| Roles of the ubiquitin/proteasome pathway in pollen tube growth with emphasis on MG132-induced alterations in ultrastructure, actin cytoskeleton, and cell wall components. |
| Sheng X, Hu Z, Lü H, Wang X, Šamaj J, Baluška F, Lin J |
| Plant Physiol 141, 1578-1590 (2006) |
| |
| The ubiquitin/proteasome pathway represents one of the most important proteolytic systems in eukaryotes and has been proposed as being involved in pollen tube growth, but the mechanism of this involvement is still unclear. Here, we report that proteasome inhibitors MG132 and epoxomicin significantly prevented Picea wilsonii pollen tube development and markedly altered tube morphology in a dose- and time-dependent manner; while hardly similar effects were detected when Cys-protease inhibitor E-64 was used. Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins was also observed using immunoblotting. TEM revealed that MG132 induces ER-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of ubiquitinated proteins in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with FITC-phalloidin and β-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules, and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca2+ gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with anti-pectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by FTIR analysis, thus demonstrating for the first time the inhibitor-induced weakening of tube walls. Taken together, these findings suggest that MG132 treatment promotes the accumulation of ubiquitinated proteins in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of the cytoskeleton. Finally, MG132 affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes. |
| |
| |
