Preparation of rhodamine-labelled tubulin and its reassembly in guard cells in leaves of Vicia faba L.

Abstract

AbstractTubulin from fresh pig brain was extracted through two cycles of polymerization and depolymerization, purified by chromatography on an ion-exchange column and labelled with 5(6)-carboxytetramethylrhodamine succinimidyl ester. We prepared 10.8 mg/ml rhodamine-labelled tubulin probe (dye/protein=0.6) and then microinjected it into living guard cells in leaves of Vicia faba L. After 5–10 min, a network of microtubules (MTs) could clearly be observed in the guard cells under a confocal laser scanning microscope. In opening guard cells, the cortical MTs radiated from the ventral wall to the dorsal wall and in closed guard cells the radial MTs were completely depolymerized into random and diffuse fragments. This shows that pig brain tubulin can easily be incorporated into MTs in guard cells, and that the dynamic behaviour of MTs can be examined directly in vivo during stomatal movement.