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ORIGINAL RESEARCH
published: 24 June 2020
doi: 10.3389/fpls.2020.00849
Edited by:
Yoshitaka Nishiyama,
Saitama University, Japan
Reviewed by:
Seiji Akimoto,
Kobe University, Japan
Stefano Santabarbara,
National Research Council (CNR), Italy
*Correspondence:
Petar H. Lambrev
lambrev.petar@brc.hu
Specialty section:
This article was submitted to
Plant Physiology,
a section of the journal
Frontiers in Plant Science
Received: 31 March 2020
Accepted: 26 May 2020
Published: 24 June 2020
Citation:
Lingvay M, Akhtar P,
Seb ˝
ok-Nagy K, Páli T and
Lambrev PH (2020) Photobleaching
of Chlorophyll in Light-Harvesting
Complex II Increases in Lipid
Environment. Front. Plant Sci. 11:849.
doi: 10.3389/fpls.2020.00849
Photobleaching of Chlorophyll in
Light-Harvesting Complex II
Increases in Lipid Environment
Mónika Lingvay1,2 , Parveen Akhtar1, Krisztina Seb ˝
ok-Nagy3, Tibor Páli3and
Petar H. Lambrev1*
1Institute of Plant Biology, Biological Research Centre, Szeged, Hungary, 2Doctoral School of Physics, Faculty of Science
and Informatics, University of Szeged, Szeged, Hungary, 3Institute of Biophysics, Biological Research Centre, Szeged,
Hungary
Excess light causes damage to the photosynthetic apparatus of plants and algae
primarily via reactive oxygen species. Singlet oxygen can be formed by interaction of
chlorophyll (Chl) triplet states, especially in the Photosystem II reaction center, with
oxygen. Whether Chls in the light-harvesting antenna complexes play direct role in
oxidative photodamage is less clear. In this work, light-induced photobleaching of Chls in
the major trimeric light-harvesting complex II (LHCII) is investigated in different molecular
environments – protein aggregates, embedded in detergent micelles or in reconstituted
membranes (proteoliposomes). The effects of intense light treatment were analyzed
by absorption and circular dichroism spectroscopy, steady-state and time-resolved
fluorescence and EPR spectroscopy. The rate and quantum yield of photobleaching
was estimated from the light-induced Chl absorption changes. Photobleaching occurred
mainly in Chl aand was accompanied by strong fluorescence quenching of the
remaining unbleached Chls. The rate of photobleaching increased by 140% when
LHCII was embedded in lipid membranes, compared to detergent-solubilized LHCII.
Removing oxygen from the medium or adding antioxidants largely suppressed the
bleaching, confirming its oxidative mechanism. Singlet oxygen formation was monitored
by EPR spectroscopy using spin traps and spin labels to detect singlet oxygen directly
and indirectly, respectively. The quantum yield of Chl aphotobleaching in membranes
and detergent was found to be 3.4 ×10−5and 1.4 ×10−5, respectively. These
values compare well with the yields of ROS production estimated from spin-trap
EPR spectroscopy (around 4 ×10−5and 2 ×10−5). A kinetic model is proposed,
quantifying the generation of Chl and carotenoid triplet states and singlet oxygen.
The high quantum yield of photobleaching, especially in the lipid membrane, suggest
that direct photodamage of the antenna occurs with rates relevant to photoinhibition
in vivo. The results represent further evidence that the molecular environment of LHCII
has profound impact on its functional characteristics, including, among others, the
susceptibility to photodamage.
Keywords: electron paramagnetic resonance, non-photochemical quenching, photoinhibition, photosystem II,
reconstituted membranes, singlet oxygen
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Lingvay et al. Photobleaching in Light-Harvesting Complex II
INTRODUCTION
Plants have to cope with variable light conditions – maintaining
efficient light harvesting while avoiding photodamage (Li et al.,
2009). Prolonged exposure to excess light causes photoinhibition,
that is decrease in photosynthetic activity, followed by chlorosis –
bleaching of chlorophylls (Chl) – and ultimately death. The
primary site of photoinhibition is PSII (Aro et al., 1993) and the
major route of PSII photoinactivation involves ROS, especially
singlet oxygen (1O2), formed by the interaction of molecular
oxygen with the Chl triplet (3Chl) states (Triantaphylides et al.,
2008;Vass, 2011;Fischer et al., 2013). Most of the Chls are located
in the light-harvesting antenna, including the core antenna, CP43
and CP47, and LHCII monomers and trimers (van Amerongen
and Croce, 2013). However, it is believed that the antenna has
negligible role in the production of ROS because the 3Chl states
are effectively quenched by carotenoids (Cars) bound to the
complexes (Breton et al., 1979;Sonneveld et al., 1979;Frank
and Cogdell, 1996). In contrast, 3Chl states in the PSII RC are
readily formed following charge recombination (Vass and Cser,
2009;Vass, 2011) and, because they are relatively far from the
nearest Cars, quenching is less efficient. The formation of 1O2
during light exposure of chloroplast thylakoid membranes has
been directly followed by spin-trapping EPR spectroscopy and
associated with the acceptor-side inhibition of PSII and the D1
protein degradation (Hideg et al., 1994a,b).
Despite the abundance of Cars, 3Chl have been detected in
isolated core antenna (Carbonera et al., 1992b;Groot et al.,
1995) and peripheral antenna complexes (Carbonera et al., 1992a;
Peterman et al., 1995;Barzda et al., 1998) and found to sensitize
the formation of ROS, including 1O2(Rinalducci et al., 2004). As
a result, Chl PB has been observed in native and recombinant
LHCII exposed to strong illumination in aerobic conditions
(Formaggio et al., 2001;Zhang et al., 2008) and found to depend
on the Car composition of the complex (Croce et al., 1999).
Mozzo et al. (2008) studied the quenching capacity of individual
Cars in LHCII and concluded that about 5% of Chl triplets are not
quenched by Cars in contrast to the earlier results (Siefermann-
Harms and Ninnemann, 1982;Peterman et al., 1995). Using
optical magnetic resonance, Santabarbara et al. (2002a) detected
3Chl in thylakoid membranes generated far from the PSII RC.
Together with the observed inefficiency of excitation quenching
to protect from the loss of PSII activity and the blue-shifted action
spectrum of photoinhibition, they proposed the involvement of
weakly coupled Chls in PSII photoinhibition (Santabarbara et al.,
2001b, 2002b;Santabarbara, 2006).
