1. Organelle pH in the Arabidopsis Endomembrane System
Jinbo Shen, Yonglun Zeng, Xiaohong Zhuang, Lei Sun, Xiaoqiang Yao, Peter Pimpl, Liwen Jiang 
Molecular Plant 
Volume 6, Issue 5, Pages (September 2013)
DOI: /mp/sst079
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Spectral Analysis of the Plant-Solubility-Modified pHluorins (PpHluorins) PEpHluorin and PRpHluorin.
(A, B) Excitation spectra of PEpHluorin (A) and PRpHluorin (B) in various pH-adjusted buffers. Samples containing purified PRpHluorin in a solution were excited from λ = 350–500nm, and the fluorescence emission was collected at λ = 515nm. The ordinate scales reflect normalized differences in emitted fluorescence intensity.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2 In Situ Calibration of PEpHluorin and PRpHluorin.
(A) Representative confocal images of Arabidopsis PSB-D protoplasts expressing PEpHluorin at a series of pH as indicated. Notice that the fluorescence was not imaged below pH 6.2, which could be used as threshold.
(B) A plot of the fluorescence intensity versus intracellular pH between 5.5 and 8.5. The fluorescence intensity after excitation λ = 488nm for the cytoplasm was calculated and fitted to the curve.
(C) Pseudocolored images of Arabidopsis PSB-D protoplasts expressing PRpHluorin at a series of pH as indicated taken using wide-field epifluorescence microscopy.
(D) A plot of the fluorescence excitation ratio (F380/F480) versus intracellular pH between 5.0 and 8.5. The ratio for each pH condition in the cytoplasm was calculated and fitted to the curve.
Scale bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3 pH Measurement of the Cytoplasm and Nucleus.
(A, B) Confocal images of NLS–PEpHluorin (A) and NLS–PRpHluorin (B) showing the pH indicator signals and colocalization with the nucleus marker (NLS–mRFP) in Arabidopsis PSB-D protoplasts. The strong signal of NLS–PEpHluorin indicates the pH value in the nucleus is above 6.2.
(C, D) Representative fluorescent image (C) and pseudocolored ratio image (D) of PRpHluorin after a pixel-by-pixel calculation. Region corresponding to the pH measurements is enlarged. The pseudo-color look-up table shown was adjusted to span the entire ratio signal range as determined in Figure 2D.
(E, F) Representative fluorescent image (E) and pseudocolored ratio image (F) of NLS–PRpHluorin after a pixel-by-pixel calculation.
Nuc, nucleus; DIC, differential interference contrast. Bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
CLSM Analysis of Arabidopsis Protoplasts Expressing pH Sensors for ER and cis-Golgi.
(A, B) Protoplasts of Arabidopsis cells transiently coexpressing PEpHluorin–HDEL (A) or PRpHluorin–HDEL (B) with the ER marker CNX–mRFP show colocalization of fluorescent signals. The strong signal of PEpHluorin–HDEL indicates the pH value in the ER is above 6.2.
(C, D) Protoplasts of Arabidopsis cells transiently coexpressing ManI–PEpHluorin (C) or ManI–PRpHluorin (D) with the cis-Golgi marker mRFP–EMP12 show colocalization of fluorescent signals. The strong signal of ManI–PEpHluorin indicates the pH value in the cis-Golgi is above 6.2.
DIC, differential interference contrast. Bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
CLSM Analysis of the TGN, MVB/PVC, and Lytic Vacuole Targeted pH Sensors in Arabidopsis Protoplasts.
(A, B) Protoplasts of Arabidopsis cells transiently expressed PEpHluorin–BP-80 (Y612A) (A) or PRpHluorin–BP-80 (Y612A) (B) show colocalization with TGN marker mRFP–SYP61. The weak signal of PEpHluorin–BP-80 (Y612A) indicates the pH value in the TGN is around 6.2.
(C, D) PEpHluorin–AtVSR2 (C) or PRpHluorin–AtVSR2 (D) co-localized with MVB/PVC marker mRFP–AtVSR5. Note that PEpHluorin–AtVSR2 lacks some punctate dots, indicateing the pH value in the MVB/PVC is around 6.2.
(E, F) Coexpression of aleurain–PEpHluorin (E) or aleurain–PRpHluorin (F) with aleurain–mRFP. Note that aleurain–PEpHluorin gave rise to punctate fluorescence within the cytoplasm, but lacks a diffuse pattern in the vacuole as shown in aleurain–PRpHluorin (F). The lack of signal in the vacuole indicates that the pH in the vacuole is below 6.2.
Vac, vacuole; DIC, differential interference contrast. Bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Topology Analysis of pH Sensors in the Endomembrane Compartments.
Membrane vesicles were isolated from Arabidopsis PSB-D protoplasts expressing the indicated constructs for trypsin digestion, followed by protein extraction and separation via SDS–PAGE for immunoblot analysis using GFP antibody.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

