pHusion

Gjetting, S. K., Ytting, C. K., Schulz, A., & Fuglsang, A. T. (2012). Live imaging of intra-and extracellular pH in plants using
pHusion, a novel genetically encoded biosensor. Journal of experimental botany, 63(8), 3207-3218.

EGFP mRFP
Dye Cell compart. Excitation Emission Excitation Emission
pHusion cytoplasm
488 500-550 558-585 600-630
Apo-pHusion apoplast

Confocal channels: EGFP (Laser line 488- dye EGFP)

mRFP1( Laser line 561- dye mRFP1.2)
Ratio: EGFP/mRFP1

Reuse settings from control pHusion

(DON’T CHANGE LASER SETTINGS, only averaging-scan speed if necessary)
Doherty, G. P., Bailey, K., & Lewis, P. J. (2010). Stage-specific fluorescence intensity of GFP
and mCherry during sporulation in Bacillus subtilis. BMC Research notes, 3(1), 1-8.
 pHluorin

Shen, J., Zeng, Y., Zhuang, X., Sun, L., Yao, X., Pimpl, P., & Jiang, L. (2013). Organelle pH in the Arabidopsis
endomembrane system. Molecular plant, 6(5), 1419-1437.
Martinière, A., Bassil, E., Jublanc, E., Alcon, C., Reguera, M., Sentenac, H., ... & Paris, N. (2013). In
vivo intracellular pH measurements in tobacco and Arabidopsis reveal an unexpected pH gradient
in the endomembrane system. The Plant Cell, 25(10), 4028-4043.

smGFP EclGFP
Dye Cell compart. Excitation Emission Excitation Emission
pHluorin cytoplasm
380-400 520 470-480 520
pHluorin-HDEL ER

Confocal channels: smGFP (Laser line 405- dye wtGFP)

EclGFP( Laser line 488- dye EGFP)
Ratio: smGFP/EclGFP

Reuse settings from control pHluorin/control pHluorin-HDEL

(DON’T CHANGE LASER SETTINGS, only averaging-scan speed if necessary)
Differentiation
zone
• Για να μετρήσουμε το κύτταρο
πρέπει το confocal να το έχει
«κόψει» στη μέση (να μην
μετράμε την πάνω ή κάτω
επιφάνεια του κυττάρου.
• Ανά κύτταρο 2-3 ορθογώνια, στην
ίδια περιοχή της ρίζας
Elongation τουλάχιστον 3-4 φυτά. (n=25)
zone

Meristematic
zone
1) Take czi images, drag & drop in FiJi (https://imagej.net/software/fiji/downloads)
2) Import images as Hyperstack
3) Image will open with ALL channels stacked together
4) Image -> Color -> Split Color channels
5) Keep the two channels of interest (#2 & #4 OR Green and Red if they are stacked)
6) Click & save each image as jpeg (File -> Save As -> Jpeg)
7) Make a collage of images in CorelDRAW with rectangles,
IN THE SAME positions between images

A) Place both images in one corel project

B) With the rectangle tool draw rectangles on the cell you want to measure
C) Select all the rectangles (Shift AND Click) and group them (Right Click-> Group objects)
 D) Copy

E) Paste
F) Now there are 3 layers of pictures 1st and 2nd are the two groups of rectangles and the 3rd
is the Green picture.
G) Bring the red picture exactly on the green picture. ( “C” for center and “E” button for
equal) OR Copy and Paste the coordinates
H) Now there are 4 layers pictures 1st and 2nd are the two groups of rectangles, the 3rd is the
Red picture and the 4th is the Green picture.
I) Select the rectangles (click on them, only the first layer will be selected) and the red
picture (Shift AND Click).
J) Group them (Right Click-> Group objects OR Control+G). Now there are 3 layers 1 st is red
picture and rectangles grouped together, 2nd is the other rectangles and 3rd is the green
picture.
K) Click and drag the red picture (grouped with the rectangles) on the other side
8) Export as jpeg (File-> Export)
9) Export as jpeg (Color mode -> RGB color, Quality-> Custom 100%)
9) Open image with FiJi
10) Φτιάχνεις τα measurements (Analyze -> Set Measurements)
11) Tick Area, Mean Gray Value, Integrated density
12) Με το polygon tool επιλέγεις την περιοχή μέσα σε ένα πολύγωνο
12) Πατάς Analyze -> Measure ή στο πληκτρολόγιο το “M”

