Two Quenchers Formed During Photodamage of Phostosystem II and The Role of One Quencher in Preemptive Photoprotection

The quenching of chlorophyll fluorescence caused by photodamage of Photosystem II (qI) is a well recognized phenomenon, where the nature and physiological role of which are still debatable. Paradoxically, photodamage to the reaction centre of Photosystem II is supposed to be alleviated by excitation quenching mechanisms which manifest as fluorescence quenchers. Here we investigated the time course of PSII photodamage in vivo and in vitro and that of picosecond time-resolved chlorophyll fluorescence (quencher formation). Two long-lived fluorescence quenching processes during photodamage were observed and were formed at different speeds. The slow-developing quenching process exhibited a time course similar to that of the accumulation of photodamaged PSII, while the fast-developing process took place faster than the light-induced PSII damage. We attribute the slow process to the accumulation of photodamaged PSII and the fast process to an independent quenching mechanism that precedes PSII photodamage and that alleviates the inactivation of the PSII reaction centre.

changes in the τ AV in Panel c were fitted to a double exponential. The criteria to fit each signal were based on using the best fitting for the obtained results.
In order to facilitate a comparison between different treatments, the time point where 50% of the changes were observed (T 50 = ln 2/k PI ) were compared for both experimental model systems. It was observed for illuminated leaves that for 460 nm illumination, T 50 is smaller than for 660 nm (see Table 1). These data are in good agreement with the previously reported action spectra of photodamage for different species 3,12,21,25 where the blue region of the visible spectrum induced more damage than the red region. By contrast in leaves the decay of F V /F M for both wavelengths was the same in BBY's (see Table 1).
Photodamage was accompanied by shortening of τ AV in both leaves and BBY's, as presented in Fig. 1c,d. In the case of leaves (Fig. 1c) illumination with 460 nm light induced a faster shortening of τ AV than illumination with 660 nm light. This shortening of τ AV can be described by a double-exponential decay and the fitting parameters are presented in Table 1 (and Supplementary Table 2).
BBY's do not show a statistically significant difference in τ AV for 460 nm and 660 nm light ( Table 1). The observed time course does not follow a simple first-order rate law; at least two components are needed (Eq. 3, see Supplementary Information). This can be interpreted as two quenching processes occurring in BBY's. It is remarkable that for both wavelengths most of the changes in τ AV occur during the first minutes of illumination.
It has been hypothesized that PSII photodamage increases the yield of fluorescence in the F O state 24,26 . An increase of F O corresponds to an increase of the average fluorescence lifetime of PSII. However, it was reported 23 that even in the absence of NPQ, τ AV decreased with illumination time when measured in the F M state (closed PSII RCs). In the dark-adapted state, as measured in present study, the τ AV is expected to be shortest under control conditions since all RCs are expected to be open. Energy cannot be used more efficiently by the RCs than in the dark adapted state. Therefore, the observed shortening of τ AV must be interpreted as an energy dissipation event which is different from primary photochemistry. It is worth noting that under control conditions (darkness across the time course) no significant changes in τ AV and F V /F M were observed in vivo or in vitro ( Supplementary Fig. 2).
In Supplementary Fig. 3 (shifting of histograms) and 4 (representative micrographs) the changes in τ AV of leaves after 460 and 660 nm illumination are shown and the values of τ AV are colour coded. The various images taken at different time points show that τ AV shortens during the illumination experiment for both illumination wavelengths. We observed that a change in τ AV occurred in the whole chloroplast and that there was heterogeneity among chloroplasts within the same focal plane.

The decrease in average fluorescence lifetime is faster than the increase in PSII photodamage.
