in cell wall preparations from Nicotiana de elandii, we have used 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)), a cationic amphiph ilic fluorescent probe, widely employed for general studies of membrane str ucture and dynamics. Punctate fluorescent staining was readily seen in pit fields, small depressions within the cell wall known to be rich in plasmode smata. Scanning electron microscopy was used to demonstrate that the puncta te staining corresponded to plasmodesmata. Treatment of cell wail fragments with chloroform-methanol to remove lipids did not alter the staining of pl asmodesmata. In contrast, pronase E-sodium dodecyl sulfate treatment comple tely abolished staining, indicating that the DiOC(6) labelling of plasmodes mata may be protein rather than lipid specific. Although not membrane media ted, DiOC(6) staining of plasmodesmata is a simple, rapid, and specific too l for the detection of plasmodesmata in isolated cell walls and will prove useful for studies of plasmodesmal location, structure, and composition.