Jia Peng-Fei, Li Hong-Ju, Yang Wei-Cai
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, No.1 West Beichen Road, Chaoyang District, Beijing, 100101, China.
Methods Mol Biol. 2017;1669:173-180. doi: 10.1007/978-1-4939-7286-9_14.
Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).
过氧化物酶体是大多数真核细胞中一种重要的单层膜细胞器，在多种细胞过程中发挥作用。过氧化物酶体的从头形成、分裂和周转有助于其生物发生、形态和数量调控。在植物中，过氧化物酶体发挥多种作用，包括代谢、发育和应激反应。过氧化物酶体生物发生和发育缺陷会阻碍植物的生长、适应和繁殖。通过追踪过氧化物酶体荧光报告标记的基质蛋白的亚细胞定位，荧光显微镜是检测过氧化物酶体生物发生的一种可靠且快速的方法。通过透射电子显微镜（TEM）对过氧化物酶体进行进一步的精细结构观察，使研究人员能够观察其形态的详细超微结构以及与其他细胞器的空间接触。花粉粒是一种特殊结构，两个小精子细胞被包裹在一个大营养细胞的细胞质中。花粉粒具有两个特点，使其成为研究过氧化物酶体生物发生的良好系统：过氧化物酶体对柱头萌发不可或缺，且具有同质性。在这里，我们描述了通过共聚焦激光扫描显微镜（CLSM）进行荧光实时成像以及基于DAB染色的透射电子显微镜（TEM）研究拟南芥花粉粒中过氧化物酶体生物发生的方法。