If the changes in τ AV are a consequence of photodamage that leads to the decrease of F V /F M , they should occur at an equal or lower rate as changes in PSII efficiency. Which is not what is observed in the present experimental observation. Table 1 shows that τ AV in leaves (0.9 h at 460 nm and 1.6 h at 660 nm) decreased faster than PSII efficiency (1.8 h at 460 nm and 3.3 h at 660 nm) for both wavelengths (T 50 for decrease in τ AV is 2 times smaller than the T 50 for photodamage, see Table 1). This implies that the decrease in τ AV cannot be solely explained by loss of maximum PSII efficiency. Similarly, BBY's showed a faster decrease in τ AV (4.5 min at 460 nm and 4.9 min at 660 nm) than PSII efficiency (∑28 min for both wavelengths); T 50 (τ AV ) is 3.4 times smaller than T 50 (F v /F m ) in the case of 460 nm excitation, while for 660 nm it is 6.1 times smaller. Despite the differences in complexity between in vivo and in vitro (BBY's do not have an unlimited source of electron acceptors, PSI is absent, and other molecular pathways are not present), both experimental model systems showed: (1) photodamage shows single exponential behaviour; (2) higher rate of photodamage induced by 460 nm than by 660 nm light; (3) shortening of τ AV ; (4) a faster decrease of τ AV than of PSII efficiency. All these observations support the idea that the mechanism(s) of photodamage and quenching in vitro and in vivo have a common origin.
Analysis of the fluorescence lifetime components and populations shows that there are at least two types of quenchers formed, one dependent on and one independent of photodamage.
In leaves, decrease in τ AV can be satisfactorily fitted with a single exponential, so only one population of quenched PSII was observed. However, it is possible to analyse the three individual lifetime components of the τ AV (each lifetime component, a i *τ i , has its own amplitude and lifetime) to establish the relative contributions of individual quenching events to the overall decrease in τ AV . It is possible to discern the presence of either one or several quenchers by comparison between the three individual lifetime components of τ AV to T 50 of loss of F V /F M www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 2a,b). It was observed that the first component (C1 = a 1 *τ 1 , which had the shortest fluorescence lifetime) has a T 50 value similar to that of the loss of F V /F M ) for both illumination wavelengths. The second component of the fluorescence lifetime (C2) had shorter T 50 values (faster development) than that of photodamage but similar to the T 50 of the decrease of F M at both wavelengths. The T 50 of the third component (C3) was statistically similar to that of F M decrease for 460 nm but that was not the case at 660 nm. This suggests that there are at least two distinct quenching processes occurring in parallel in leaves, one where the quencher is formed as a result of photodamage and another where the quencher (represented by C2) is dependent only on illumination. The latter was formed faster and explains why the change in τ AV has shorter T 50 values than the T 50 of the loss of F V /F M . It is worth noting that the decrease of F O was very slow and cannot be satisfactorily fitted appropriately to our single exponential model (See Supplementary Fig. 5).
In in vitro measurements, decrease in τ AV can be satisfactorily fitted as double exponential indicating two populations of quenched PSII being formed in response to illumination (decrease of τ AV ). The first (fast) population was quenched almost immediately after illumination started (0.3 and 1.1 min for 460 and 660 respectably) and the second population formed in a slower manner (approximately 8 min). The decay in F O was remarkably faster and it was not possible to fit the data points in a satisfactory manner (see Supplementary Fig. 5). By contrast, the T 50 for the changes in F M is statistically similar to the T 50 of the slow population. None of the two quenched populations seemed to correspond to photodamaged PSII as none of them matched the T 50 of decrease of PSII maximum efficiency. The analysis of individual components of BBY's was not possible as each component followed a complex kinetics that did not adjust to first or second order rate laws and for this reason were not taken into account for interpretation.
photodamage to the Rc is slower than total pSii inactivation and dependent on excessive pigment excitation. Three independent reports ( 3,4,12 ) have shown that RC photoinactivation is slower than Total PSII inactivation. Also, it was shown than RC inactivation is dependent on excessive pigment excitation. We have shown that quenchers can be formed before Total PSII inactivation takes place, so it is within reason to hypothesize that the observed quenchers photoprotect the RC to some extent. For this reason, we have estimated the effect on the electron transport rate (ETR) activity of PSII at the time point corresponding to T 50 of photodamage (which was estimated by PSII efficiency). www.nature.com/scientificreports www.nature.com/scientificreports/ Due to the difference between in vivo and in vitro two approaches were used to estimate the effect on ETR. For leaves the relative ETR was estimated using the modulated reflectance changes at 820 nm of P700 by measuring the amplitude between 0.014 s to the maximum reflectance intensity usually located around 0.2 s (during illumination). In Fig. 3a, a comparison between the efficiency to pump electrons from PSII to PSI (re-reduction of P700 + ) is presented for both wavelengths at the T 50 time point (2 h for 460 nm and 3 h for 660 nm). As expected from the F V /F M kinetics, at T 50 of both conditions PSII activity dropped by the same magnitude, indicating that the electron flow between both systems (ETR) was impaired. Figure 3b displays the net amplitude of the P700 accumulation at 220 ms (maximum P700 value for the control), for 460 nm a decrease in the activity of ∑60% was observed while for 660 nm the decrease was ∑50%. One should note that the difference between illuminations is not statistically significant, but both are statistically different from the control condition. This confirms that PSII of illuminated leaves were indeed photoinactivated.