When exposed to light, especially in the presence of oxygen,
free Chls undergo PB or photomodification by a variety of
mechanisms (Bonnett et al., 1999). Cars are also sensitive to
Abbreviations: 1O2, singlet oxygen; 4-oxo-TEMPO, (4-oxo-2,2,6,6-
tetramethylpiperidin-1-yl)oxyl; 5-SASL, 5-doxyl-stearic acid spin label; Car,
carotenoid; CD, circular dichroism; Chl, chlorophyll; EPR, electron paramagnetic
resonance; LHCII, light-harvesting complex II; NPQ, non-photochemical
quenching; PAR, photosynthetically active radiation; PB, photobleaching;
PFD, photon flux density; PSII, photosystem II; RC, reaction center; ROS,
reactive oxygen species; TCSPC, time-correlated single-photon counting;
TEMPD ×H2O, 2,2,6,6-tetramethyl-4-piperidone monohydrate; TEMPO,
(2,2,6,6-tetramethylpiperidin-1-yl)oxyl; β-DDM, n-dodecyl-β-maltoside.
oxidative photodamage and appear to be bleached faster than
Chls upon irradiation of thylakoid membranes or PSII-enriched
membranes (Yamashita and Butler, 1969;Yamashita et al., 1969;
Klimov et al., 1990), which in turn accelerates the PB of Chls
(Santabarbara, 2006). Also, Chls absorbing at longer wavelengths
are bleached before those absorbing at shorter wavelengths
(Zucchelli et al., 1988;Miller and Carpentier, 1991). PB of Chl
boccurs at a much slower rate than Chl a– due to fast energy
transfer between them (Carpentier et al., 1986;Peterman et al.,
1997). These findings point toward the role of antenna Chls in
photodamage. Several studies have followed the photodamage in
isolated light-harvesting complexes (Zucchelli et al., 1988;Croce
et al., 1999;Formaggio et al., 2001;Olszówka et al., 2003;Zhang
et al., 2008;Zubik et al., 2011) but a quantitative analysis of the
kinetics and quantum yield of pigment PB and its relevance to
photoinhibition is lacking.
Not only the pigments but also the apoprotein is vulnerable to
degradation by ROS, in addition to the proteolytic degradation of
photosynthetic proteins known to occur during photoinhibition
(Li et al., 2018). Zolla and Rinalducci (2002) reported the direct
photodegradation of LHCII without the involvement of proteases
(Rinalducci et al., 2004). Using spin trapping EPR spectroscopy,
the group detected the generation of ROS in isolated LHCII upon
irradiation with visible light and correlated it with fragmentation
of the polypeptide. It was also pointed out that the cleavages
take place in the hydrophilic portion of the N-terminal region.
On the other hand, the protein secondary structure was not
affected by PB of the bound pigments (Olszówka et al., 2003).
Zubik et al. (2011) also followed changes in LHCII upon exposure
to strong light and postulated the photoisomerization of Cars,
particularly neoxanthin.
LHCII is known to have both structural and functional
flexibility (Lambrev and Akhtar, 2019). It plays a crucial role
in photoprotection by NPQ. The purpose of NPQ is precisely
to minimize photodamage of the system by ROS generated
under excess light. The reasoning is that when LHCII is in
its quenched state, i.e., singlet excitations rapidly decay via
thermal deactivation, the formation of ROS and the photodamage
should be reduced; however, no quantitative experimental data
exists to confirm this. The switch between light-harvesting and
energy-dissipating mode involves changes in the molecular and
supramolecular structure of the pigment–protein complexes
(Ruban, 2016). This may include aggregation or clustering of
LHCII (Horton et al., 2005), which is well known to induce strong
excitation quenching – both in lipid-free aggregates (Ruban
and Horton, 1992, 1994) and in protein-dense reconstituted
membranes (Natali et al., 2016;Crisafi and Pandit, 2017;Akhtar
et al., 2019). In addition, we have observed characteristic changes
in the pigment–protein and protein–protein interactions in
LHCII upon changing its molecular environment – in aggregates
and reconstituted membranes – some of which are not associated
with NPQ (Akhtar et al., 2015). It is not clear how these changes
might affect the susceptibility to photodamage.
The aim of this work is to quantify Chl PB in isolated LHCII
in different molecular environments – detergent-solubilized
LHCII trimers, quenched LHCII-aggregates and reconstituted
membranes. The effects of intense light treatment were analyzed
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Lingvay et al. Photobleaching in Light-Harvesting Complex II
by absorption and CD spectroscopy, steady-state and time-
resolved fluorescence, and EPR spectroscopy. One could naively
presume that LHCII is more stable in the quenched aggregates
but also in lipid membranes, which are closer to the native
environment. For example, higher thermostability of the complex
has been shown in reconstituted lipid membranes (Yang et al.,
2006). On the contrary, the results presented here reveal a
markedly increased oxidative PB of Chls when LHCII is in a lipid
environment. Further, we estimated the rate and quantum yield
of PB of Chl a, compared it with the yield of ROS formation
detected by EPR spectroscopy and also with predictions from
theoretical modeling.
MATERIALS AND METHODS
Preparation of LHCII
LHCII trimers were purified by solubilization of PSII-enriched
membrane fragments isolated from 14-days-old greenhouse
grown pea (Pisum sativum) leaves with 0.7% n-dodecyl-β-
maltoside (β-DDM, Cube Biotech, Germany) followed by
sucrose gradient ultracentrifugation. Reconstituted membranes
of LHCII and plant thylakoid lipids (lipid:protein ratio
100:1) were prepared using the protocol described previously
(Akhtar et al., 2016). LHCII aggregates were prepared by
removal of the detergent from suspension of solubilized
complexes with polystyrene adsorbent beads (Bio-Beads SM-2
Resin, Bio-Rad). The Chl and Car contents were determined
spectrophotometrically from 80% acetone extracts using molar
absorption coefficients from Lichtenthaler (1987) and are
presented in Supplementary Table 1.
Photooxidation of LHCII Pigments
For the comparative photostability tests, samples were diluted to
absorbance of 0.4 at the red maximum and placed in a glass cell of
1-cm optical pathlength. White light from a KL 2500 LED lamp
(Schott, Germany) was used for irradiation, with incident PFD
on the cuvette of 3000 µmol photons m−2s−1PAR. This PFD
is equivalent to an average of ca. 2000 µmol photons m−2s−1in
the whole sample volume. For testing the wavelength dependence
of PB, the actinic light was passed through either a Schott FS red
(630 nm) or a Schott FS blue (525 nm) filter, and the intensity
was adjusted to obtain an equal fluorescence emission from the
sample. A set of experiments was performed with light from a KL
1500 electronic lamp (Schott, Germany) passed through an SZS–
22 glass cutoff filter (580 nm) to an incident PFD of 500 µmol
photons m−2s−1PAR.
