A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants

Yang, Hong; Pu, Xiaojun; Wang, Lu; Liu, Li; Theg, Steven M.

doi:10.1016/j.bbrc.2016.12.032

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. Here, 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 ATPmore » synthesis and protein transport.« less

Authors:

Yang, Hong ^[1];

^[1]; Wang, Lu ^[1]; Liu, Li ^[2];

^[3]

Chinese Academy of Sciences, Kunming (China)
Chinese Academy of Sciences, Kunming (China); University of California, Davis, CA (United States)
University of California, Davis, CA (United States)

Publication Date:: Thu Dec 08 00:00:00 EST 2016

Research Org.:: Univ. of California, Davis, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences & Biosciences Division; USDOE

OSTI Identifier:: 1490415

Alternate Identifier(s):: OSTI ID: 1413373

Grant/Contract Number:: FG02-03ER15405

Resource Type:: Accepted Manuscript

Journal Name:: Biochemical and Biophysical Research Communications

Additional Journal Information:: Journal Volume: 486; Journal Issue: 1; Journal ID: ISSN 0006-291X

Publisher:: Elsevier

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; chloroplast; GFP; genetically encoded biosensor; thylakoid lumen pH; proton dynamics

Citation Formats


                    Yang, Hong, Pu, Xiaojun, Wang, Lu, Liu, Li, and Theg, Steven M. A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants.  United States: N. p., 2016. 
Web.  doi:10.1016/j.bbrc.2016.12.032.

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                    Yang, Hong, Pu, Xiaojun, Wang, Lu, Liu, Li, & Theg, Steven M. A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants.  United States.  https://doi.org/10.1016/j.bbrc.2016.12.032

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                    Yang, Hong, Pu, Xiaojun, Wang, Lu, Liu, Li, and Theg, Steven M. Thu .  
"A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants".  United States.  https://doi.org/10.1016/j.bbrc.2016.12.032.  https://www.osti.gov/servlets/purl/1490415.

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@article{osti_1490415,

  title        = {A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants},

  author       = {Yang, Hong and Pu, Xiaojun and Wang, Lu and Liu, Li and Theg, Steven M.},

  abstractNote = {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. Here, 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.},

  doi          = {10.1016/j.bbrc.2016.12.032},

  journal      = {Biochemical and Biophysical Research Communications},

  number       = 1,

  volume       = 486,

  place        = {United States},

  year         = {Thu Dec 08 00:00:00 EST 2016},

  month        = {Thu Dec 08 00:00:00 EST 2016}

}

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Works referenced in this record:

Chemiosmotic coupling in oxidative and photosynthetic phosphorylation
journal, December 2011

Mitchell, Peter
Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol. 1807, Issue 12
DOI: 10.1016/j.bbabio.2011.09.018

Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation
journal, August 1966

Mitchell, Peter
Biological Reviews, Vol. 41, Issue 3
DOI: 10.1111/j.1469-185X.1966.tb01501.x

On Why Thylakoids Energize ATP Formation Using Either Delocalized or Localized Proton Gradients – A Ca ²⁺ Mediated Role in Thylakoid Stress Responses
journal, January 2004

Dilley, Richard A.
Photosynthesis Research, Vol. 80, Issue 1-3
DOI: 10.1023/B:PRES.0000030436.32486.aa

Protein Transport into Chloroplasts
journal, June 2010

Li, Hsou-min; Chiu, Chi-Chou
Annual Review of Plant Biology, Vol. 61, Issue 1
DOI: 10.1146/annurev-arplant-042809-112222

Mechanisms of Protein Import and Routing in Chloroplasts
journal, December 2004

Jarvis, Paul; Robinson, Colin
Current Biology, Vol. 14, Issue 24
DOI: 10.1016/j.cub.2004.11.049

Targeting of nucleus‐encoded proteins to chloroplasts in plants
journal, June 2008

Jarvis, Paul
New Phytologist, Vol. 179, Issue 2
DOI: 10.1111/j.1469-8137.2008.02452.x

In vivo transport of folded EGFP by the pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana
journal, July 2004

Marques, J. P.
Journal of Experimental Botany, Vol. 55, Issue 403
DOI: 10.1093/jxb/erh191

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana
journal, May 2001

Moseyko, N.; Feldman, L. J.
Plant, Cell & Environment, Vol. 24, Issue 5
DOI: 10.1046/j.1365-3040.2001.00703.x

Engineered GFP as a vital reporter in plants
journal, March 1996

Chiu, Wan-ling; Niwa, Yasuo; Zeng, Weike
Current Biology, Vol. 6, Issue 3
DOI: 10.1016/S0960-9822(02)00483-9

Abscisic acid and other plant hormones: Methods to visualize distribution and signaling
journal, November 2015

Waadt, Rainer; Hsu, Po‐Kai; Schroeder, Julian I.
BioEssays, Vol. 37, Issue 12
DOI: 10.1002/bies.201500115

Quantitative Imaging with Fluorescent Biosensors
journal, June 2012

Okumoto, Sakiko; Jones, Alexander; Frommer, Wolf B.
Annual Review of Plant Biology, Vol. 63, Issue 1
DOI: 10.1146/annurev-arplant-042110-103745

Fluorescent proteins as a toolkit for in vivo imaging
journal, December 2005

Chudakov, Dmitriy M.; Lukyanov, Sergey; Lukyanov, Konstantin A.
Trends in Biotechnology, Vol. 23, Issue 12
DOI: 10.1016/j.tibtech.2005.10.005

Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
journal, July 1998

Miesenböck, Gero; De Angelis, Dino A.; Rothman, James E.
Nature, Vol. 394, Issue 6689
DOI: 10.1038/28190

