EPR Spin-Trapping for Monitoring Temporal Dynamics of Singlet Oxygen during Photoprotection in Photosynthesis

A central goal of photoprotective energy dissipation processes is the regulation of singlet oxygen (1O2*) and reactive oxygen species in the photosynthetic apparatus. Despite the involvement of 1O2* in photodamage and cell signaling, few studies directly correlate 1O2* formation to nonphotochemical quenching (NPQ) or lack thereof. Here, we combine spin-trapping electron paramagnetic resonance (EPR) and time-resolved fluorescence spectroscopies to track in real time the involvement of 1O2* during photoprotection in plant thylakoid membranes. The EPR spin-trapping method for detection of 1O2* was first optimized for photosensitization in dye-based chemical systems and then used to establish methods for monitoring the temporal dynamics of 1O2* in chlorophyll-containing photosynthetic membranes. We find that the apparent 1O2* concentration in membranes changes throughout a 1 h period of continuous illumination. During an initial response to high light intensity, the concentration of 1O2* decreased in parallel with a decrease in the chlorophyll fluorescence lifetime via NPQ. Treatment of membranes with nigericin, an uncoupler of the transmembrane proton gradient, delayed the activation of NPQ and the associated quenching of 1O2* during high light. Upon saturation of NPQ, the concentration of 1O2* increased in both untreated and nigericin-treated membranes, reflecting the utility of excess energy dissipation in mitigating photooxidative stress in the short term (i.e., the initial ∼10 min of high light).


Contents
Characterization of oxygen evolution capabilities of thylakoid membrane preparation showing a comparison of fresh thylakoids (1 day old) and 2-month-old frozen thylakoids in the presence and absence of electron accepting molecules (250 µM DCBQ and 1 mM ferricyanide) in glycerol resuspension buffer (pH 6).All O2 evolution curves were measured using a Unisense Oxy-NP probe with a 2-point O 2 concentration calibration, ranging from 0 to 278 μM.Prior to measurement, the buffer was bubbled with N 2 for at least 30 min to minimize the concentration of O 2 .Immediately prior to measurement, thylakoid stock was diluted into buffer at a concentration of 80 µg Chl/mL in a septa-capped cuvette equipped with a stir bar and stored in the dark.For each experiment, the LED was turned ON at 1 min.As a control experiment to assess the effect of pre-illumination on the apparent spin concentration, thylakoids were incubated with TEMPO for exactly 1 minute in the light following durations of 0, 15, or 60 minutes of preillumination.The concentration of TEMPO measured for all durations of illumination was within ~10% of each other.While it is possible that this small change reflects differences in the concentration or composition of reductants during illumination of membranes, it is more likely representative of the uncertainty (~10%) associated with the EPR measurements especially given that each thylakoid sample was measured using a different capillary.Therefore, the concentration of thylakoid reducing equivalents produced during a short 1-minute incubation is mostly independent of the duration of prior HL illumination of the membranes.

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S9 A B S10

Figure S1 :
Figure S1: Characterization of O 2 evolution capability of thylakoids.

Figure S2 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S3 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S4 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S5 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S6 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S7 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S8 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S9 :
Figure S2: Spectrum of light source.Figure S3: Fluorescence lifetime measurements of thylakoids with MV and DCBQ.Figure S4: Absorption spectra of photosensitizers (TB and RB).Figure S5: Light intensity dependence of 1 O 2 * photosensitization by TB.Figure S6: Additional replicates for the kinetics of 1 O 2 * in thylakoids.Figure S7: EPR control measurements with ATP.Figure S8: Freeze-thaw effect on TEMPO EPR signal.Figure S9: Spin concentrations measured in snapshot EPR spectroscopy during illumination of thylakoid membranes.Figure S10: Kinetic analysis of changes in TEMPO concentration during illumination.

Figure S1.
Figure S1.Characterization of oxygen evolution capabilities of thylakoid membrane preparation showing a comparison of fresh thylakoids (1 day old) and 2-month-old frozen thylakoids in the presence and absence of electron accepting molecules (250 µM DCBQ and 1 mM ferricyanide) in glycerol resuspension buffer (pH 6).All O2 evolution curves were measured using a Unisense Oxy-NP probe with a 2-point O 2 concentration calibration, ranging from 0 to 278 μM.Prior to measurement, the buffer was bubbled with N 2 for at least 30 min to minimize the concentration of O 2 .Immediately prior to measurement, thylakoid stock was diluted into buffer at a concentration of 80 µg Chl/mL in a septa-capped cuvette equipped with a stir bar and stored in the dark.For each experiment, the LED was turned ON at 1 min.

Figure S2 .
Figure S2.Spectrum of the daylight white LED (SOLIS-3C, ThorLabs) used as a light source in this study.

Figure S3 .
Figure S3.Effect of methyl viologen (MV, 50 uM), DCBQ (DCBQ, 250 uM), and buffer pH on fluorescence lifetime measurements of NPQ.Similar NPQ magnitude and kinetics were observed in the presence and absence of MV, independent of buffer composition and pH.Inclusion of DCBQ, an artificial electron acceptor for PSII, resulted in significantly shorter lifetimes presumably because DCBQ replenishes the depleted plastoquinone pool leading to higher activity of PSII reaction centers and associated photochemical quenching of Chl fluorescence.

Figure S10 .
Figure S10.Kinetic analysis of changes in TEMPO concentration during illumination of (A) untreated and (B) nigericintreated thylakoid membranes.The initial time points were fitted with an exponential to extract the approximate timescale of the decrease in singlet oxygen production, suggesting faster photoprotection in the absence of nigericin (5.7 min vs. 12.0 min).Error bars designate standard error of the mean (n=3).