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\begin{document}

\title{Exclusive photosystem II photoinhibition derived from photoinactivation
of oxygen-evolving complex in the marine angiosperm \emph{Zostera
marina}}



\author[1]{Wei Zhao}%
\author[2]{Xiaoqi Yang}%
\author[2]{Ying Tan}%
\author[2]{Zhe Liu}%
\author[2]{Ming Yu Ma}%
\author[2]{Meng Xin Wang}%
\author[2]{Zimin Hu}%
\author[2]{Quan sheng Zhang}%
\affil[1]{Affiliation not available}%
\affil[2]{Yantai University}%


\vspace{-1em}



  
  \date{May 18, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Photoinhibition is the popular topic in plant photosynthesis. However,
restricted to experimental systems of \emph{in vitro} membranes,
knowledge of photosystem II (PSII) donor-side photoinhibition remains
limited. Here, we report the first \emph{in vivo} study of the mechanism
in the marine higher plant \emph{Zostera marina}. Preferential
oxygen-evolving complex photoinactivation decreased the light-harvesting
capacity and enhanced photosystem I cyclic electron flow (CEF).
Non-photochemical quenching was inefficient and alternative electron
flows, e.g. chlororespiration, Mehler reaction, malic acid synthesis,
and photorespiration, remained unactivated, thereby reducing the
unnecessary consumption of limited electron resources and maintaining a
well carbon assimilation level. At variance with the PSII acceptor-side
photoinhibition, the PSII photodamage of Z. marina was not attributed to
\textsuperscript{1}O\textsubscript{2} but was associated with the
long-lived P680\textsuperscript{+} resulted from the photoinactivated
OEC. Furthermore, we provided the novel insights into the PSII
donor-side photoinhibition that rare PSII-CEF and ascorbate assumed
photoprotective roles in \emph{Z. marina}, which could donate electrons
to the PSII reaction center to prevent the oxidative damage by
P680\textsuperscript{+}. This study addressed an important knowledge gap
in PSII donor-side photoinhibition, providing a novel understanding of
photosynthetic regulation mechanism responding to light stress.%
\end{abstract}%



\sloppy


\textbf{Abstract}

Photoinhibition is the popular topic in plant photosynthesis. However,
restricted to experimental systems of\emph{in vitro} membranes,
knowledge of photosystem II (PSII) donor-side photoinhibition remains
limited. Here, we report the first \emph{in vivo} study of the mechanism
in the marine higher plant\emph{Zostera marina} . Preferential
oxygen-evolving complex photoinactivation decreased the light-harvesting
capacity and enhanced photosystem I cyclic electron flow (CEF).
Non-photochemical quenching was inefficient and alternative electron
flows, e.g. chlororespiration, Mehler reaction, malic acid synthesis,
and photorespiration, remained unactivated, thereby reducing the
unnecessary consumption of limited electron resources and maintaining a
well carbon assimilation level. At variance with the PSII acceptor-side
photoinhibition, the PSII photodamage of \emph{Z. marina} was not
attributed to\textsuperscript{1}O\textsubscript{2}but was associated
with the long-lived P680\textsuperscript{+} resulted from the
photoinactivated OEC. Furthermore, we provided the novel insights into
the PSII donor-side photoinhibition that rare PSII-CEF and ascorbate
assumed photoprotective roles in \emph{Z. marina} , which could donate
electrons to the PSII reaction center to prevent the oxidative damage by
P680\textsuperscript{+}. This study addressed an important knowledge gap
in PSII donor-side photoinhibition, providing a novel understanding of
photosynthetic regulation mechanism responding to light stress.

\textbf{Keywords:} alternative electron donor; oxygen-evolving complex;
P680\textsuperscript{+}; PSII donor-side photoinhibition; \emph{Zostera
marina}

\textbf{Introduction}

Photosystem II (PSII) photoinhibition is commonly expected to occur when
photosystems cannot sufficiently utilize the energy absorbed by their
antenna system, its occurrence depends on the redox state of PSII
acceptor components (Gururani et al., 2015). Strong illumination creates
excessive excitation, which in turns leads to over-reduction of
plastoquinone (PQ) acceptors, thus rendering the PSII reaction center
(RC) inactive (Barber and Andersson, 1992; Melis, 1999). In contrast to
acceptor-side photoinhibition, PSII donor-side photoinhibition is caused
by water-splitting dysfunction, e.g., chemical damage like the removal
of the Mn cluster by NH\textsubscript{2}OH, Tris or high-salt washing
(Callahan and Cheniae, 1985; Yadav and Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il, 2012), and light damage
like the photoinactivated oxygen-evolving complex (OEC) caused by direct
UV illumination absorption (Yadav and Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il, 2012; Havurinne and
Tyystj\selectlanguage{ngerman}ärvi, 2017). These can be observed early in the photosynthetic
membrane system\emph{in vitro} . Recently, vulnerable OEC under visible
wavelengths have also been reported \emph{in vivo}in specific unicelluar
algae including the diatom \emph{Phaeodactylum tricornutum} and the
cyanobacteria\emph{Synechocystis} , which are likely the result of a
lack of photoprotective sunscreen compounds such as non-photosynthetic
pigments or divinyl chlorophyll (Havurinne and Tyystjärvi, 2017; Soitamo
et al., 2017). So far,\emph{in vivo} PSII photoinhibition, derived from
photoinactivated OEC, has not been demonstrated with direct experimental
evidence in higher plants under visible light.