In order to estimate ETR in vitro a colorimetric DCPIP assay was used (Fig. 3c,d). This method allows the two activities to be probed separately (total PSII activity and RC activity, see methods). Photodamaged samples illuminated in the absence of electron acceptors showed that RC inactivation was significantly lower than the loss of total activity (t-test 460nm < 0.1 and t-test 660nm < 0.05) at T 50 . This effect was observed to a greater extent if the sample was exposed under the presence of artificial electron donor (DPC) and acceptor (PPBQ), where RC inactivation was only approx. 20% and significantly lower (t-test < 0.001 at both wavelengths). This indicates that RC damage is clearly dependent on excitation pressure and slower than total PSII inactivation.
Comparison of the ETR measurements and PSII efficiency for dark controls and photodamage are presented in Supplementary Fig. 6.

Discussion
In this work, we address the issue that photodamage is accompanied by a decrease in τ AV in two experimental model systems under excessive excitation. We will analyse four possible scenarios that can explain the observed quenching due to PSII photodamage and shortening of τ AV : www.nature.com/scientificreports www.nature.com/scientificreports/ (1) Quenching represents the first alterations in the PSII functionality (there is no photoprotective effect) 27,28 ; (2) Long lasting qE, qZ, and qT 29-32 ; (3) Dissipation of excessive light energy from active PSII by neighboring inactivated PSII RCs 23,33 , implying that photodamaged PSII centres have a photoprotective role 23 ; (4) Accumulation of a photoproduct, not related to photodamaged PSII itself 34 .
Scenario 1 would imply that the observed quenching effect is a direct consequence of photodamage that inactivates PSII. Here, we demonstrate that the overall quenching in the two model systems occurred faster than the accumulation of PSII photodamage. It has been hypothesized that PSII photodamage should increase the yield of fluorescence in the F O state 26 , but this should be manifested as an increase of τ AV which is contrary to experimental observations in both model systems.
Scenario 2 would link some components of qI with residual of qE, qZ, and qT mechanisms. One possible explanation of the long lasting quenching is a manifestation of qI at the antenna 35 , and the decrease of F V /F M is due to long lasting qZ. In both in vivo and in vitro models, F M decreased faster than F V /F M . In vivo the quencher that is accumulated faster seems to be correlated with F M which in turn reflects the changes in the efficiency of energy dissipation 35 . However, this process should not occur in vitro, where the decrease in F M is also faster than F V /F M . For this reason, component 2 cannot be directly related to a long lasting qZ mechanism.
Also, it has been reported that a non-linear relationship of photoprotection and quenching is possible 22 . However, in this work samples were measured several hours after photodamage, indicating that the observed quenching is a long-lived component of qI. It was demonstrated 23 that quenching during photodamage occurs regardless of the use of DTT and nigericin in leaves which discards the role of qE, qZ, and qT as the sole explanation of the observed quencher. The alternative is that dark-sustained xanthophyll would be accumulated, but this type of quenching has τ AV longer than F O 36 , which is contrary to present observations. Furthermore 37,38 , demonstrated that violaxantin conversion occurs very slowly in leaves not acclimated to chilling temperatures, as in the present study, so the contributions of qZ in this particular case would not explain alone the strong quenching effect. Finally, these types of quenching are not present in BBY's. Scenario 3 attributes the origin of the quencher to inactive photodamaged RCs 33 , that concomitantly protect neighbouring active PSII by dissipating excessive excitation. This scenario is dependent on PSII photodamage, as the quenchers are the inactivated PSII. In leaves, a direct relation between photoinactivated PSII and formation of quenchers could be established because of the similarities of T 50 of decrease in the second component of the fluorescence lifetime. However, this scenario is only applicable in vivo.
