Roles Of The Ubiquitin/proteasome Pathway In Picea Wilsonii Pollen Germination And Tube Growth

The Ubiquitin/Proteasome Pathway (UPP) is one of the most important proteolytic systems in eukaryotes. Ubiquitinated proteins and the degradation of substrate proeins by proteasome have been detected in Actinidia deliciosa pollen tubes. Besides, it was reported that inhibition of proteasome activity strongly affects pollen germination and pollen tube growth, as well as polarized tip growth. However, the data available at present appear insufficient to provide complete knowledge of the functions of the UPP during pollen tube development. Particularly, no attention has been paid to the possible functions of the UPP in cytoskeleton organization, the polarized distribution of organdies, or the deposition of cell wall components, all of which are closely linked to tip growth in pollen tubes. Furthermore, different angiosperm species show quite different pollen germination dynamics, let alone gymnosperm pollen grains that are characterized by prolonged lag period and far slow tube growth rate. It remains questionable whether current ideas about the roles of UPP in pollen germination and tube growth are valid also in gymnosperms since little information is available. In the present paper, the effects of MG132 and epoxomicin on pollen germination and tube growth, ultrastructure, cytoskeleton, and cell wall were studied using statistic analysis, ultrastruction observation, immunogold labeling, immunofluorescent labeling, immunoblotting, as well as FTIR analysis. The results revealed that, in addition to the inhibition of pollen germination and tube elongation, treatment of pollen tubes with proteasome inhibitor MG132 reversibly induced significant morphological alterations, including tip swelling, cytosolic vacuolization, and irregularly broadened diameters in a dose- and time-dependent manner. Similar effects were observed when epoxomcin, a more specific inhibitor for the proteasome, was added to culture medium, while neither germination rate nor tube morphology was affected by Cys-protease inhibitor E-64,Fluorogenic kinetic assays using fluorogenic substrate sLLVY-AMC confirmed MG132-induced inhibition of proteasome activity. The inhibitor-induced accumulation of ubiquitinated proteins in pollen tubes was also observed using immunoblotting. TEM demonstrated that MG132 induces ER-derived cytosolic vacuolization in pollen tubes in a time-dependent manner. Immunogold-labeling analysis revealed a significant accumulation of ubiquitinated proteins in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescent labeling with FITC-phalloidin and anti-P-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules, and consequently affects cytoplasmic streaming in pollen tubes in a time-dependent manner. Finally, fluorescent labeling with anti-pectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. The decline of cell wall components responded to MG132 treatment was confirmed by FTIR analysis, thus demonstrating for the first time the inhibitor-induced weakening of cell walls in pollen tubes. This study provided two novel findings: (i) inhibition of proteasome activity disrupted cytoskeleton, especially the actin cytoskeleton in a time-dependent manner;and (ii) inhibitor treatment causes a sharp decline in the main cell wall components of pollen tubes, such as cellulose and pectins, especially in the apical and subapical regions. The ER-derived cytoplasmic vacuolization in gymnosperm pollen tubes in response to proteasome inhibitor treatment is also a novel result. Taken together, these findings suggest UPP may involve in ER-associatted protein degradation (ERAD), as well as the regulation of the proteins essential for cytoskeleton dynamics. Inhibition of proteasome activity in pollen tubes promotes the accumulation of ubiquitinated proteins in pollen tubes, which induces ER-derived cytoplasmic vacuolization and disruption of the cytoskeleton, and consequently strongly affects the deposition of cell wall components. Given that plant cell growth is driven by internal turgor pressure and propulsion force of the cytoskeletal elements, and is restricted by the ability of the cell wall to extend under this pressure, we conclude here that inhibitor-induced cytoplasmic vacuolization and weakening of cell wall are the most important factors in the formation of swollentube tips and irregularly broadened tube diameters, thus providing a mechanistic framework for the functions of proteasomes in the polarized tip growth of pollen tubes.