Several explicit characteristics of the mechanism can be summarized as
follows: (1) the mechanism even occurs under low light conditions (Keren
and Krieger-Liszkay, 2011); (2) non-photochemical quenching (NPQ) has
low protective efficiency (Tyystjärvi, 2013); (3) the photoinhibition
rate constant (K\textsubscript{PI}) and light intensity are directly
proportional (Tyystjärvi and Aro, 1996); (4) light leads to primary
inactivation of OEC and secondary damage of PSII RC (Hakala et al.,
2005; Tyystjärvi, 2008). The \emph{in vitro} occurrence of donor-side
photoinhibition, although mimicking, might not completely reflect
\emph{in vivo} phenomena (Dall'Osto et al., 2017) because of the
incomplete photosynthetic apparatus. Restricted to imperfect
experimental systems of photosynthetic membranes\emph{in vitro} ,
knowledge of the mechanism remains limited and fragmented (Zavafer et
al., 2015, 2017).

During PSII donor-side photoinhibition, the PSII RC may be susceptible
to damage by both P680\textsuperscript{+} and
\textsuperscript{1}O\textsubscript{2} (Aro et al., 1993; Vass, 2011).
When the electron donation of OEC is inferior to the rate of electron
withdrawal by P680\selectlanguage{english}\textsuperscript{+*} (Vass, 2012), the long-lived
P680\textsuperscript{+}, a strong oxidant damaging component of PSII RC,
is assumed to be a source of damage in this mechanism, which appears to
be uncontroversial. However, whether the highly reactive
\textsuperscript{1}O\textsubscript{2} is generated in the mechanism
remains unclear, since the relevant results are mainly based on
speculation and \emph{in vitro} experiments (Nishiyama et al., 2011;
Yadav and Pospi\selectlanguage{polish}š\selectlanguage{english}il, 2012; Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il, 2016). It has been considered that
peroxidation of lipids by the highly oxidizing P680\textsuperscript{+}
and TyrZ\textsuperscript{+}could cause
\textsuperscript{1}O\textsubscript{2} formation (Yadav and Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il,
2012; Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il, 2016). The impaired OEC might damage specific oxygen
channels that block oxygen molecules to P680 and conduct formative
oxygen molecules outward, thus resulting in an accumulation of
\textsuperscript{1}O\textsubscript{2}(Nishiyama et al., 2011). However,
it has also been proposed
that\textsuperscript{1}O\textsubscript{2}formation only occurs in
preparations that contain functional OEC (Hideg et al., 1994; Johnson et
al., 1995). When OEC is damaged, the redox potential of the
Q\textsubscript{A}/Q\textsubscript{A}\textsuperscript{-} pair shifts to
a higher value, thus decelerating the conversion from
P680\textsuperscript{+}Q\textsubscript{A}\textsuperscript{-}
to\textsuperscript{3}{[}P680\textsuperscript{+}Phe\textsuperscript{-}{]}
(Ivanov et al., 2008). Even though the inactive OEC first
promotes\textsuperscript{1}O\textsubscript{2} generation, conformational
changes induced by the persistent OEC inactivity protects
against\textsuperscript{1}O\textsubscript{2} formation (Tyystj\selectlanguage{ngerman}ärvi,
2008).

Ascorbate (AsA), as an alternative PSII electron donor, exerts a
photoprotective role by continually supporting electron transport
through PSII (Tóth et al., 2011). Although the amount of AsA and its
affinity to PSII varies with species, this alternative electron
transport appears to be ubiquitous in both plants and green alga, and
serves a more vital protection in heat-stressed plants (Tóth et al.,
2009). When the OEC in thylakoids is impaired, either by acidic pH or by
UV-B exposure, AsA is also photooxidized at the donor side of PSII (Mano
et al., 2004). During normal OEC function, no electrons are donated from
AsA to PSII. It has thus been considered that the AsA acts as emergency
electron donor when water oxidation is impaired (Mano et al., 2004). As
another electron donation event, PSII cyclic electron flow (PSII-CEF), a
pathway by which electrons on the Q\textsubscript{B} site of PSII are
returned to P680\textsuperscript{+} via cytb559, has been reported to
exert a species-dependent role in photoprotection (Ananyev et al.,
2017). Typically, strong light would lead to the over-reduction of
Q\textsubscript{A} through linear electron transfer, thereby enhancing
the activity of PSII-CEF to consume the excess energy (Feikema et al.,
2006; Lavaud, 2007). Other factors, such as nitrogen limitation which
caused over-reduction of PQ pool, can stimulate the electron flow, thus
preventing PSII RC photodestruction (Wagner et al., 2016). PSII-CEF also
plays a vital role in drought-tolerant species via proton gradient
formation as a contribution to ATP production and photoprotection
without consuming the limited water supply (Ananyev et al., 2016, 2017).
Electron transport from PSII-CEF to PSII RC occurs when suppressed
transfer of electrons from OEC to P680 extend the life time of
P680\textsuperscript{+} (Thompson and Brudvig, 1988; Miyake, 2002).
Hence, it is reasonable to assume that both AsA and PSII-CEF can donate
electrons to prevent the accumulation of the long-lived
P680\textsuperscript{+}, exerting photoprotective roles during the PSII
donor-side photoinhibition.

