Immunogold Labeling of terminal cellulose-synthesizing complexes

More than 40 years have passed since the concept of a multimeric enzyme complex for cellulose biosynthesis was first proposed by Roelofsen in 1958. However, no direct evidence has emerged and its existence remains one of the great enigmas of plant biology. We have overcome two difficulties in visualizing rosette terminal complexes (TCs) labeled with antibodies to cellulose synthases. Freeze-fracture electron microscopy is the only way to visualize rosette TCs. However, the technique is not suitable for immuno- gold labeling. We successfully applied SDS-solubilized freeze-fracture replica labeling (SDS-FRL), first developed by Fujimoto (1995), to plant cells. The purification of cellulose synthases has not succeeded in higher plant cells. We cloned GhCesA using AxCesA from cotton cDNA libraries, and prepared polyclonal antibodies against GhCesA protein. Using SDS-FRL, the antibodies of cellulose synthases were specifically localized to rosette TCs in the plasma membrane of higher plant cells. This provides direct evidence that the rosettes contain the catalytic subunit of the cellulose synthase.