13) Μεταφέρεις το πολύγωνο που μετράς και κάνεις τα ίδια για το

ΙΔΙΟ πολύγωνο στο κόκκινο πάνελ
14) Copy/paste σε ένα excel (Το RawIntDen δε σε ενδιαφέρει)

15) Μετράς διάφορες περιοχές του background της φωτογραφίας

(το μέγεθος του κύκλου δεν έχει σημασία)

16) Copy/paste στο excel

17) Βγάζεις τον μέσο όρο του mean gray value

18) Υπολογίζεις το background για κάθε πολύγωνο που μέτρησες ( Avg * Area του πολυγώνου)

19) Υπολογίζεις το normalized intensity για κάθε πολύγωνο που μέτρησες

(IntDen-Background)

20) Υπολογίζεις το ratio που θες (πχ pHusion = GFP/RFP)

 270
trh1 x pHluorin trh1 x pHluorin-
250 HDEL
Relative smGFP/EclGFP intensity (%)

230

210

190

170

150

130

110

90

70
pHluorin pHluorin-HDEL
 130
trh1 x pHusion trh1 x Apo-pHusion
120
Relative EGFP/mRFP1 intensity (%)

110

100

90

80

70

60

50
pHusion Apo-pHusion
 180
pHusion 4.5 2h pHusion 4.5 4h Apo-pHusion 4.5 Apo-pHusion 4.5
160 2h 4h
Relative EGFP/mRFP1 in
-

140
120
tensity (%)

100
80
60
40
20
0
pHusion 5.7 2h pHusion 5.7 4h Apo-pHusion 5.7 Apo-pHusion 5.7
2h 4h
 Fig2.
The csi1-3 mutants are deficient in auxin- or FC-induced hypocotyl
elongation. (A) Auxin-induced hypocotyl segment elongation of the
WT (Col-0) and csi1-3 and jia1-1 mutants. One out of three
reproducible independent experiments was used in the analysis.
The lengths of the hypocotyls were measured at 0 min and 150
min of each treatment. **P<0.01, ***P<0.001 (n=5 in mock
treatment for each genotype, n=6 in IAA treatment for each
genotype). Statistical analysis was performed by two-tailed
Student’s t-test. (B) FC-induced hypocotyl segment elongation of
the WT (Col-0) and csi1-3 and jia1-1 mutants. One out of three
reproducible independent experiments was used in the analysis.
The lengths of the hypocotyls were measured at 0 min and 150
min of each treatment. **P<0.01 (n=5 in mock treatment for each
genotype, n=6 in FC treatment for each genotype). Statistical
analysis was performed by two-tailed Student’s t-test. (C)
Apoplastic pH change in hypocotyl segments of the WT (Col-0) and
csi1-3 in response to IAA treatment. Images were taken at 0 min
and 150 min of IAA treatment. The apoplastic pH was reflected by
the GFP/RFP intensity ratio. *P<0.05 (n=12 individual hypocotyl
segments for each genotype). Statistical analysis was performed by
two-tailed Student’s t-test. (D) Apoplastic pH change in hypocotyl
segments of the WT (Col-0) and csi1-3 in response to FC
treatment. Images were taken at 0 min and 150 min of FC
treatment. The apoplastic pH was reflected by the GFP/RFP
intensity ratio. **P<0.01, ***P<0.001 (n=12 individual hypocotyl
segments for each genotype). Statistical analysis was performed by
two-tailed Student’s t-test.