\emph{Zostera marina} (Zosteraceae), a widespread seagrass species
throughout the temperate northern hemisphere, playing ecological service
function, evolved from a freshwater ancestor of terrestrial monocots and
successfully adapted to a fully submerged marine environment (Wissler et
al., 2011) where it must deal with shifted spectral composition,
characterized by a high penetration of blue-green light (Olsen et al.,
2016). The lack of cryptochrome blue-light receptors demonstrated by
genome-based research in\emph{Z. marina} (Olsen et al., 2016) would lead
to insufficient anthocyanin levels (Li et al., 2013), thus weakening the
screening of high-energy blue-green wavelengths (Hughes et al., 2010).
Accordingly, \emph{Z. marina} would possess more susceptible OEC with
absorption peaks ranging between UV and blue-green light wavelengths
(Tyystjärvi, 2008), providing the condition for the occurrence of PSII
donor-side photoinhibition. Our recent study (Yang et al., 2017) showed
that: (1) photoinhibition of \emph{Z. marina}also occurred in low light
conditions; (2) photoinhibition is closely relevant to the inactivation
of OEC described by delayed fluorescence (DF); (3) the time-course
changes of\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m}
exhibited the deficiency of the \emph{P} \textsubscript{fast} component
of the Hanelt photoinhibition model, suggesting the slow NPQ
development. These results agreed with the characteristics of PSII
donor-side photoinhibition. Therefore,\emph{Z. marina} , a marine
angiosperm with complete photosynthetic apparatus and functional
differentiation, may be a valid model species to study PSII donor-side
photoinhibition.

In the present study, \emph{in vivo}PSII photoinhibition derived from
photoinactivated OEC under visible light was first determined in
\emph{Z. marina} , by identifying the primary target of the
light-induced impairment. The integrated characteristics of the PSII
photoinhibition associated with light absorption, electron transfer, and
energy conversion were explored. Furthermore, damaging and
photoprotective mechanisms were verified, based on the assumption that
the alternative PSII electron donation pathways were activated to
promote depletion of the long-lived P680\textsuperscript{+}.

\textbf{Materials and methods}

\textbf{Sample preparation}

Healthy\emph{Z. marina} with intact rhizomes-systems was harvested on
the sea floor at around 3 m depth near Rongcheng (37º 16'N, 122º 41'E)
in the Weihai, Shandong province, China. Samples were precultured for 3
days in an aquarium under conditions of 15 °C and a 10: 14 light: dark
cycle with a minimum saturation light intensity of 100 \selectlanguage{greek}µ\selectlanguage{english}mol photons
m\textsuperscript{-2}s\textsuperscript{-1}. Prior to each experiment,
each shoot was ``standardized'' to similar leaf morphology. Leaves for
experimental measurement were collected from 2 cm above the sheath to
keep the same age.

\textbf{Experimental treatment}

Prior to experimental treatment, pre-cultured \emph{Z. marina} plants
were dark-adapted overnight. To obtain the photoinhibition rate
constant, \emph{Z. marina} leaves were pre-incubated with lincomycin for
3 h in the dark and were then exposed to 20, 50, 100, 150, 200, 250,
300, 350, 400, 450, and 500 \selectlanguage{greek}µ\selectlanguage{english}mol photons m\textsuperscript{-2}
s\textsuperscript{-1} (\selectlanguage{greek}µ\selectlanguage{english}E) at 15 \selectlanguage{ngerman}°C (Miyata et al., 2012).\emph{Z.
marina} leaves, which were dark-adapted and exposed to 300 \selectlanguage{greek}µ\selectlanguage{english}E for 3 h at
15 \selectlanguage{ngerman}°C, were used for\textsuperscript{1}O\textsubscript{2}contents, AsA
levels and chlorophyll a fluorescence measurement, and proteome
analysis. The light intensity of 300 \selectlanguage{greek}µ\selectlanguage{english}E was approximated the maximum
midday light intensity at the site of collection. The home-built LED
lamps with color temperature of 6000 K and adjustable illumination
intensity up to 1000 \selectlanguage{greek}µ\selectlanguage{english}mol photons m\textsuperscript{-2}
s\textsuperscript{-1} were used for the sample preculture and light
treatments. The spectrum of the home-built 6000 K LED lamp measured by
HR-450 (HiPoint, China, Taiwan) has the main emission bands at around
450 nm (Fig. S2), similar with the spectrum of the 3 m depth under
seawater where the plants are naturally located (Kirk, 2010; Olsen et
al., 2016).

