期刊
PLANT CELL
卷 26, 期 4, 页码 1729-1745出版社
AMER SOC PLANT BIOLOGISTS
DOI: 10.1105/tpc.113.122358
关键词
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资金
- National Basic Research Program of China (973 Program) [2011CB944600, 2011CB809103]
- National Natural Science Foundation of China [31121065, 31170163, 31270224]
- Program of Introducing Talents of Discipline to Universities (111 project) [B13007]
- Program for Changjiang Scholars and Innovative Research Team in University [IRT 13047]
Arabidopsis thaliana respiratory burst oxidase homolog D (RbohD) functions as an essential regulator of reactive oxygen species (ROS). However, our understanding of the regulation of RbohD remains limited. By variable-angle total internal reflection fluorescence microscopy, we demonstrate that green fluorescent protein (GFP)-RbohD organizes into dynamic spots at the plasma membrane. These RbohD spots have heterogeneous diffusion coefficients and oligomerization states, as measured by photobleaching techniques. Stimulation with ionomycin and calyculin A, which activate the ROS-producing enzymatic activity of RbohD, increases the diffusion and oligomerization of RbohD. Abscisic acid and flg22 treatments also increase the diffusion coefficient and clustering of GFP-RbohD. Single-particle analysis in clathrin heavy chain2 mutants and a Flotillin1 artificial microRNA line demonstrated that clathrin-and microdomain-dependent endocytic pathways cooperatively regulate RbohD dynamics. Under salt stress, GFP-RbohD assembles into clusters and then internalizes into the cytoplasm. Dual-color fluorescence cross-correlation spectroscopy analysis further showed that salt stress stimulates RbohD endocytosis via membrane microdomains. We demonstrate that microdomain-associated RbohD spots diffuse at the membrane with high heterogeneity, and these dynamics closely relate to RbohD activity. Our results provide insight into the regulation of RbohD activity by clustering and endocytosis, which facilitate the activation of redox signaling pathways.
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