Absorption, CD and Fluorescence
Spectroscopy
Absorption and CD spectra were recorded using an Evolution 500
dual-beam spectrophotometer (Thermo Scientific, United States)
and a J-815 (Jasco, Japan) spectropolarimeter in the visible range,
at room temperature, with spectral bandwidth of 1.5 nm and
3 nm, respectively. The absorbance of the samples was 0.4 at
the red maximum in a 1-cm pathlength cuvette. Synchrotron-
radiation UV CD spectra were recorded at the B23 CD beamline
of the Diamond synchrotron (United Kingdom). Fluorescence
emission spectra in the visible range were measured from the
same samples, at room temperature, on a FP-8500 (Jasco, Japan)
spectrofluorometer.
Time-Resolved Fluorescence
Spectroscopy
Room temperature fluorescence decays were recorded by
TCSPC using a FluoTime 200/PicoHarp 300 spectrometer
(PicoQuant, Germany) as described elsewhere (Akhtar et al.,
2016). A WhiteLase Micro supercontinuum fiber laser (NKT
Photonics, United Kingdom) at 20 MHz repetition rate was
used to generate excitation pulses. Excitation wavelength of
633 nm was selected by a monochromator, and the pulse energy
was attenuated to approximately 0.1 pJ with neutral density
filters. Fluorescence photons were detected by a microchannel-
plate detector (R3809, Hamamatsu, Japan) and timed with
4-ps resolution. The fluorescence decays were recorded from
untreated LHCII samples and after 30 min of light treatment.
The samples were placed in a 1.5 mm pathlength quartz cell
without further dilution. The total instrument response (IRF)
width was ∼50 ps (FWHM), measured using 1% Ludox as
scattering solution. The fluorescence lifetimes were determined
by multiexponential fitting of the fluorescence decay kinetics
combined with iterative reconvolution with the IRF. The average
fluorescence lifetime was calculated as τav =P
i
aiτi/P
i
ai.
Electron Paramagnetic Resonance
Spectroscopy
The principle of the experiments was similar to the one described
by Rinalducci et al. (2004). Samples for EPR measurements were
prepared under dim light and contained detergent-solubilized
LHCII trimers or reconstituted LHCII membranes diluted to
0.1 mg Chl/mL in case of hydrophilic spin label and spin trap,
and ca. 0.3 mg Chl/mL in case of lipophilic spin label. 5 µL
sample aliquots were added to glass capillaries (with ca. 1 mm
internal diameter), which were irradiated for 30 min in the EPR
resonator (after tuning the instrument) with the same lamp as
above, with PFD of 4800 µmol photons m−2s−1PAR incident
on the illumination grid (front window of the resonator), directly
during measurements (assuming 50% cut off by the grid and
efficient reflection in the resonator (Rinalducci et al., 2004),
relative PFD hitting the sample was approximately same as in the
optical spectroscopy experiments). Individual scans were started
at different time points of irradiation.
Singlet oxygen production upon irradiation was followed in
samples with 100 mM TEMPD ×H2O (Fischer et al., 2007).
TEMPD ×H2O traps 1O2resulting in the 4-oxo-TEMPO, which
is paramagnetic and hence detectable by EPR. Spectra of dark
and light-treated blank sample (only spin trap, no LHCII) were
also measured to exclude contributions from or effects by other
possible sensitizers from the buffer or impurities of the spin trap.
Indirect measurement of the production of singlet oxygen
and other light-induced radicals was performed by following
the consumption of spin labels in irradiated samples containing
0.5 mM TEMPO – giving signal only from the aqueous phase –
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Lingvay et al. Photobleaching in Light-Harvesting Complex II
or 50 µM 5-SASL (spin label:lipid molar ratio = ∼2:100) –
giving signal primarily originating from the hydrophobic region
of the vesicles/micelles (Kóta et al., 2002). For reference EPR
spectra, the stable nitroxide radicals (TEMPO and 5-SASL) were
measured in buffer solution at same concentrations as in samples
in dark and after 30 min light treatment.
All EPR spectra of the above nitroxide radicals (spin trap
adducts and spin labels) were recorded with a Brucker ELEXSYS-
II E580 X-band spectrometer at room temperature, with the
following instrument settings: microwave frequency of 9.38 GHz;
microwave power attenuation of 10 dB (12 dB in case of TEMPO);
field modulation of 1 G (3 G in case of 5-SASL); scan range
of 100 G, and conversion time of 40.96 s. To obtain the best
possible signal-to-noise ratio, spectra in the dark were measured
before and after illumination (after 10 min dark incubation),
whereby the final spectra were averages of 20, 10, and 4 scans,
for 4-oxo-TEMPO, 5-SASL and TEMPO, respectively.
In order to determine the concentration of the nitroxide
radicals (the spin labels TEMPO and 5-SASL and the trapping
adduct 4-oxo-TEMPO), reference spectra were recorded from
samples lacking LHCII using the same instrument settings as
for LHCII-containing samples but with known concentrations
of spin labels (5-SASL or TEMPO). (It should be noted that the
spectrum of TEMPO and 4-oxo-TEMPO are indistinguishable
as concerns intensity calibration.) A linear fit to the plot
of the integrated EPR absorptions (second integrals of the
spectra) versus the known spin label concentrations served as
a calibration to calculate nitroxide radical concentrations from
the EPR spectra.
Data Analysis
All data processing, statistical analyses and theoretical
computations were done in MATLAB using the Spectr-O-
Matic toolbox (available at the MATLAB File Exchange) and
homebuilt routines.
RESULTS
Photobleaching Kinetics
Photobleaching of Chls in LHCII in different molecular
environments was observed by monitoring the changes in
absorption in the course of irradiation with intense white light.
Absorption spectra of LHCII solubilized with β-DDM and
reconstituted lipid membranes before and after 30 min light
exposure are shown in Figure 1. Upon light illumination a
marked decrease in the absorption of Chl band Chl awas
observed at 652 and 675 nm, respectively, accompanied by
similar changes in the Soret region. Across the visible wavelength
region, the degree of PB was significantly higher in reconstituted
membranes than in detergent solution (β-DDM). Qualitatively
the changes are similar in all sample types (for LHCII aggregates,
see Supplementary Figure 1a). As seen in the difference spectra,
the Chl abands at 675, 436 nm undergo the most bleaching, Chl
bbands 652, 485 nm are less bleached and no appreciable PB of
Cars is observed (450, 500 nm).