Xiaoran Xin, Lei Lei, Yunzhen Zheng, Tian Zhang, Sai Venkatesh Pingali, Hugh O’Neill, Daniel J Cosgrove,
Shundai Li, Ying Gu, Cellulose synthase interactive1- and microtubule-dependent cell wall architecture is
required for acid growth in Arabidopsis hypocotyls, Journal of Experimental Botany, Volume 71, Issue 10,
30 May 2020, Pages 2982–2994, https://doi.org/10.1093/jxb/eraa063
Figure 7
In vivo tenuazonic acid (TeA)-induced alkalization of the apoplast in elongating root cells of 4- to 5-d-old Arabidopsis thaliana roots expressing apo-
pHusion. (a) Seedlings were mounted with agar on a Teflon-coated microscope slide and covered with a droplet of bath solution. Root cells in the
elongation zone were viewed on an SP5-X confocal laser scanning microscope, using a ×20 dipping objective and a perfusion setup for addition of TeA.
After c. 2 min, 500 µl of 10 µM TeA (black curve) or bath solution (control, red curve) was added (black arrow) and the experiment was run for 10 min in
total. Normalized ratio measurements of EGFP/mRFP1 are given as means ± SEM of n = 4 replicates. pH changes in response to TeA treatment were
highly significant (***, P < 0.001) in comparison with controls (Student's t-test). Inset shows in planta pH calibration curve using 10 mM MES, 10 mM
MOPS, and 10 mM citrate buffer with pH adjusted to pH 5–7. (b) Confocal overlay images of EGFP (green) and mRFP1 (red) channels at three time
points during the experiment: t = 0 before addition of TeA, t = 4:30 at the peak of the response, and t = 10:00 after 10 min. Bar, 50 µm.

Bjørk, P.K., Rasmussen, S.A., Gjetting, S.K., Havshøi, N.W., Petersen, T.I., Ipsen, J.Ø., Larsen, T.O. and Fuglsang, A.T.
(2020), Tenuazonic acid from Stemphylium loti inhibits the plant plasma membrane H+-ATPase by a mechanism
involving the C-terminal regulatory domain. New Phytol, 226: 770-784. https://doi.org/10.1111/nph.16398
 Figure 3
RALFs cause transient, highly localized and isoform-specific Ca2+
influx and suppress H+ efflux.
Rapid RALF-induced Ca2+ and pH responses observed using
genetically encoded biosensors for Ca2+ (YC3.6) and apoplastic
pH (apo-pHusion) and perfusion with RALF peptides.
(a) Confocal overlay images of yellow fluorescent protein/cyan
fluorescent protein (in pseudocolors green and red), with red
indicating an increase in [Ca2+] of selected time points of the
Ca2+ response in (b) (see also Movie S1).
(b) Cytosolic Ca2+ response to perfusion with RALF33 peptide
(10 µm). Peptide was added at time = 0. Graph shows responses
in five regions of the root elongation zone, corresponding to
regions of interest 1–5 on the overlay image shown in the inset.
(c) Perfusion with the RALF36 peptide, showing the response in
the same five regions of interest as in (b) of the root elongation
zone.
(d) Apoplastic pH response to perfusion with RALF 33, RALFL8 or
RALFL36 peptides, shown as normalized enhanced green
fluorescent protein/monomeric red fluorescent protein ratio.
Time of RALF peptide addition is indicated by arrows.
(e) Plot combining the Ca2+ response shown in (b) with the pH
response shown in (d).

