Sheng Xianyong, Hu Zhenghai, Lü Hongfei, Wang Xiaohua, Baluska Frantisek, Samaj Jozef, Lin Jinxing
Institute of Botany, Chinese Academy of Sciences, Key Laboratory of Photosynthesis and Molecular Environment Physiology, Beijing 100093, China.
Plant Physiol. 2006 Aug;141(4):1578-90. doi: 10.1104/pp.106.081703. Epub 2006 Jun 15.
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 beta-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.
泛素/蛋白酶体途径是真核生物中最重要的蛋白水解系统之一，有人提出该途径参与花粉管生长，但其参与机制仍不清楚。在此，我们报告蛋白酶体抑制剂MG132和埃坡霉素以剂量和时间依赖性方式显著阻止了青杄花粉管发育并明显改变了花粉管形态，而使用半胱氨酸蛋白酶抑制剂E-64时几乎未检测到类似效果。使用荧光底物sLLVY-AMC的荧光动力学分析证实了MG132诱导的蛋白酶体活性抑制。使用免疫印迹也观察到了抑制剂诱导的泛素化蛋白（UbP）积累。透射电子显微镜显示MG132诱导内质网（ER）衍生的细胞质空泡化。免疫金标记分析表明，在MG132处理的花粉管中，UbP在降解的细胞质和扩张的内质网中大量积累。用异硫氰酸荧光素-鬼笔环肽和β-微管蛋白抗体进行荧光标记显示，MG132破坏了F-肌动蛋白和微管的组织，从而影响了花粉管中的细胞质流动。然而，尽管MG132处理后尖端聚焦的Ca2+梯度有所降低，但似乎仍然存在。最后，用抗果胶抗体和荧光增白剂进行荧光标记表明，MG132处理导致果胶和纤维素急剧减少。傅里叶变换红外分析证实了这一结果，从而首次证明了抑制剂诱导的花粉管壁弱化。综上所述，这些发现表明MG132处理促进了花粉管中UbP的积累，这诱导了内质网衍生的细胞质空泡化和细胞骨架解聚，从而强烈影响细胞壁成分的沉积，为蛋白酶体在花粉管顶端生长中的功能提供了一个机制框架。