Figure 2 shows the degree of PB of Chls in LHCII in different
molecular environments – in detergent (β-DDM), aggregates
and reconstituted membranes during 30 min of irradiation.
The bleaching is quantified as the relative irradiation-induced
absorption difference 1A/A. The time courses reinforce the
finding that reconstituted membranes are significantly more
susceptible to PB than either detergent-solubilized or aggregated
LHCII. The transients at 675 and 652 nm, mainly associated
with the Qytransitions of Chl aand b, respectively, fit
well to monoexponential kinetics, especially for the Chl a
band (R2>0.99). This indicates that PB is a (pseudo) first-
order process: 1A/A=1−e−kpbt, parametrized by the PB rate
constant kpb (Croce et al., 1999).
First-order PB rate constants and the respective quantum
yields of PB, ϕpb, for LHCII in different environments are shown
in Table 1. The quantum yield was calculated as the ratio
ϕpb =kpb/kabs with kabs being the absorbed photon flux per
Chl (at the beginning of irradiation). The latter was estimated
by integrating over the entire wavelength region, taking into
account the product of the wavelength-dependent light intensity
and absorption cross-section (assuming it does not vary among
sample types). The quantum yield ϕpb in detergent-solubilized
trimers is more than an order of magnitude lower than that
of free Chls (Aronoff and Mackinney, 1943) but comparable
to the PB of various porphyrins (Spikes, 1992;Bonnett et al.,
1999). In LHCII aggregates, prepared by removing the detergent
from the medium, the degree of PB was about 20% higher.
Even more notably, we found that the PB yield was two- to
three-fold higher in reconstituted membranes than in detergent
micelles. To test whether PB in membranes is oxygen-dependent,
we performed experiments in anoxic environment (continuously
bubbling the reaction mixture with N2gas) and in the presence
of sodium ascorbate as an antioxidant. In both cases the PB was
reduced to values comparable with those of detergent-solubilized
LHCII (Table 1).
A separate set of experiments was conducted on all LHCII
sample types listed above with a different light treatment
regime – using a tungsten halogen light source through a blue
colored-glass filter and an incident PFD of 500 µmol photons
m−2s−1(Supplementary Figure 2). Under these conditions,
PB was substantially slower but qualitatively the results were
similar; more importantly, ϕpb was comparable as with high-
intensity LED irradiation (Supplementary Table 2). Further,
we performed treatment with red and blue actinic light with
intensities adjusted to achieve identical excitation flux. The
fluorescence intensity was measured from the sample excited by
either red or blue light to confirm the equal absorbed photon
flux. The PB rate was identical in both cases (Supplementary
Figure 3), therefore ϕpb is wavelength-independent.
CD Spectral Changes
We employed CD spectroscopy to monitor the
structural/conformational changes in LHCII induced by
intense irradiation. The CD spectra of complexes in detergent
and reconstituted membranes (Figure 3) show significant
changes both in the Soret as well as the Chl Qyregion; the same
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FIGURE 1 | Photobleaching in LHCII. (A) Absorption spectra of LHCII solubilized in detergent (β-DDM) and reconstituted membranes before (solid lines) and after
30 min of irradiation (dotted lines). (B) Absorption difference spectra (dark–minus–irradiated sample).
FIGURE 2 | Time course of LHCII photobleaching in detergent (β-DDM), reconstituted membranes and aggregates during 30 min of irradiation (2000 µmol photons
m−2s−1). (A) Absorbance changes at 675 nm and (B) at 652 nm. Circles and lines represent experimental data points and monoexponential fits, respectively.
TABLE 1 | Photobleaching rate constants and quantum yields for LHCII in different environments.
LHCII environment PB after 30 min 1A675/A675 (%) PB rate constant kpb (s−1) Quantum yield ϕpb
β-DDM 29 ±2* (1.9 ±0.1) ×10−4(1.4 ±0.1) ×10−5
Aggregates 34 ±2 (2.3 ±0.2) ×10−4(1.7 ±0.1) ×10−5
Reconstituted membranes 54 ±2 (4.5 ±0.3) ×10−4(3.4 ±0.3) ×10−5
Anoxic 34 ±5 (2.3 ±0.4) ×10−4(1.7 ±0.3) ×10−5
10 mM Na-ascorbate 32 ±3 (2.1 ±0.2) ×10−4(1.6 ±0.2) ×10−5
20 mM Na-ascorbate 29 ±2 (1.8 ±0.1) ×10−4(1.3 ±0.1) ×10−5
*Values represent standard error (n = 3–9).
applies for LHCII aggregates (Supplementary Figure 1b). The
CD amplitude in the Chl Qyregion decreased proportionally to
the decrease in the absorption (PB) and the shape of the spectra
remained unchanged – indicating that the general structure of
the pigment–protein complex remains intact even though a large
part of the chromophores are lost (Olszówka et al., 2003). In
the Soret region, there were additional changes – especially at
494 nm in β-DDM – which were not only caused by the loss of
absorbance. This is better illustrated in the spectra of the CD/A
ratio (Supplementary Figure 4). Significant loss of CD amplitude
at these wavelengths occurred already after 15 min of irradiation.
The changes could be due to a disruption of excitonic couplings
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FIGURE 3 | CD spectra of LHCII before, and after 15 and 30 min of irradiation. (A) LHCII in detergent (β-DDM) and (B) in reconstituted membranes. The spectra
correspond to absorbance 0.4 at 675 nm.
between (Chl and Car) transitions in the blue wavelength range
or due to changes in the induced CD of Cars. The negative
CD bands at 438 and 460 nm in reconstituted membranes and
aggregates, which are associated with inter-trimer interactions,
rapidly diminished upon irradiation.
Fluorescence Quenching
Room-temperature fluorescence emission spectra of LHCII in
detergent micelles and reconstituted membranes recorded with
436 nm excitation before and after 30 min of irradiation are
shown in Figure 4. The fluorescence emission was strongly
reduced compared to the unexposed samples. Even after
correcting for the loss of absorption at the excitation wavelength,
the fluorescence yield was reduced by a factor of 2.2 in detergent-
solubilized LHCII and 5–7 in reconstituted membranes and
aggregates. In all sample types, the degree of fluorescence
quenching substantially exceeded the PB (loss of absorption),
suggesting that irradiation induced non-radiative dissipation in
the partially photobleached complexes. Some spectral changes
can also be noted. The relative fluorescence intensity in the Chl
bregion (650–660 nm) was enhanced, especially in reconstituted
membranes. The shape and width of the main emission band
at 680 nm remained almost the same, except for a slight
(∼1 nm) red shift of the maximum. The normalized spectra
of irradiated reconstituted membranes (Figure 4B) as well
as aggregates (Supplementary Figure 5a) showed enhanced
fluorescence emission in the far-red region (700–720 nm).