Due to lack of genetic operating system, the physiological roles of AsA
and PSII-CEF were examined using inhibitors. PSII-CEF was inhibited or
enhanced via incubation of 2,5-dimethyl-p-benzoquinone (DMBQ, TCI,
Tokyo, Japan) at the concentration of 125 or 62 \selectlanguage{greek}µ\selectlanguage{english}M, respectively
(Ananyev et al., 2016, 2017). AsA synthesis were inhibited via 50 \selectlanguage{greek}µ\selectlanguage{english}M
rotenone purchased from Sigma-Aldrich (Millar et al., 2003; Garmier et
al., 2008). Although the chloroplast NADPH dehydrogenase-like complex
was also sensitive to rotenone (Garmier et al., 2008), no significant
side effect of 50 \selectlanguage{greek}µ\selectlanguage{english}M rotenone was observed on PSI-CEF of \emph{Z.
marina} showed by the initial rate of
P700\textsuperscript{+}re-reduction (\emph{V} \textsubscript{re-red},
Fig. S3). To test the roles of AsA and PSII-CEF, four different
combinations of AsA and PSII-CEF inhibitions were conducted: Light,
Light + Rotenone, Light + DMBQ, and Light + Rotenone + DMBQ, forming the
base of a factorial experimental design. Prior to exposure,\emph{Z.
marina} leaves saturated with filtered seawater or with the seawater
solution containing inhibitors were incubated in the dark for 10 min at
15 \selectlanguage{ngerman}°C. Moreover, leaves treated with 125 \selectlanguage{greek}µ\selectlanguage{english}M DMBQ or 50 \selectlanguage{greek}µ\selectlanguage{english}M rotenone under
darkness for 190 min showed no significant changes in\emph{F}
\textsubscript{v}/\emph{F} \textsubscript{m} (Fig. S4). A stock solution
of 10 mM DMBQ and 25 mM rotenone was prepared with dimethyl sulfoxide
whose concentration used in this study had no significant effect on
\emph{Z. marina} as indicated by\emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m} (Fig. S5).

\textbf{Absorption spectrum and oxygen evolution measurements}

To monitor the contents of AsA which could directly protect
photosystems, AsA levels in chloroplasts were monitored by using vitamin
C assay kit (A009, Nanjing Jiancheng Bioengineering Institute, Nanjing,
China).\textsuperscript{1}O\textsubscript{2}in chloroplasts contents
were measured by using plant ROS singlet oxygen assay kit (GMS10150.4,
GENMED, Boston, MA, USA). Chloroplasts were isolated from \emph{Z.
marina} leaves, which were dark-adapted and exposed to 300 \selectlanguage{greek}µ\selectlanguage{english}E for 3 h,
by using the plants leaf chloroplast rude divide kit (GMS16004.1,
GENMED). To monitor change in carbon fixation ability during 300 \selectlanguage{greek}µ\selectlanguage{english}E
light exposure, the Rubisco carboxylase activity was determined by using
ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity detection
kit (BC0440, Solarbio, Beijing, China). Absorption measurements were
performed using a multifunctional enzyme-labeled instrument (Tecan,
M\selectlanguage{ngerman}ännedorf, Switzerland). O\textsubscript{2}evolution rate used to
reflect the leaves photosynthetic rate was measured at 15 °C with a
liquid-phase oxygen electrode system (Chlorolab2+, Hansatech, Norfolk,
UK).

\textbf{Chlorophyll a fluorescence and 820 nm modulated reflection
(MR\textsubscript{820nm}) measurements}

A pulse-amplitude modulated fluorometer (Mini-PAM; Walz, Germany) was
used to measure NPQ. During the course of measurements, the PAM fiber
was placed in a fixed position at 60° in relation to \emph{Z. marina}
leaves to avoid shading or darkening.

