title: A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants.
abstract: The ΔpH-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 ΔpH developed across the thylakoid membrane in the light. However, this method suffers from limited sensitivity to low ΔpH 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 ΔpH on ATP synthesis and protein transport.
authors.affiliation: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Yang
authors.affiliation: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Pu
authors.affiliation: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China; Department of Plant Biology, University of California - Davis, Davis, CA 95616, USA. Electronic address: liulia@mail.kib.ac.cn.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Plant Biology, University of California - Davis, Davis, CA 95616, USA. Electronic address: smtheg@ucdavis.edu.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Theg
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008027:Light; D018613:Microscopy, Confocal / Q000379:methods; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D021381:Protein Transport / Q000528:radiation effects; D013050:Spectrometry, Fluorescence / Q000379:methods; D020524:Thylakoids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
chemical_list: C485187:PHluorin; D010455:Peptides; D010940:Plant Proteins; D049452:Green Fluorescent Proteins
keywords: Chloroplast; GFP; Genetically encoded biosensor; Proton dynamics; Thylakoid lumen pH