Further we performed picosecond time-resolved fluorescence
measurements of the LHCII samples by TCSPC. The fluorescence
recorded at 680 nm after 30 min of irradiation showed an
initial phase of rapid decay in all the tested environments
(Supplementary Figures 5b,6), confirming the light-induced
quenching observed by steady-state fluorescence. For a
quantitative analysis, the fluorescence decay curves were
subjected to multiexponential fitting. The resultant decay
lifetimes, their relative amplitudes and the average fluorescence
lifetimes at 680 nm are shown in Table 2. The average lifetime
τav =P
i
aiτi/P
i
aidecreased by a factor of 2.3 for detergent-
solubilized LHCII, in good agreement with the steady-state
fluorescence data, and by a factor of 3–4 for reconstituted
membranes and aggregates. The somewhat lower quenching
factors estimated from time-resolved fluorescence suggest the
presence of fast decay components that are below the time
resolution of the measurement.
The fluorescence of LHCII in detergent decayed almost
monoexponentially, as it is well known, with a lifetime of
3.8 ns and a very small (5%) contribution from a shorter, 0.8-
ns component. After 30 min irradiation, at least two additional
shorter decay lifetimes were observed – about 70 and 300 ps –
with a combined amplitude of approximately 50%. Similar decay
lifetimes (80 and 300 ps) appeared after irradiation of LHCII
in reconstituted membranes, in this case having a combined
amplitude of 80%, at the expense of the nanosecond decay
components. In irradiated aggregates, 80% of the excitations
decayed with a lifetime of 60 ps. The absence of blue-shifted
emission components in the decay-associated spectra (data not
shown) and long lifetimes shows that no free/uncoupled Chls
were present in the irradiated samples.
Electron Paramagnetic Resonance
To identify and quantify the ROS formed during irradiation of
LHCII, EPR measurements were performed at different intervals
after light exposure of samples containing either the spin trap
TEMPD or the spin labels TEMPO and 5-SASL. The EPR-silent
spin trap 4-oxo-TEMP (TEMPD) converts to the paramagnetic
nitroxide radical 4-oxo-TEMPO upon reaction with 1O2(Lion
et al., 1976), yielding a specific EPR spectrum almost identical
to that of the TEMPO spin label (Marshall et al., 2011). A dose-
dependent EPR signal, typical for 4-oxo-TEMPO, was detected
after irradiation of LHCII-containing samples (Figure 5A).
No signal was observed in samples kept in the dark or
after illumination of the spin-label-containing buffer/liposomes
without LHCII (data not shown).
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FIGURE 4 | Fluorescence emission spectra of LHCII before and after 30 min irradiation recorded with 436 nm excitation light. (A) LHCII in detergent (β-DDM) and (B)
in reconstituted membranes. Note the separate intensity axes (right side) for irradiated samples.
TABLE 2 | Fluorescence lifetime analysis of LHCII in different environments.
Sample Irradiation τ1(ns) a1(%) τ2(ns) a2(%) τ3(ns) a3(%) τ4(ns) a4(%) τav (ns)
β-DDM – 0.8 5 3.8 95 3.6
30 min 0.07 27 0.27 21 1.3 16 3.6 35 1.6
Membranes – 0.30 16 1.1 60 2.9 24 1.4
30 min 0.08 44 0.28 35 0.9 17 2.5 4 0.4
Aggregates – 0.11 61 0.33 33 1.0 6 3.1 1 0.26
30 min 0.06 81 0.19 18 0.6 2 3.0 0.1 0.10
FIGURE 5 | EPR spectra of 4-oxo-TEMPO, generated during illumination of LHCII membranes in the presence of 100 mM 4-oxo-TEMP (TEMPD ×H2O).
(A) Spectra recorded before and after 7 and 30 min of irradiation of reconstituted membranes. (B) Time course of singlet oxygen trapping by 4-oxo-TEMP
(TEMPD ×H2O) during 30 min irradiation. Circles and lines represent experimental data points and monoexponential fits, respectively.
The dependence of the 4-oxo-TEMPO concentration on
illumination time, estimated from the intensity of the central
EPR band, is plotted in Figure 5B, along with exponential fits
for LHCII in reconstituted membranes and β-DDM. The 4-
oxo-TEMPO signal nearly saturated after 30 min irradiation
and the total detected concentration was about 4-fold higher
(6 µM) in detergent-solubilized LHCII than in reconstituted
membranes (1.5 µM). On the other hand, for the initial
exponential phase of the curves, the fitted time constant of
radical formation was shorter for membranes than detergent (5
vs. 10 min) – thus the initial rate of 1O2generation was higher in
reconstituted membranes.
Figure 6 shows the EPR spectra and illumination time
course of samples containing 5-SASL. Stearic acid spin labels,
such as 5-SASL, partition between the membrane and the
aqueous buffer with very high preference toward membranes.
The spectra of thylakoid lipid vesicles, LHCII proteoliposomes
as well as of detergent-solubilized LHCII all showed features
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typical for 5-SASL in membrane phase (see, e.g., Kóta et al.,
2002;Páli and Kóta, 2019), whereas the aqueous-phase EPR
signature was less than 1% and could be neglected. 5-SASL
can react with various radicals thereby losing its EPR signal,
via either one electron oxidation or reduction. As Figure 6B
shows, after the onset of illumination, the 5-SASL concentration
decayed approximately exponentially in both LHCII-containing
samples but the rate of quenching was approximately double
in reconstituted membranes than in detergent solution. The
same result was obtained when using TEMPO instead of 5-SASL
(Supplementary Figure 7).
DISCUSSION
The Degree of PB Does Not Correlate
With the Excited-State Lifetime
The investigation of Chl PB in LHCII presented here confirms
earlier observations that the light-harvesting antenna complexes
are sensitive to photodamage when they are not coupled to
active RCs (Siefermann-Harms, 1990;Zolla and Rinalducci, 2002;
Rinalducci et al., 2004;Zhang et al., 2008). The quantum yield
of Chl PB, ϕpb, ranges between 1 ×10−5and 4 ×10−5.