Multi-function plant efficiency analyzer 2 (M-PEA-2; Hansatech,
Hercules, UK) was used to simultaneously detect the kinetics of prompt
fluorescence (PF), delayed fluorescence (DF), and
MR\textsubscript{820nm} and thus to monitor photochemical activity and
dissect events in the electron transfer chain (Strasser et al., 2010;
Gururani et al., 2015)\textsuperscript{10,62}. Changes in the OJIP
fluorescence rise kinetics were assessed by calculating the difference
value of variable fluorescence curves as \selectlanguage{greek}Δ\selectlanguage{english}\emph{V} t = \selectlanguage{english}\selectlanguage{greek}Δ{[}(\emph{F}
\textsubscript{t} \emph{- F} \textsubscript{O}) / (\emph{F}
\textsubscript{m} \emph{- F} \textsubscript{O}){]}. The relative
variable fluorescence at the K-step (\emph{W} \textsubscript{K}) was an
indicator of the PSII electron donation capacity (Strasser, 1997;
Brestic et al., 2012)\{Brestic, 2012 \#265;Strasser, 1997 \#269\}. The
decay kinetics of DF at maximum \emph{I} \textsubscript{1} were fitted
with the function DF(\emph{t} ) = \emph{L} \textsubscript{1} \selectlanguage{ngerman}× exp(-t
/\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{1}) + \emph{L} \textsubscript{2} \selectlanguage{ngerman}× exp(-t
/\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{2}) + \emph{L} \textsubscript{3}, where\emph{L}
\textsubscript{1} and \emph{L} \textsubscript{2}represent the amplitudes
of the decay kinetics component and \selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{1}
and\selectlanguage{greek}\emph{τ} \selectlanguage{english}\textsubscript{2} represent their lifetimes (in ms),
respectively. \emph{L} \textsubscript{1} corresponded to the electron
transport capacity at PSII donor side (Goltsev et al., 2009; Gao et al.,
2013). MR\textsubscript{820nm} obtained by a modulated measuring light
at 820 nm was measured after far-red light was closed (Zhang et al.,
2011).\emph{V} \textsubscript{re-red}, the initial rate of
P700\textsuperscript{+}re-reduction, was calculated to quantify PSI-CEF
activity. Detailed calculations and physiological explanations of
parameters are listed in Table S2.

\textbf{Protein extraction, digestion, and tandem mass tag (TMT)
labeling}

TMT proteome of chloroplasts was adopted to identify differential
proteins for achieving high-throughout and high-resolution screening.
Chloroplasts were isolated from \emph{Z. marina} leaves, which were
dark-adapted (Dark) and exposed to 300 \selectlanguage{greek}µ\selectlanguage{english}E for 3 h (Light). The
chloroplasts were suspended in BPP and Tris-saturated phenol, and then
vortexed for 10 min. Proteins in the phenol phase collected via
centrifugation at 12,000 g for 20 min were precipitated with ice-cold
ammonium acetate, and then washed three times with 90\% cooled acetone.
The precipitate was dissolved in protein lysate (8 M urea, 1\% SDS
containing protease inhibitor), and the solution was then centrifuged as
above to collect the protein supernatant. The above operations were
performed at 4 \selectlanguage{ngerman}°C. The protein concentration was measured by BCA.

Protein digestion was performed according to the standard procedure and
the resulting peptide mixture was labeled using the 6-plex TMT reagents
(ThermoFisher Scientific, Waltham, MA, USA). The peptides from Dark and
Light samples were both labeled with TMT tags for three biological
replicates. Briefly, each tube with 100 \selectlanguage{greek}μ\selectlanguage{english}g protein, containing 10 mM
tris-(2-carboxyethyl)phosphine hydrochloride, was incubated at 37 \selectlanguage{ngerman}°C for
60 min. The appropriate volume of iodoacetamide was added to a final
concentration of 40 mM, and the solutions were incubated for 40 min in
the dark. The samples were mixed with six volumes of cold acetone and
the precipitates collected by centrifugation at 10,000 g for 20 min were
resuspended with 100 \selectlanguage{greek}µ\selectlanguage{english}l 50 mM triethylammonium formate buffer. Trypsin
solution was added to each sample tube according to a proportion of 1 :
50 (trypsin : protein) and the tubes were incubated overnight at 37 \selectlanguage{ngerman}°C.
The TMT reagent was added to corresponding peptide mixture respectively.
All samples were then combined and vacuum dried.

\textbf{High pH reversed-phase liquid chromatography separation}

Samples were fractionated to increase the proteomic depth, using high pH
reverse phase separation. The peptides resuspended in buffer A (2\%
acetonitrile, containing ammonium hydroxide solution, at pH 10) were
separated by ACQUITY UPLC (Waters, Milford, MA, USA) connected to a high
pH UPLC column. Gradient elution was achieved at a rate of 200 \selectlanguage{greek}μ\selectlanguage{english}l
min\textsuperscript{-1} for 66 min with buffer B (80\% acetonitrile,
containing ammonium hydroxide solution, at pH 10). Twenty fractions were
collected from each sample and were pooled into 10 total fractions.

\textbf{Liquid chromatography-mass spectrometry (LC-MS/MS) analysis}

Mass spectrometry analysis was performed on a Q Exactive mass
spectrometer coupled with Easy-nLC 1200 (ThermoFisher Scientific). The 2
\selectlanguage{greek}μ\selectlanguage{english}g peptides loaded onto the C18 column in buffer A (2\% acetonitrile,
0.1\% formic acid) were separated with a linear gradient of buffer B
(80\% acetonitrile and 0.1\% formic acid) at a flow rate of 300 nl
min\textsuperscript{-1} in Easy-nLC 1200. The eluted peptides were
injected into a Q Exactive mass spectrometer (ThermoFisher Scientific),
using 1.8 kV electrospray voltage. The acquisition of survey full-scan
MS spectra (m/z 350-1300) was attributed to a mass resolution of 70,000,
followed by 20 sequential high energy collisional dissociation MS/MS
scans at a resolution of 35,000.

