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题名: A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants
作者: Yang, Hong1; Pu, Xiaojun1; Wang, Lu1; Liu, Li1, 2; Theg, Steven M.2
刊名: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
出版日期: 2017-04-22
卷号: 486, 页码:1-5
关键词: Chloroplast ; GFP ; Genetically encoded biosensor ; Thylakoid lumen pH ; Proton dynamics
DOI: 10.1016/j.bbrc.2016.12.0320006-291X
文章类型: Article
英文摘要: The ApH-dependent/Tat pathway is unique for using only the proton motive force for driving proteins transport across the thylakoid membrane in chloroplasts. 9-aminoacridine fluorescence quenching is widely used to monitor the Delta pH developed across the thylakoid membrane in the light. However, this method suffers from limited sensitivity to low ApH values and to spurious fluorescence signals due to membrane binding. In order to develop a more sensitive method for monitoring the real pH of the thylakoid lumen without these problems we transformed Arabidopsis thaliana with a ratiometric pH sensitive GFP variant (termed pHluorin) targeted to the lumen by the prOE17 transit peptide. Positive transgenic plants displayed localization of pHluorin in the chloroplast by confocal microscopy, and fractionation experiments revealed that it is in the lumen. The pHluorin signal was the strongest in very young plants and diminished as the plants matured. The pHluorin released from the lumen displayed the expected fluorescence intensity changes in response to pH titration. The fluorescence signal in isolated chloroplasts responded to illumination in a manner consistent with light-dependent lumen acidification. Future experiments will exploit the use of this new pH-indicating probe of the thylakoid lumen to examine the influence of the thylakoid ApH on ATP synthesis and protein transport. (C) 2016 Elsevier Inc. All rights reserved.
类目[WOS]: Biochemistry & Molecular Biology ; Biophysics
研究领域[WOS]: Biochemistry & Molecular Biology ; Biophysics
关键词[WOS]: ARABIDOPSIS-THALIANA ; PHOTOSYNTHETIC PHOSPHORYLATION ; CELL PHOTOSYNTHESIS ; CHLOROPLASTS ; PROTEIN ; PLANTS ; STRESS ; CA2+ ; TRANSPORT ; RESPONSES
收录类别: SCI
项目资助者: Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy(DE-FG0203ER15405) ; 100 Talents Program of The Chinese Academy of Sciences ; National Natural Science Foundation of China(31571262)
语种: 英语
WOS记录号: WOS:000398880900001
Citation statistics:
内容类型: 期刊论文
URI标识: http://ir.kib.ac.cn/handle/151853/51066
Appears in Collections:中国科学院东亚植物多样性与生物地理学重点实验室_期刊论文

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作者单位: 1.Chinese Acad Sci, Kunming Inst Bot, Key Lab Plant Divers & Biogeog East Asia, Kunming 650201, Peoples R China
2.Univ Calif Davis, Dept Plant Biol, Davis, CA 95616 USA
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