A key experimental result is the non-trivial dependence of
ϕpb on the molecular environment of LHCII. Firstly, higher-
order aggregation of LHCII trimers leads to effective quenching
of singlet excited states, diminishing the singlet lifetime by a
factor of 20 in accord with numerous studies (Horton et al.,
1991;Mullineaux et al., 1993;Miloslavina et al., 2008). One
would expect a proportional reduction in ϕpb, assuming that
the photosensitizer agent is Chl∗. The results, however, show
that ϕpb is slightly increased instead. In this sense LHCII
aggregation, which is considered as a model of NPQ, does
not seem to have a photoprotective effect on LHCII itself,
although evidently any quenching mechanism will relieve the
excitation pressure on PSII and have a photoprotective role
in vivo. Lack of expected correlation between the excited-
state lifetime and photoinactivation has also been noticed in
thylakoid membranes. The loss of PSII activity or D1 degradation
showed no or only mild correlation with the excitation
quenching induced by spillover or the addition of exogeneous
quenchers (Tyystjärvi et al., 1999;Santabarbara et al., 2001a,
2002a). Exogeneous quenchers exerted a modest protection
from PB of Chls in light-exposed thylakoids (Santabarbara,
2006). The dose response of both photoinhibition and PB,
the lack of a linear relationship, and the blue-shifted action
spectrum of photoinhibition led to the suggestion that a
small population of antenna complexes in which Chl–Car
coupling is impaired, are mainly responsible for ROS generation
and photodamage in the thylakoid membranes (Santabarbara
et al., 2001b;Santabarbara, 2006). In the following sections
we focus on the formation of 3Chl and their quenching
by Cars in LHCII.
Perhaps the most striking result of the current investigation
is that LHCII in reconstituted membranes is significantly
(nearly three-fold) more sensitive to photodamage
than either LHCII aggregates or detergent-solubilized
trimers. To understand these results, it is demanding
to comprehend the specific photochemical mechanisms
of photodamage.
Fluorescence Quenchers Are Generated
in the Course of PB
Both steady-state and time-resolved fluorescence measurements
revealed that the Chl fluorescence yield ϕFand lifetime τF
are significantly reduced upon irradiation in all types of
samples, indicating that PB is associated with the generation
of quenchers. Light-induced fluorescence quenching has been
known to occur in isolated LHCII and especially in lamellar
LHCII aggregates (Jennings et al., 1991;Barzda et al., 1996).
Quenching in irradiated LHCII liposomes has been reported by
Zubik et al. (2011), who ascribed it to Car photoisomerization
and formation of long-lived quencher states, particularly Chl–
Car charge-transfer states, owing to the increased absorption
and Stark effect around 900 nm. These results are consistent
with a more general interpretation that the photoproducts,
be it long-lived Chl radicals or other derivates, possibly
bilinone analogs (Jose et al., 1990), may act as fluorescence
quenchers in the photodamaged complexes. Upon prolonged
irradiation this fluorescence quenching might have a self-
protecting role; however, further quantitative analysis would be
necessary to test this.
Photobleaching Is Caused by Singlet
Oxygen Produced by Chl Triplets
Chlorophyll PB in LHCII in reconstituted membranes was
effectively suppressed in anaerobic environment (Table 1) or by
adding ascorbate in aerobic conditions, as has been shown for
isolated LHCII (Siefermann-Harms, 1990;Croce et al., 1999),
confirming that it is, for the main part, oxidative. The quantum
yield ϕpb was independent of the intensity and wavelength of the
actinic light (Supplementary Figures 2,3and Supplementary
Table 2), indicating that the reaction is one-photon and initiated
by the lowest-lying singlet-excited state of Chl. Moreover, very
little PB of Chl bwas observed, which is consistent with results
on solubilized LHCII (Croce et al., 1999;Olszówka et al., 2003;
Zhang et al., 2008) and the fact that Chl btransfers energy
to Chl aon a much shorter timescale than the formation of
triplets (Connelly et al., 1997). All these data corroborate that
the PB occurs via a type II reaction photosensitized by Chl
triplet states (3Chl), which has been thoroughly demonstrated
for Chls (Krasnovskii Jr., 1994). Moreover, the sensitizer is
Chl aas Chl btriplets have not been detected in LHCII
(Peterman et al., 1997). Presumably, the triplet Chl areacts with
molecular oxygen producing singlet oxygen (1O2) which then
attacks the Chl directly or is transformed to another ROS, e.g.,
a hydroxyl radical.
The formation of 1O2in reconstituted LHCII membranes
was confirmed directly and indirectly by EPR in agreement with
the experiments of Zolla and Rinalducci (2002) and Rinalducci
et al. (2004). The spin trap 4-oxo-TEMP, directly sensing 1O2,
produced 4-oxo-TEMPO radicals only in irradiated samples
containing Chl. In principle, the EPR analysis is quantitative,
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FIGURE 6 | (A) EPR spectra of 5-SASL in detergent micelles and lipid membranes. (B) Time course of the 5-SASL concentration estimated from the EPR signal
intensity during 30 min of irradiation of the reaction mixture containing LHCII with 50 µM 5-SASL. Circles and lines represent experimental data points and
monoexponential fits, respectively.
meaning that we should be able to estimate the 1O2yield from
the time-dependent concentration of the spin labels. However,
the hydrate form of 4-oxo-TEMP (TEMPD ×H2O) is water-
soluble and partitioned entirely in the aqueous phase whereas
1O2is produced in the hydrophobic lipid/protein phase. The 4-
oxo-TEMPO concentration then depends not only on the rate of
1O2formation but also on its solubility, diffusion and lifetime in
the different phases. For this reason, quantifying the 1O2yield
in different environments is not straightforward. The reaction
mixture of LHCII–lipid membranes contains substantial amount
of lipids (0.9 mM). The lipids are at the same time solvent for the
oxygen and substrate for lipid peroxidation, which may explain
why 4-oxo-TEMP reported less overall amount of 1O2in the
reconstituted membranes than in detergent LHCII. On the other
hand, the initial rate of 4-oxo-TEMPO formation was higher in
reconstituted membranes but the signal saturated at a lower level.
This is probably because of heterogeneity of the sample with only
a fraction of the LHCII complexes exposed to the spin trap, e.g.,
those on the outer sheet of multilamellar vesicles.
In contrast to 4-oxo-TEMP, the EPR spectrum of the spin label
5-SASL evidenced its incorporation into the lipid phase (Kóta
et al., 2002;Páli and Kóta, 2019) which is the site where ROS
are formed (we observe negligible aqueous 5-SASL signal, with
sharp lines). In principle, the loss of 5-SASL EPR intensity over
time should reflect the ROS produced during irradiation of the
samples – 0.06 and 0.11 mol/mol Chl for LHCII in detergent and
membranes, respectively, after 30 min. With a large excess of free
spin label, we can approximate the kinetics to be first-order with
rate constants of 4.3 ×10−4s−1and 6.3 ×10−4s−1, respectively.