It should be noted that a photoinactivated RC is not equivalent to a closed RC. While both closed RC and photoinactivated RC cannot reduce Q A , a closed RC has τ AV in the range of ns and remains active. In contrast, a photoinactivated RC is chemically modified (due to photodamage), it is inactive and we have demonstrated that its lifetime becomes shorter, rather than longer as it is seen in the closed RC.
Scenario 4 considers a photoproduct generated under high light that acts as a quencher of PSII fluorescence, which is not related to PSII photodamage. This scenario is a good candidate to explain the fast quenching observed in vivo and in vitro, as the observed decrease of τ AV is not related to the accumulation of photodamaged PSII. It has been suggested that long-lived quenchers can be formed in LHCII complexes, originating from chlorophyll cations or other radicals produced from chlorophyll triplets 33 . As these long-lived quenchers in LHCII are expected to be homogeneously distributed across the PSII population and that LHCII is functionally connected to PSII, they would be able to quench excessive excitations efficiently. Thus, we hypothesize that an unidentified quencher located in the LHCII is a reasonable explanation for the fast quenching component.
Such quencher can be present in leaves and BBY's as both contain functional LHCII. The difference in the lifetime kinetics of shortening of lifetime between the two model systems is due to the difference in the organizational level of both systems (i.e. ability to change stacking or presence of other possible quenchers in vivo). This could be the reason why two quenched PSII populations were observed in BBY's.
Since damage to the RC 3,4 and the repair mechanism 15 is dependent on excitation pressure, the formation of the quenchers before PSII photodamage operates as a pre-emptive photoprotective mechanism that dissipates the accumulated excess energy. This is consistent with the observations that the quencher(s) is (are) accumulated faster (faster shortening of τ AV ) than photodamaged PSII (loss of F V /F M ), thereby slowing down photoinactivation significantly.
The assays to estimate the electron transport rate (ETR) confirmed that PSII was photoinactivated in both model systems. However, the amount of RC damage can only be estimated in vitro. RC damage is greater under limited electron transport at the acceptor and donor side, and vice versa, which indicates that RC is susceptible to damage under excessive light excitation. We hypothesize that PSII photodamage is slower than quencher accumulation because the quencher(s) relieve(s) excessive excitation as soon as it is formed.
In summary, the quenchers photoprotect the RC because its photodamage is dependent on excessive excitation and its damage is slower than the loss of the total PSII activity. We hypothesize that the quenching observed in both experimental model systems has the same nature as both models are photodamaged, both models present a quencher that accumulates faster than the photodamaged PSII and the lifetime of both models is shorter than in F O state. This faster-developing quenching process has two implications: (1) the quencher acts as a "pre-emptive" photoprotection mechanism and (2) quencher formation depends mostly on illumination.

Materials and Methods
Plant material and sample preparation. Fresh and intact spinach leaves were purchased at local markets www.nature.com/scientificreports www.nature.com/scientificreports/ PSII efficiency measurements). BBY's were prepared from fresh market spinach as described (see Supplementary Methods). photodamage in vivo. Before illumination, to inhibit photorepair, leaves were infiltrated with 5 mM lincomycin by passive petiole infiltration for 12 hours in order to ensure its uptake by the leaf tissue 23,39 . The irradiance at the surface of the leaf during illumination was 1300 µmol photons m −2 s −1 of narrow band light (460 ± 10 nm or 660 ± 10 nm). The sample was illuminated for 30, 60, 120, 180 and 300 min. The control group was kept under identical conditions in darkness inside the photoinhibition boxes (see Supplementary Methods). The experiment was repeated three times. photodamage in vitro. Samples were illuminated with either 460 or 660 nm light for 1, 2.5, 5, 20 or 60 min (1300 µmol photons m −2 s −1 ). BBY's were then centrifuged at 20,000 × g, after which they were re-suspended in standard buffer for cryogenic storage. Aliquots of the sample then were flash frozen in liquid nitrogen and stored at −80 °C until use. The experiment was repeated three times. PSII activity measurements. Leaves were dark adapted for at least 15 min at room temperature. Each leaf was measured from petiole to tip in at three different regions. After the measurements, the leaves were transferred back to the LED photoinhibition boxes. The PSII efficiency was measured at room temperature with an M-PEA fluorometer according to Strasser et al. 40 (Hansatech Instruments). The actinic light intensity was 3000 photons m −2 s −1 for 10 s. All sample manipulation occurred under low light of less than 1 µmol photons m −2 s −1 .