For protein identification, MS/MS spectra were searched using Protein
Discoverer\textsuperscript{TM}Software 2.2 and parent proteins were
identified by the highest score for a given peptide mass. The following
settings were used: a maximum of two miscleavages; carbamidomethylation
of cysteines as fixed modification; protein N-terminal acetylation and
oxidation of methionines as variable modifications. At least one unique
peptide containing a minimum of six amino acids sequence was required
for per protein. The false discovery rate (FDR) was set to 1\% to
validate peptide spectra.

\textbf{Bioinformatics analysis}

BLASTP was used to search homologous proteins for uncharacterized
proteins defined by the \emph{Z. marina} information category in
Uniprot. Proteins with\emph{P} -value \textless{} 0.05 and fold change
(FC) \textgreater{} 1.2 were defined as DEPs. GO enrichment analysis of
DEPs was performed using the Fisher's exact test and FDR correction.
Protein-protein interaction (PPI) network was constructed using the
STRING database with the Cytoscape software.

\textbf{Western blotting}

Western blotting was used to quantify OEC peripheral proteins PsbO,
PsbP, PsbQ, as well as the terminal enzyme l-galactono-1,4-lactone
dehydrogenase (GLDH) of AsA biosynthesis and PSII RC proteins D1, CP43.
GLDH located in mitochondrion was detected using the proteins in leaves
which were isolated as previously described (Jorrin-Novo, 2014). The
other proteins located in photosynthetic membranes were detected using
chloroplasts. To determine the loading quantity of sample, the protein
and chlorophyll contents were measured as previously published (Porra et
al., 1989). Solubilized materials were fractionated using sodium dodecyl
sulfate--polyacrylamide gel electrophoresis. Western blot assays with
antibodies (Agrisera, Sweden) were performed as previously described
(Fristedt et al., 2009). As equal loading control, the Rubisco large
subunit (RbcL) antibody was used. The chemiluminescent bands visualized
on a Gel Doc XR+ system (Bio-Rad, CA, USA) were quantified with the
program Quantity One software. For each case, the density of the samples
was standardized based on the RbcL density.

\textbf{Statistical Analysis}

Statistical analyses were performed using SPSS 22.0 statistical software
package. All parameters were analyzed by one-way analysis of variance
(ANOVA). Tukey's tests were used to make post hoc comparisons. Repeated
measures ANOVA was adopted to examine the effect of rotenone and DMBQ
on\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m} and\emph{W}
\textsubscript{k}.

\textbf{Data and materials availability}

Raw MS and Proteome Discoverer output data have been uploaded to the
iProX with ID: IPX0002022001. All other data needed to evaluate the
conclusions in the paper are present in the paper and/or the
Supplementary Materials. Additional data related to this paper may be
requested from the authors.

\textbf{Results}

\textbf{The category of PSII photoinhibition}

The amplitude \emph{L} \textsubscript{1} of the DF decay kinetics
component declined significantly during the initial illumination already
and continuously decreased with exposure duration (Fig. 1A), suggesting
the decreased electron transport capacity of the PSII donor side
following light exposure. As shown in Fig. 1B and C, the relative
variable fluorescence at the K-step (\emph{W} \textsubscript{K}) was
significantly increased and OEC peripheral proteins including the
manganese-stabilizing protein PsbO and the
Ca\textsuperscript{2+}-binding protein PsbP were significantly
downregulated, confirming the partial impairment of OEC during the
initial illumination. This decrease of OEC activity was accompanied by
the decrease of PSII photochemical activity characterized by the
decrease of \emph{F} \textsubscript{P}following light exposure (Fig.
1D). In the presence of lincomycin inhibiting D1 protein synthesis, the
time-course changes in\emph{F} \textsubscript{v}\emph{/F}
\textsubscript{m} under various photosynthetic photon flux density
(PPFD) values fitted the first-order kinetics well (Fig. 1E). Both the
decreased \emph{F} \textsubscript{v}\emph{/F} \textsubscript{m}even
under ultra-dim light during the initial illumination and the direct
proportionality of K\textsubscript{PI} to PPFD from dim light to
supersaturating light intensities further supported the occurrence of
PSII donor-side photoinhibition in \emph{Z. marina}(Fig. 1F).

\textbf{Overview of quantitative proteome analysis}

Among 2613 protein groups, a total of 2187 proteins were quantified with
at least one unique peptide sequence (Fig. S1A). \emph{Z. marina}samples
were clustered and distinguished from each other based on light
treatment (Fig. S1B). The 127 differentially expressed proteins (DEPs)
were determined, which included 89 upregulated proteins and 38
downregulated proteins (Fig. S1C). Based on Gene ontology (GO)
enrichment analysis, DEPs were classified into the following several
biological processes: ``photosystem II assembly'', ``chlorophyll
biosynthetic process'', ``photosynthetic electron transport in
photosystem I'', ``regulation of photosynthesis, light reaction'',
``PSII associated light-harvesting complex II catabolic process'',
``response to light intensity'', and so on (Fig. 2A). In the
Protein-protein interaction (PPI) network of the DEPs, OEC peripheral
protein PsbO had a high degree of connectivity and strong interactions
with other proteins (Fig. 2B), indicating that thte photoinactivated OEC
played a central role in the photosynthetic regulation of \emph{Z.
marina} .

