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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Roles of the Ubiquitin/Proteasome Pathway in Pollen Tube Growth with Emphasis on MG132-Induced Alterations in Ultrastructure, Cytoskeleton, and Cell Wall Components^1,[W]

Xianyong Sheng, Zhenghai Hu, Hongfei Lü, Xiaohua Wang, Frantiek Baluka, Jozef amaj and Jinxing Lin^*

Institute of Botany, Chinese Academy of Sciences, Key Laboratory of Photosynthesis and Molecular Environment Physiology, Beijing 100093, China (X.S., H.L., X.W., J.L.); College of Life Science, Northwest University, Xi'an 710069, China (X.S., Z.H.); Institute of Cellular and Molecular Botany, Rheinische Friedrich-Wilhelms-University Bonn, Department of Plant Cell Biology, D–53115 Bonn, Germany (F.B., J..); Institute of Botany, Slovak Academy of Sciences, SK–84223, Bratislava, Slovak Republic (F.B.); and Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, SK–95007, Nitra, Slovak Republic (J..)

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 cysteine-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 (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 isothiocyanate-phalloidin and -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 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.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jinxing Lin (linjx{at}ibcas.ac.cn).

^[W] The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.081703.

^* Corresponding author; e-mail linjx{at}ibcas.ac.cn; fax 0086–10–62590833.

Received April 7, 2006; returned for revision June 6, 2006; accepted June 6, 2006.

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