To measure the PSII photochemical efficiency of BBY's, an aliquot of the sample was defrosted at 4 °C and then diluted with Buffer A and centrifuged at 20,000 × g. Then the concentration of BBY's was adjusted to 150 µg of Chl mL −1 . PSII efficiency was measured by Chl a fluorescence using the same protocol described above. 30 µL of the sample was transferred to wet filter paper before measurement.
Colorimetric measurements for BBY's were carried down as reported 4 . Additional PSII activity measurements were done using P700 oxidation kinetics as 820 nm reflectance changes with the method of 41 . P700 signal was recorded simultaneously with the chlorophyll fluorescence using a Multifunctional Plant Efficiency Analyser M-PEA (built by Hansatech Instrument Ltd., King's Lynn, Norfolk, PE30 4NE, UK). The M-PEA sensor unit is designed to detect 820 ± 25 nm modulated light from an LED and the detector is shielded with band pass filters at 820 ± 20 nm.
Time correlated single photon counting (TCSPC). Excitation was carried out by 0.2 ps excitation pulses (412 nm) at a repetition rate of 3.8 MHz. The excitation power was reduced with neutral density filters and the detection rate was kept below 30,000 photons s −1 . The diameter of the excitation spot was 2 mm, and the excitation laser power was kept at 1-5 µW to keep PSII RCs (RC) in the open state. The sample was kept in a flow cuvette (flowing speed ~2.5 mL s −1 ) connected to a sample reservoir (7.5 mL), kept at 20 °C. The optical path length of the cuvette was 3 mm and the optical density of the sample was 0.1 per cm. Fluorescence was collected at right angle to the excitation beam, under magic angle (54.7°) polarization with a 680 nm interference filter (15 nm bandwidth), as described previously 42 .
The instrument response function (IRF) was obtained from the 6 ps decay of pinacyanol iodide in methanol 43 (the full width at half maximum of the IRF was 35 ps and a resolution of 2 ps per channel was used). Data analysis was performed with a home-built program 44,45 . For the global analysis the decay lifetime components were kept equal for each run of measurements on the sample exposed to a certain illumination time. The fit quality was judged from the residuals of the fit. Fluorescence decay curves were fitted to a sum of exponentials with the amplitudes a i and fluorescence decay times τ i , convoluted with the IRF 46 .
Average lifetimes were calculated as: , a i is the amplitude of the i-th component and τ i is the lifetime of the i-th component.

Two-photon fluorescence lifetime imaging microscopy (FLIM). Time resolved measurements on
leaves were performed on a FLIM setup described 47 . Fluorescence was selected using a 680 nm interference filter (13 nm bandwidth) and a 770 nm cut-off filter was used to prevent detection of the excitation beam. A neutral density filter was used in the excitation path to reduce the excitation light intensity. The output of the detector was coupled to a Becker & Hickl single-photon counting module (SPC 830) 48 . The time window was set to 256 channels and fluorescence was recorded for 5 min at a count rate of 10,000 counts per second. The IRF was obtained from decay of pinacyanol iodide in methanol.
SPCImage software from Becker&Hickl was used to process FLIM images. Fluorescence decay curves were fitted to a sum of N exponentials, convoluted with the IRF, the lifetime components were fitted individually for each pixel. Average lifetimes were calculated using Eq. 1.

Data availability
The datasets generated during and/or analysed during the current study are available in supplementary data and from the corresponding author on reasonable request.