8. Figure 7
pH Analysis of the Endomembrane Compartments in Arabidopsis Protoplasts Using Wide-Field Epifluorescence Microscopy.
(A) ER, pH = 7.1±0.4.
(B) cis-Golgi, pH = 6.8±0.2.
(C) TGN, pH = 6.3±0.3.
(D) MVB/PVC, pH = 6.2±0.3.
(E) Vacuole, pH = 5.2±0.2.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Aleurain–PEpHluorin Is Quenched Because of the Acidic Lytic Vacuole Environment.
(A) Aleurain–PEpHluorin shows strong fluorescence signals in the ER. Aleurain–PEpHluorin and aleurain–mRFP were coexpressed together with ER export inhibitor Sar1(H74L), which traps both aleurain–PEpHluorin and marker protein aleurain–mRFP and co-localized in the ER.
(B) PEpHluorin–AtVIT1 were coexpressed together with mRFP–AtVIT1. PEpHluorin–AtVIT1 with the pH indicator facing the cytoplasm shows strong fluorescence signal.
(C) Aleurain–PEpHluorin shows fluorescence signals in the vacuole, while the SCAMP–mRFP can reach the plasma membrane after low concentration of ConcA treatment (200nM).
(D) The aleurain–PEpHluorin shows fluorescence signals in the vacuole after ConcA treatment (200nM). Arabidopsis PSB-D protoplasts coexpressing aleurain–PEpHluorin with the lytic vacuole marker aleurain–mRFP were treated with ConcA immediately after transformation, followed by incubation for 8 h before confocal imaging.
DIC, differential interference contrast. Bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

10. Figure 9
pH Analysis of the Endomembrane Compartments in Arabidopsis Protoplasts after ConcA Treatment (200nM) Using Wide-Field Epifluorescence Microscopy.
(A) ER, pH = 7.2±0.3.
(B) cis-Golgi, pH = 6.7±0.2.
(C) TGN, pH = 6.9±0.4.
(D) MVB/PVC, pH = 6.1±0.1.
(E) Vacuole, pH = 7.0±0.3.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

11. Figure 10
CLSM Analysis of the pH Indicators in Peroxisome, Mitochontria Matrix, and Plastid Stroma.
Protoplasts of Arabidopsis cells transiently expressed PEpHluorin–SRL (A) or PRpHluorin–SRL (B) show colocalization with the mRFP–SRL. Mito-PEpHluorin (C) or Mito-PRpHluorin (D) co-localized with the MitoTracker Red. RecA–PEpHluorin (E) or RecA–PRpHluorin (F) co-localized with the chlorophyll autofluorescence from chloroplasts. The strong signals of PEpHluorin in the organelles indicate the pH value is above 6.2.
DIC, differential interference contrast. Bar = 50 μm.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

12. Figure 11
Representative Pseudocolored Ratio Images of the pH Indicators in the Peroxisome, Mitochontria Matrix, and Plastid Stroma of Arabidopsis Protoplasts Using Wide-Field Epifluorescence Microscopy.
(A) Peroxisome, pH = 8.4±0.3.
(B) Mitochontria matrix, pH = 8.1±0.2.
(C) Plastid stroma, pH = 7.2±0.3.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

13. Figure 12
The Comparison of the pH of Intracellular Organelles in Arabidopsis and Mammalian Cells.
ER, endoplasmic reticulum; TGN, trans-Golgi network; MVB, multivesicular body; PVC, prevacuolar compartment.
Molecular Plant 2013 6, DOI: ( /mp/sst079)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions