Roles of the Ubiquitin/Proteasome Pathway in Pollen Tube Growth with Emphasis on MG132-Induced Alterations in Ultrastructure, Cytoskeleton, and Cell Wall Components

Abstract

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 cysteineprotease 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 (UbPs) was also observed using immunoblotting. Transmission electron microscopy revealed that MG132 induces endoplasmic reticulum (ER)-derived cytoplasmic vacuolization. Immunogold-labeling analysis demonstrated a significant accumulation of UbPs in degraded cytosol and dilated ER in MG132-treated pollen tubes. Fluorescence labeling with fluorescein isothiocyanatephalloidin and b-tubulin antibody revealed that MG132 disrupts the organization of F-actin and microtubules and consequently affects cytoplasmic streaming in pollen tubes. However, tip-focused Ca 21 gradient, albeit reduced, seemingly persists after MG132 treatment. Finally, fluorescence labeling with antipectin antibodies and calcofluor indicated that MG132 treatment induces a sharp decline in pectins and cellulose. This result was confirmed by Fourier transform infrared 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 UbPs in pollen tubes, which induces ER-derived cytoplasmic vacuolization and depolymerization of cytoskeleton and consequently strongly affects the deposition of cell wall components, providing a mechanistic framework for the functions of proteasome in the tip growth of pollen tubes.Most cellular proteins are continuously synthesized and degraded within the lifespan of a cell. Efficient protein turnover is essential for many aspects of cell physiology and development (Hellmann and Estelle, 2002). As one of the most important proteolytic pathways in eukaryotic cells, the ubiquitin/proteasome pathway (UPP) is believed to be involved in the degradation of the bulk of intracellular proteins, including misfolded proteins and short-and long-lived regulatory proteins (Glickman and Ciechanover, 2002;Ciechanover, 2005;Hershko, 2005;Varshavsky, 2005). Therefore, the biological functions of the UPP in both animal and plant systems have received considerable attention.Pollen tubes are responsible for delivering sperm cells to the egg for fertilization and are therefore essential for higher plant sexual reproduction. The emergence and elongation of pollen tubes is a complex and intriguing example of cell morphogenesis. The control of this type of growth requires a number of factors and activities to be integrated in space and time

Cytoskeleton-Associated Large RNP Complexes in Tobacco Male Gametophyte (EPPs) Are Associated with Ribosomes and Are Involved in Protein Synthesis, Processing, and Localization

“…50 The importance of the selective proteolytic activity associated with 26S proteasome system during pollen germination and tube growth was repeatedly demonstrated. [51][52][53] Moreover, a number of transcripts encoding proteasome-associated and F-box proteins were identified in Arabidopsis developing and germinating pollen. 42,54 Proteasomes were also demonstrated to perform other nonproteolytic functions including transcriptional regulation, [55][56][57] RNase activity, 58,59 translational regulation, 60,61 and possessing cytoskeleton-binding properties.…”

“…42,54 Proteasomes were also demonstrated to perform other nonproteolytic functions including transcriptional regulation, [55][56][57] RNase activity, 58,59 translational regulation, 60,61 and possessing cytoskeleton-binding properties. 52,62 With regard to all abovementioned alternative functions, the presence of proteasome subunits within EPP complexes is natural in relation to complex EPP-associated spatially and temporarily regulated activities.…”

Salt Stress–Induced Disassembly ofArabidopsisCortical Microtubule Arrays Involves 26S Proteasome–Dependent Degradation of SPIRAL1

“…Studies using MG132 have shown that the UPS is involved in the regulation of plant cell microtubule (MT) networks (Yanagawa et al, 2002;Oka et al, 2004;Sheng et al, 2006;S. Wang et al, 2011).…”

Combined Proteomic and Cytological Analysis of Ca2+-Calmodulin Regulation inPicea meyeriPollen Tube Growth

“…Pollen grains were kept at room temperature for 30 min and then suspended in the germination medium containing 12% Suc, 0.01% H 3 BO 3 , and 0.01% CaCl 2 at pH 6.8 as documented previously (Sheng et al, 2006). All of the samples were incubated on a shaker (121 rpm) at 24°C in the dark.…”