\textbf{Regulation of light capture}

ATP-dependent zinc metalloprotease (FtsH) 4 and FtsH 2, both involved in
``PSII associated light-harvesting complex II (LHCII) catabolic
process'', were significantly upregulated (Fig. 2A). Three proteins
responsible for antenna assembly, including LHCII protein Lhcb3, low
PSII accumulation 3 (LPA3) assisting chlorophyll a binding protein CP43
assembly within PSII, and PsaH binding light-harvesting complex I
protein Lhca2 to PSI core complex (Table S1), were downregulated,
suggesting a decrease of antenna size following light exposure. The
distinct interactions of PsbO with Lhcb3, PsaH were shown in the PPI
network, indicating the potential of OEC to regulate antenna assembly
(Fig. 2B).

\textbf{Regulation of electron transport}

Proteome data revealed that the proteins involved in PSII assembly, such
as high chlorophyll fluorescence phenotype244 (HCF244), domain of
unknown function (DUF1995) and thylakoid formation 1 (THF1), displayed
upregulation expression, indicating that continuous repair was required
to retain PSII functionality. The proteins involved in electron donating
to PSI reaction center P700\textsuperscript{+}, such as proton gradient
regulation 5 (PGR5) and PsaN, were also upregulated, indicating the
activation of the PGR5-dependent PSI-CEF (Fig. 2A). In addition, the
protein called acclimation of photosynthesis to environment was
upregulated, contributing to the acclimation of photosynthetic electron
transfer to illumination (Table S1). The interactions between PsbO and
HCF244, as well as PGR5 and PsaN, shown in the PPI network, suggested
the need of electron transport regulation due to the partially impaired
OEC (Fig. 2B).

\textbf{Regulation of carbon fixation}

Biological process enrichment analysis based on DEPs showed no
significant change in the carbon fixation pathway at the protein level.
However, both Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
phosphoribulokinase (PRK), involved in carbon fixation pathway, were
dissociated from the GAPDH/CP12/PRK complex by the downregulated CP12,
indicating the activation of GAPDH and PRK. Moreover, thioredoxin
reductase (NTRC), which is associated with the activation of
thioredoxins-regulated enzymes in carbon fixation pathway, was
upregulated (Table S1). The good function of the carbon fixation pathway
during photoinhibition was supported by the significantly increased
carboxylase activity of ribulose bisphosphate carboxylase/oxygenase
(Rubisco) (Fig. 3A). Further supporting this function, photosynthetic
rate characterized by O\textsubscript{2} evolution rate exhibited a
typical photoinduction process followed by a normal level (Fig. 3A).

\textbf{Energy dissipation}

NPQ exhibited a decreasing tendency following a slow increase (Fig. 3B).
The slow NPQ development followed a single exponential function and as a
consequence fast-activated qE component was absent, while the
NPQ\textsubscript{max}showed a low value (Fig. 3C). Moreover, proteome
data showed that the luminal pH sensor PsbS protein, which was required
for qE, remained unchanged in response to light exposure (Table S1).
Slightly upregulated zeaxanthin epoxidase (ZEP) and violaxanthin
de-epoxidase (VDE) levels indicated a low level of xanthophyll
cycle-dependent energy dissipation (Table S1). The two key enzymes of
chlororespiration, post-illumination chlorophyll fluorescence increase
and ubiquinol oxidase, remained unchanged. Similarly, D-glycerate
3-kinase, the core enzyme in photorespiration pathway, both ascorbate
peroxidase (APX) and superoxide dismutase, the key enzymes of the Mehler
reaction, and malate dehydrogenase, the key enzyme of malic acid
synthesis, remained unchanged (Table S1). These results suggested that
alternative electron flows associated with energy dissipation were not
significantly activated.

\textbf{Antioxidant system defense}

Both the chlorophyll synthesis pathway and the early light-induced
protein were not significantly induced by light exposure (Fig. 2A and
Table S1), suggesting that there was no accumulation of free chlorophyll
which would have acted as a generator of reactive oxygen species.
Although Delta-aminolevulinic acid dehydratase, the enzyme committed to
tetrapyrrole biosynthesis, and geranylgeranyl diphosphate reductase,
which provides phytol, were upregulated, the terminal key enzyme
NADPH-protochlorophyllide oxidoreductase of chlorophyll synthesis was
downregulated (Fig. 2A). The expression of the stromal antioxidant
enzymes glutathione peroxidase (GPX) and APX remained unchanged (Table
S1), further displaying that the antioxidant ability was not
significantly enhanced following light exposure.

