Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
0.6
Absorbance
0.5
0.4
0.3
0.2
0.1
0
300
400
500
600
700
Wavelength (nm)
Supplemental Figure 1: Absorption spectra of dark orange OCP (black)
and after 20 min illumination with orange-red light (1000 µmol quanta m-2
s-1) (red). White light was passed via a cut-on 600 nm filter. This picture
clearly shows that OCP is not converted to its red active form under orange-red
light. These light conditions were used in Figures 1 and 2.
Rate of 1O2 production (µmol1O2/mgChl/h)
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
30
WT
∆OCP
∆psbA2
25
OE-OCP
20
15
10
5
0
DCMU
DCMU+NaN3
Supplemental Figure 2: Effect of sodium azide (NaN3) on the extent of singlet oxygen
production as measured by His-mediated O2 uptake. 1O2 production was quantified by
measuring the rate of His-mediated oxygen uptake in OCP-containing WT (grey), OCP
deficient ∆OCP (right hatch), OCP-overexpressing OE-OCP strain (cross hatch) and the
∆psbA2 strain (left hatch). The measurements were performed either in the presence of 10
µM DCMU or in the presence of DCMU plus 5 mM NaN3, a known quencher of singlet
oxygen. The measurements were performed using visible light (2300 µmol quanta m-2s-1).
Each bar represents the mean of three independent experiments, with the indicated
standard errors.
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
A
Control
OCP 5 µM
2 mT
EPR signal decrease (%)
B
100
80
60
40
20
0
1
2
3
4
5
OCP (µM)
6
7
8
Supplemental Figure 3. 1O2 quenching activity of OCP. In A and B, singlet oxygen was
produced by illumination (1000 µmol quanta m-2 s-1) of 10 µM Rose Bengal during 8 min in
the presence of TEMPD and in the absence (control) or in the presence of different
concentrations of OCP. In A, the EPR signal (32 scans) observed in the absence of OCP
(control) or in the presence of 5 µM OCP are shown. In B, the percentage of EPR signal
decrease versus OCP concentration is shown. 50% decrease was achieved at 1.5 µM OCP.
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
A
M
1
kDa
25
20
15
B
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0.8
0.6
0.4
0.2
0
350
All1123 ∆CrtR
1
Absorbance
Absorbance
1.2
All1123 WT
400
450
500
550
600
Wavelength (nm)
650
700
350
400
450
500
550
600
Wavelength (nm)
650
700
Supplemental Figure 4. Characterization of the All1123 RCP protein. (A) Detection
of RCP by OCP antibody. The purified RCP (All1123) isolated from ∆CrtR
Synechocystis cells was recognized by the antibody against Arthrospira OCP.
Immunodetection with anti-Arthrospira OCP antibody (line 1). Molecular mass marker
(line M). (B) Absorbance spectra of All1123 isolated from Synechocystis WT and
from the ∆CrtR mutant.
Figure 5
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
zea
9.57
28%
MXP
a)
8.28
44%
OHechi
carot
10.35
13%
echi
20.14
13%
16.02
2%
DeoxyMXP
14.14
56%
b)
16.03
20%
cantha
11.05
8%
20.11
16%
11.03
63%
Chl
15.57
c)
echi
zea
MXP 9.60 OH8.59 17% echi
16%
16.08
22%
10.37
3%
15.49
DeoxyMXP
d)
carot
20.24
42%
14.02
20%
20.15
51%
15.99
29%
Supplemental Figure 5: Carotenoid analysis. UV Chromatograms (450 nm) of
pigment
extracted from All1123 WT (A), All1123 ∆CrtR (B), methanolic extract of Synechocystis 6803
WT cells (C) and ∆CrtR mutant cells (D). Retention time (min) and relative integration of peak
area indicated (% of shown). MXP: myxoxanthophyll, zea: zeaxanthin, OH-echi: 3’hydroxyechinenone,
echi:
echinenone,
cantha:
canthaxanthin,
DeoxyMXP:
deoxymyxoxanthophyll, Chl a: Chlorophyll a, carot: β-carotene.
Carotenoids were separated on a 150 × 4.6 mm i.d., 5 µm, reverse-phase VisionHT C18 HL
C18 column (Grace, Epernon, France) using solvent A, ethyl acetate 100% (0.5% acetic acid),
and solvent B, water/acetonitrile (10:90, v/v, 0.5% acetic acid), with a gradient profile (from 10
to 95% A) and 0.5 mL/min flow rate. UV detection was set at 450 nm. Cone voltage was set at
30V, and collision energy at 30-40 eV for MS-MS experiments.
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
0,2
Absorbance
0,15
0,1
0,05
0
350
400
450
500
550
600
650
700
Wavelength (nm)
Supplemental Figure 6. Absorbance spectra of OCP WT in 50% ethanol in dark
conditions (black) and under illumination (red). The maximum of the peak is at
495 nm while the maximum of the red form of the OCP is at 510 nm. This spectra
strongly suggested that the protein is partially denaturated and almost not
photoactive. This could explain the lower activity as singlet oxygen quencher
observed for the OCP in 50% ethanol.
Supplemental data. Sedoud et al. (2014) Plant Cell 10.1105/tpc. 114.123802
0.5
Absorbance
0.4
0.3
0.2
0.1
0
300
400
500
600
700
Wavelength (nm)
Supplemental Figure 7: Changes in absorption spectra of OCP under illumination.
The black trace corresponds to the spectrum of OCPo. The OCP was illuminated 5 min
with 5000 µmol quanta m-2s-1 white light (red trace) or with 1000 µmol quanta m-2s-1
white light (blue trace).