These values correspond to quantum yields of radical formation
of 2 ×10−5(β-DDM) and 4 ×10−5(lipid membranes),
comparing well with ϕpb. However, these values must also be
taken with caution because in detergent micelles, a large fraction
of 5-SASL must be incorporated in micelles that do not contain
any LHCII, whereas the majority of lipid vesicles contain more
than one LHCII trimer (Tutkus et al., 2018). For a more accurate
modeling of the ROS dynamics in such a heterogeneous system,
the partitioning and mobility of both the ROS and the spin probe
in all phases must be accounted for.
A Kinetic Model of Singlet Oxygen
Formation
The rate of the photosensitization reaction is proportional to
the concentration of 3Chl states, which in turn depends on the
ability of Cars in LHCII to quench 3Chls. Several studies have
shown that the triplet–triplet (T–T) energy transfer from Chls
to Cars in LHCII occurs with near 100% efficiency (Siefermann-
Harms and Ninnemann, 1982;Peterman et al., 1995) – which is
the very reason why antenna PB should be negligible in the first
place. Even if that is the case, low transient concentration of 3Chl
may still generate 1O2. To address this question quantitatively,
having in mind the considerations above, we can construct a
simplified kinetic model of the PB reaction (Scheme 1). The
relevant kinetic parameters are the rate constants of Chl singlet
and triplet decay, kDand kT, intersystem crossing, kISC, T–T
transfer to Cars, kT−T, the sensitization rate constant kox and
the local oxygen concentration [O2]. The 3Chl yield ϕTcan be
calculated as
ϕT=kISC
kD+kISC
.
The rate constant kISC for Chl ais 0.1 ns−1(Bowers
and Porter, 1967) and the denominator is equal to the
inverse fluorescence lifetime – (3.8 ns)−1for detergent-
solubilized LHCII (Table 2). The triplet yield is then
ϕT= 0.38. The 1O2yield is in turn given by the expression
ϕox =ϕT
kox[O2]
kox [O2]+kT+kT−T
.
In anaerobic environment, kT, which is equal to the inverse
triplet lifetime, kT=1/τT, is vanishingly small, in the
range of 1–2.5 ms−1(Peterman et al., 1997;Niedzwiedzki
and Blankenship, 2010). The Car quenching rate constant
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SCHEME 1 | Kinetic scheme of the photosensitization of singlet oxygen in
LHCII.
kT−Thas been estimated in the range of 2–10 ns−1
(Schödel et al., 1998). Finally, for estimating kox we refer to
measurements on free Chl, where we can make use of the
relation
τT=1
kT+kox[O2].
In aerated organic solvents τTis about 0.3 µs (Drzewiecka-
Matuszek et al., 2005;Niedzwiedzki et al., 2014). Consequently,
kox ≈2×109M−1s−1(calculating the oxygen concentration in
water at 20◦C to be 1.4 mM), which is consistent with the values
reported for Chl aand its derivatives (Mathis and Kleo, 1973;
Fiedor et al., 1993).
Using these rate parameters and the equations above, we
calculate values of ϕox in the range of 1 ×10−4to 5 ×10−4. The
time dependence of 3Chl and 1O2upon Chl excitation, obtained
by solving the kinetic model, is given in Supplementary Figure 8.
The theoretically estimated quantum yield is comparable to the
yield of ROS formation reported by EPR (see above). Therefore,
the calculations demonstrate that the transient concentration
of 3Chl, which decays with the singlet excitation lifetime, is in
principle sufficient to generate ample amounts of 1O2to account
for the observed PB (ϕpb ≈10−5). The calculations also show
unequivocally that ϕox, and therefore PB, should be linearly
proportional to the fluorescence lifetime. Thus, the rate of PB in
reconstituted LHCII membranes and in aggregates should be 3-
fold and 20-fold lower, respectively, than in detergent micelles (all
other parameters being constant).
Why Is LHCII More Susceptible to PB in
Lipid Membranes?
The observed variation of ϕpb with the molecular environment
can be explained with variations in either the rates kISC,kT−T,
or kox[O2] in the kinetic model discussed above. In principle,
all of these are possible. Both ϕTand τTare shown to depend
on the solvent environment (Hurley et al., 1980). The quenching
of 3Chl by Cars strongly depends on the arrangement of the
pigments in the complex, with an exponential dependence on
the distance between them, stemming from the Dexter exchange
transfer mechanism (Siefermann-Harms, 1987). Comparatively
small structural alterations, for example induced by increasing
the detergent concentration, can affect the energetic coupling
between pigments, reducing kT−T, and in turn raising the 3Chl
yield (Naqvi et al., 1999). The CD spectra of LHCII, especially in
the Cars region, indicate that such conformational changes occur
upon aggregation and in the lipid environment (Akhtar et al.,
2015). From the kinetic model it follows that a two-fold reduction
in kT−Twill result in a corresponding two-fold increase in ϕox.
It may also be speculated that some Cars are destabilized or
missing in the artificial reconstituted membranes; however, this
seems unlikely because we do not detect a significant change in
the pigment composition (Supplementary Table 1). Moreover,
the primary quenchers of the terminal emitter Chl aare luteins
(Dall’Osto et al., 2006) and their loss would result in protein
unfolding (Formaggio et al., 2001), which has not been detected
in the UV-CD of irradiated samples in our experiments (data
not shown) or in previous studies (Olszówka et al., 2003). While
photodegradation of the LHCII apoprotein does occur, it only
involves the N-terminus (Zolla and Rinalducci, 2002).
Another potential factor affecting ϕox, and hence ϕpb is
the local O2concentration or the O2accessibility to the site
of 3Chl formation. This may well be a leading cause for the
enhanced photosensitivity of LHCII in lipid membranes, as
it has been shown that the lipid/water partitioning factor of
O2in phospholipid liposomes and lipoproteins is up to 4,
particularly in the liquid crystalline phase (Möller et al., 2005,
2016). If the O2concentration is higher in the vicinity of
the LHCII pigments, that will also affect the Chl and Car
triplet lifetimes. Careful comparison of the triplet lifetimes
in LHCII in different environments might be useful to test
this hypothesis.