Self-Reporting Arabidopsis Expressing pH and [Ca2+] Indicators Unveil Ion Dynamics in the Cytoplasm and in the Apoplast under Abiotic Stress
journal, March 2004

Gao, Dongjie; Knight, Marc R.; Trewavas, Anthony J.
Plant Physiology, Vol. 134, Issue 3
DOI: 10.1104/pp.103.032508

In Vivo Imaging of Ca ²⁺ , pH, and Reactive Oxygen Species Using Fluorescent Probes in Plants
journal, June 2011

Swanson, Sarah J.; Choi, Won-Gyu; Chanoca, Alexandra
Annual Review of Plant Biology, Vol. 62, Issue 1
DOI: 10.1146/annurev-arplant-042110-103832

Measurement of the ΔpH and Electric Field Developed Across Arabidopsis Thylakoids in the Light
book, January 2011

Theg, Steven M.; Tom, Curtis
Chloroplast Research in Arabidopsis
DOI: 10.1007/978-1-61779-237-3_18

Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly
journal, March 1997

Haseloff, J.; Siemering, K. R.; Prasher, D. C.
Proceedings of the National Academy of Sciences, Vol. 94, Issue 6
DOI: 10.1073/pnas.94.6.2122

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
journal, June 2006

Zhang, Xiuren; Henriques, Rossana; Lin, Shih-Shun
Nature Protocols, Vol. 1, Issue 2
DOI: 10.1038/nprot.2006.97

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts
journal, May 2010

Su, Pai-Hsiang; Li, Hsou-min
The Plant Cell, Vol. 22, Issue 5
DOI: 10.1105/tpc.109.071415

Guard cell photosynthesis and stomatal function
journal, December 2008

Lawson, Tracy
New Phytologist, Vol. 181, Issue 1
DOI: 10.1111/j.1469-8137.2008.02685.x

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar
journal, May 2003

Lawson, T.
Journal of Experimental Botany, Vol. 54, Issue 388
DOI: 10.1093/jxb/erg186

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Similar Records

Title: A new fluorescence-based method to monitor the pH in the thylakoid lumen using GFP variants

Abstract

Citation Formats

Chemiosmotic coupling in oxidative and photosynthetic phosphorylation journal, December 2011

Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation journal, August 1966

On Why Thylakoids Energize ATP Formation Using Either Delocalized or Localized Proton Gradients – A Ca 2+ Mediated Role in Thylakoid Stress Responses journal, January 2004

Protein Transport into Chloroplasts journal, June 2010

Mechanisms of Protein Import and Routing in Chloroplasts journal, December 2004

Targeting of nucleus‐encoded proteins to chloroplasts in plants journal, June 2008

In vivo transport of folded EGFP by the pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana journal, July 2004

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana journal, May 2001

Engineered GFP as a vital reporter in plants journal, March 1996

Abscisic acid and other plant hormones: Methods to visualize distribution and signaling journal, November 2015

Quantitative Imaging with Fluorescent Biosensors journal, June 2012

Fluorescent proteins as a toolkit for in vivo imaging journal, December 2005

Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins journal, July 1998

Self-Reporting Arabidopsis Expressing pH and [Ca2+] Indicators Unveil Ion Dynamics in the Cytoplasm and in the Apoplast under Abiotic Stress journal, March 2004

In Vivo Imaging of Ca 2+ , pH, and Reactive Oxygen Species Using Fluorescent Probes in Plants journal, June 2011

Measurement of the ΔpH and Electric Field Developed Across Arabidopsis Thylakoids in the Light book, January 2011

Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly journal, March 1997

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method journal, June 2006

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts journal, May 2010

Guard cell photosynthesis and stomatal function journal, December 2008

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar journal, May 2003

Chemiosmotic coupling in oxidative and photosynthetic phosphorylation
journal, December 2011

Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation
journal, August 1966

On Why Thylakoids Energize ATP Formation Using Either Delocalized or Localized Proton Gradients – A Ca ²⁺ Mediated Role in Thylakoid Stress Responses
journal, January 2004

Protein Transport into Chloroplasts
journal, June 2010

Mechanisms of Protein Import and Routing in Chloroplasts
journal, December 2004

Targeting of nucleus‐encoded proteins to chloroplasts in plants
journal, June 2008

In vivo transport of folded EGFP by the pH/TAT-dependent pathway in chloroplasts of Arabidopsis thaliana
journal, July 2004

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana
journal, May 2001

Engineered GFP as a vital reporter in plants
journal, March 1996

Abscisic acid and other plant hormones: Methods to visualize distribution and signaling
journal, November 2015

Quantitative Imaging with Fluorescent Biosensors
journal, June 2012

Fluorescent proteins as a toolkit for in vivo imaging
journal, December 2005

Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
journal, July 1998

Self-Reporting Arabidopsis Expressing pH and [Ca2+] Indicators Unveil Ion Dynamics in the Cytoplasm and in the Apoplast under Abiotic Stress
journal, March 2004

In Vivo Imaging of Ca ²⁺ , pH, and Reactive Oxygen Species Using Fluorescent Probes in Plants
journal, June 2011

Measurement of the ΔpH and Electric Field Developed Across Arabidopsis Thylakoids in the Light
book, January 2011

Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly
journal, March 1997

Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method
journal, June 2006

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts
journal, May 2010

Guard cell photosynthesis and stomatal function
journal, December 2008

The responses of guard and mesophyll cell photosynthesis to CO2, O2, light, and water stress in a range of species are similar
journal, May 2003