Gjetting, S.K., Mahmood, K., Shabala, L., Kristensen, A., Shabala, S., Palmgren, M. and Fuglsang, A.T. (2020),
Evidence for multiple receptors mediating RALF-triggered Ca2+ signaling and proton pump inhibition. Plant J.,
104: 433-446. https://doi.org/10.1111/tpj.14935
 Fig. 2 |N nutrition modulates apoplastic pH and radial auxin
distribution in lateral roots.a–i, DR5:GFP reporter activities
in higher-order lateral roots (LR). k, Activity of the
apoplastic pH sensor apo-pHusion in the branching zone of
second-order LR in response to localized N treatments.
Plants were grown for 12 d on vertical split-agar plates with
the first first-order LR on the HN side exposed to 0.8 mM
KCl (–N control), 0.8 mM KNO3 or 0.8 mM NH4Cl, before
fluorescence was recorded by confocal microscopy. Colour
code (black to white) indicates (low to high) fluorescence
intensity ratio of eGFP/mRFP1 and thus apoplastic pH.
Representative images from ten biological replicates per
treatment are shown. Scale bars, 100 μm. l, Quantitative
readout of the intensity ratio of eGFP/mRFP1 in inner
(vasculature and pericycle) and outer (endodermis, cortex
and epidermis) root tissues on the HN side. Bars represent
means ± s.d., n= 10 independent biological replicates.

Meier, M., Liu, Y., Lay-Pruitt, K. S., Takahashi, H., & von Wirén, N. (2020). Auxin-mediated root branching is
determined by the form of available nitrogen. Nature Plants, 6(9), 1136-1145.
Fig.4
Validation of the microchannel-device monitoring system. a Schematic of the microchannel device. b Twelve-hour time-lapse imaging of a single
root expressing Apo-pHusion. Scale bar = 100 μm. c pH monitoring of a root exposed to a series of different pH buffers. The mature region of a
root expressing Apo-pHusion was observed. The buffers were exchanged by perfusion using a peristaltic pump. The ratio of EGFP fluorescence
intensity to mRFP1 fluorescence intensity in images is expressed using 16 colors. Scale bar = 100 μm. d pH monitoring in epidermal cells and
vascular bundle cells in response to changes of external pH. The outermost layer and the fourth layer of cells were considered as epidermal cells
and vascular bundle cells, respectively. The ratio of EGFP fluorescence intensity to mRFP1 fluorescence intensity at each pH is plotted. Mean ± SE,
n=3

Kurita, K., Sakamoto, Y., Naruse, S. et al. Intracellular localization of histone deacetylase HDA6 in plants. J
Plant Res 132, 629–640 (2019). https://doi.org/10.1007
 A and B) HPTS stained Col-0-WT seedlings under standard conditions (A) and
incubated for 30 min in liquid growth medium of different pH (B). (Left)
Deprotonated (basic) version of HPTS (λex, 458 nm; λem, 514 nm). (Middle)
Protonated (acidic) version of HPTS (λex, 405 nm; λem, 514 nm). (Right)
Ratiometric image where, for each pixel, the 458 intensity has been divided
by the 405 intensity. The 458/405 ratio values correlate with the apoplastic
pH. (C) HPTS stained 4-d-old seedling root. Color code (black to white)
depicts (low to high) 458/405 intensity, and thus pH values. Error bars
represent SEM.

Barbez, E., Dünser, K., Gaidora, A., Lendl, T., & Busch, W. (2017). Auxin
steers root cell expansion via apoplastic pH regulation in Arabidopsis
thaliana. Proceedings of the National Academy of Sciences, 114(24),
E4884-E4893.
Figure 8
pH response to different nitrogen regimes. Apo-plastic pH reported by the genetically encoded pH sensor pHusion. Confocal overlay images of EGFP
(488 nm) and mRFP1 (543 nm) channels showing the sensor signal of apo-pHusion in root tips. Confocal data acquisition of root tips was performed on a
Leica SP5 confocal laser scanning microscope. Root tips were viewed with a 20× water immersion objective.

Młodzińska, E., Kłobus, G., Christensen, M.D. and Fuglsang, A.T. (2015), The plasma membrane H+-ATPase AHA2
contributes to the root architecture in response to different nitrogen supply. Physiol Plantarum, 154: 270-282.
https://doi.org/10.1111/ppl.12305