Finally, we must consider that PB may also be indirectly
caused by ROS generated in a radical chain reaction, for
example by alkoxyl radicals. In that case, lipids and lipid
peroxidation products may act as secondary sensitizers for the
PB. In support of this, we have been able to detect, although
semi-quantitatively, lipid peroxidation products in light-exposed
LHCII liposomes via a malondialdehyde–thiobarbituric acid
reactivity assay (data not shown). However, we did not observe
significant photoprotective effect of adding α-tocopherol to the
reaction mixture, in agreement with results on solubilized LHCII
(Siefermann-Harms, 1990), suggesting that alcoxyl radicals are
not a dominant trigger of Chl PB in the liposomes. Whether
lipid peroxidation is actually involved in the photodegradation
of Chls is purely a speculation but in either case our results
point to an intrinsic volatility of the lipid environment
with respect to photodamage that must not be overlooked.
Neither the local O2concentration, nor lipid peroxidation
readily explain the differences, or lack thereof, between
the Chl PB in detergent-solubilized and aggregated LHCII.
Therefore, we tend to assume that the PB dependency on
the environment is due to a combination of several factors
discussed above.
Is Chl PB in LHCII Relevant to
Photoinhibition in vivo?
So far it remains unclear whether direct photodegradation
of the antenna has a significant role in photoinhibition
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in vivo. In comparison to the values of ϕpb obtained
here, photoinhibition of PSII occurs with a significantly
lower quantum yield, in the order of 10−7(Campbell and
Tyystjärvi, 2012). In active PSII, LHCII excitations are rapidly
transferred to the RC and quenched by photochemistry, so
the PB in the functionally connected antenna will be far
less than in the isolated complexes. However, if the RCs
are closed, which can be the case under prolonged excess
light conditions, this photoprotective route is unavailable
(Lambrev et al., 2012;Ruban, 2016). Then, considering that
∼102antenna Chls are connected to one PSII RC, we can
expect that the rates of direct PB of antenna Chls and
PSII photoinhibition will have the same order of magnitude.
In native thylakoids subjected to photoinhibitory treatment,
the rate of 1O2production declined by about half after
the complete loss of oxygen evolution (Hideg et al., 1994b).
Taken together with a report of no appreciable formation of
1O2by Photosystem I (Hideg and Vass, 1995), this invites
the hypothesis that the excess 1O2is produced by the PSII
antenna. However, the same authors found no 1O2formation
in thylakoids upon donor-side inactivation of PSII electron
transport (Hideg et al., 1994a), which either invalidates the
antenna hypothesis, or it must be assumed that the oxidized RC
radical, P680+, prevents the accumulation of 1O2(for example via
efficient quenching).
Pigment PB associated with photoinhibition in thylakoid
membranes and PSII-enriched membranes has been
experimentally shown in several studies (Yamashita and
Butler, 1969;Yamashita et al., 1969;Carpentier et al., 1986;
Zucchelli et al., 1988;Miller and Carpentier, 1991). The PB
kinetics and the sensitivity of the different pigment pools,
however, appear to be markedly different in these systems
compared to isolated antenna complexes. In thylakoid
membranes, Cars were found to be the primary target of
the photooxidation reactions (Yamashita and Butler, 1969;
Yamashita et al., 1969). Cars were also the main photobleached
pigments in PSII-enriched membranes lacking manganese
(Klimov et al., 1990) and in the D1–D2–Cyt b559 complex
(Telfer et al., 1991). In a more recent study, Santabarbara
(2006) found that PB in thylakoid membranes occurred
in two distinct phases – a slow initial phase, during which
Cars were bleached at a rate three times higher than Chl
a, followed by a second phase marked by rapid PB of
Chls – evidently because the protective role of Cars was
eliminated. These results indicate that Chl PB is a late event
in photoinhibition in native thylakoid membranes and a
consequence of the disruption of T–T transfer from Chl to
Cars. As we do not observe substantial bleaching of Cars
in isolated LHCII, similar to other results (Siefermann-
Harms, 1990), it could then be postulated that T–T transfer
is disrupted in the isolated antenna complexes, making
the Chls more susceptible to PB (Naqvi et al., 1999).
According to the kinetic model, isolated LHCII should be
capable of producing enough 1O2to explain the observed
Chl PB. Obviously, there is no guarantee that the model
holds in vivo, which again exemplifies the caveats of
in vitro experiments with a flexible protein complex such
as LHCII, which is sensitive to its molecular environment
(Akhtar et al., 2015).
CONCLUSION
In this work, we have shown that light exposure of isolated
LHCII causes oxidative PB of Chl awith a quantum yield
of 1 ×10−5to 4 ×10−5, which indicates that in excess
light conditions, when the PSII RCs are predominantly closed,
direct photodamage of the antenna could occur with rates
comparable to the PSII RC photoinactivation. The sensitivity
to photodamage depends on the molecular environment of
the complex, such that PB is significantly exacerbated in
reconstituted lipid membranes. Quantitative EPR spectroscopy
analysis using spin labels confirms the increased light-induced
generation of 1O2in the membranes. This is probably a
combined effect of the solubility and diffusion of oxygen and
other factors modifying the ultimate fate of the excitation
energy. Regardless of what the exact underlying cause is,
the increased PB susceptibility of LHCII in lipid membranes
is potentially of great significance considering that this is
the native environment for the majority of photosynthetic
pigment–protein complexes. As direct photosensitization of
ROS by the light-harvesting complexes is not negligible,
ROS must be effectively scavenged in the membrane to
avoid photodamage.
DATA AVAILABILITY STATEMENT
The generated datasets for this study (absorption, CD,
fluorescence and EPR spectra) can be found online in the
Mendeley Data repository (Lingvay et al., 2020).
AUTHOR CONTRIBUTIONS
PL designed the experiments. ML and PA isolated LHCII,
prepared reconstituted membranes, and performed
optical spectroscopy measurements. ML, KS-N, and TP
performed EPR measurements. ML performed data analysis.
PA and PL did theoretical modeling. The manuscript
was written through contributions of all authors. All
authors have given approval to the final version of
the manuscript.
FUNDING
The work was supported by grants from the Hungarian Ministry
for National Economy (GINOP-2.3.2-15-2016-00001) and the
National Research, Development and Innovation Fund (NKFI
NN-124904, 2018-1.2.1-NKP-2018-00009 to PL and K-112716 to
TP). CD measurements at the B23 beamline of the Diamond
Light Source Ltd. (session 17698) were supported by the
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Lingvay et al. Photobleaching in Light-Harvesting Complex II
project CALIPSOplus under Grant Agreement 730872 from
the EU Framework Programme for Research and Innovation
HORIZON 2020. ML was supported by the ÚNKP-17-3 New
National Excellence Program of the Hungarian Ministry of
Human Capacities.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00849/
full#supplementary-material
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