摘要
Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.
Phototropin （phot）-mediated signaling initiated by blue light （BL） plays a critical role in optimizing photosyn- thetic light capture at the plasma membrane （PM） in plants. However, the mechanisms underlying the regu- lation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of photl-GFP proteins we demonstrated that in the dark photl proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of photl-GFP increased in a dose-dependent manner in response to BL. In contrast, BL did not affect the lateral diffusion of kinase-inactive photlD806N-GFP but did enhance its dimerization, suggesting that photl dimerization is independent of phosphorylation. Forster resonance energy transfer-fluorescence life- time imaging microscopy analysis revealed that the interaction between photl-GFP and a marker of sterol- rich lipid environments, AtRem1.3-mCherry, was enhanced with increased time of BL treatment. However, this BL-dependent interaction was not obvious in plants co-expressing phot1D806N-GFP and AtRem1.3- mCherry, indicating that BL facilitates the translocation of functional photl-GFP into AtRem1.3-1abeled microdomains to activate phot-mediated signaling. Conversely, sterol depletion attenuated photl-GFP dynamics, dimerization, and phosphorylation. Taken together, these results indicate that membrane micro- domains act as organizing platforms essential for the proper function of activated photl at the PM.
基金
This work is supported by the National Natural Science Foundation of China （31530084, 31270224） and the Program of Introducing Talents of Discipline to Universities （111 project, B13007）.