\textbf{Damaging mechanism}

Although the reduction of Q\textsubscript{A} represented by 1 -\emph{V}
\textsubscript{J} was significantly increased, the reduction degree of
Q\textsubscript{A} was low (by \textasciitilde{}4.5\%) (Fig. 4A).
Consistently, no significant increases in
chloroplast\textsuperscript{1}O\textsubscript{2} content and, the
quencher of\textsuperscript{1}O\textsubscript{2}, GPX were observed
following light exposure (Fig. 4A and Table S1), while PsbH, which
stabilized the combination of PSII and \selectlanguage{greek}β-\selectlanguage{english}carotene associated with
scavenging \textsuperscript{1}O\textsubscript{2}, was significantly
downregulated (Table S1). Nevertheless, PSII photochemical activity
significantly decreased, indicated by a decrease in \emph{F}
\textsubscript{P} and an increase in \emph{F} \textsubscript{O}
following light exposure (Fig. 4B). This suggested the existence of an
additional source of damage other than
\textsuperscript{1}O\textsubscript{2}.

\textbf{Photoprotective mechanisms}

l-galactono-1,4-lactone dehydrogenase (GLDH), the terminal enzyme in the
major Smirnoff-Wheeler pathway for AsA biosynthesis, together with the
AsA level in the chloroplast were significantly upregulated after light
exposure (Fig. 5A), providing a clue to AsA being the alternative
electron donor. Additionally, analysis of the K step in \selectlanguage{greek}Δ\selectlanguage{english}\emph{V}
\textsubscript{t} curves and\emph{F} \textsubscript{P} in OJIP curves
showed: there was the lower PSII electron donation capacity and PSII
photochemical activity induced by 125 \selectlanguage{greek}μ\selectlanguage{english}M DMBQ binding at the PQ site to
uncouple PSII-CEF, compared to 62 \selectlanguage{greek}μ\selectlanguage{english}M DMBQ binding at the
Q\textsubscript{B}site to enhance PSII-CEF (Fig. 5B). This suggested
that PSII-CEF serves as a possible alternative electron donation
pathway.

To test the roles of AsA and PSII-CEF in safeguarding PSII function, a
factorial design experiment with different combinations of rotenone and
DMBQ, the inhibitors of AsA and PSII-CEF, was conducted. Compared
with\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m} values, the
order of PSII activity was: Light \textgreater{} Light + Rotenone
\textgreater{} Light + DMBQ \textgreater{} Light + Rotenone + DMBQ (Fig.
6A). Simultaneously, inhibition of AsA synthesis or uncoupling of
PSII-CEF led to the further decreased PSII electron donation capacity
indicated by \emph{W} \textsubscript{K}, and the decrease was more
obvious during the late illumination (Fig. 6A). Furthermore,
Western-blotting analyses showed no visible changes of D1, CP43 proteins
after light exposure and their degradation was detected after both
inhibitor treatments, indicating that AsA and PSII-CEF prevented the
damage of PSII RC via donating electrons (Fig. 6B). The suppression of
temporary electron donor and the alternative electron flow also led to a
further impairment of OEC, as characterized by the distinct degradation
of PsbO and PsbP proteins (Fig. 6B).

\textbf{Discussion}

The evidence presented here, at both spectral and protein levels,
confirmed that OEC of \emph{Z. marina} was preferentially inactivated in
response to light exposure: (1) PSII electron donation capacity
characterized by \emph{L} \textsubscript{1} and \emph{W}
\textsubscript{K}was significantly decreased already during initial
illumination; (2) OEC activity represented by the contents of OEC
peripheral proteins (Bricker and Frankel, 2008; Popelkova et al., 2011)
greatly decreased, while the reduction degree of Q\textsubscript{A} at
the acceptor-side, represented by 1 - \emph{V} \textsubscript{J},
slightly increased; (3) PSII photoinhibition occurred upon light
exposure and the photoinhibition rate constant, determined via
first-order kinetic fits, was positively correlated with PPFD at all
tested light intensities. This was the first \emph{in vivo} study of
PSII donor-side photoinhibition with the direct experimental evidence in
higher plants under visible light. The \emph{in vivo} occurrence of this
mechanism in higher plants enabled the study of both integrated
photosynthetic characteristics and photoprotective mechanisms, which
involved multiple coordinated systems.

The present study investigated the integrated photosynthetic regulatory
mechanisms involved in light absorption, transfer and conversion, and
energy dissipation via chloroplast proteome combined with chlorophyll
fluorescence phenotype. The strong interactions of PsbO with Lhcb3,
PGR5, HCF244, Psb29, PsaN, and PsaH, shown in the PPI network, suggested
that OEC played a central role in the regulation of photosynthesis. The
decreased size of the PSI and PSII antennas indicated a decline of the
light-harvesting capacity in\emph{Z. marina} . The interactions of PsbO
with PsaH and Lhcb3 were interpreted as a need to decrease the
light-harvesting capacity due to the partially impaired OEC. It should
be realized that a decrease of PSII antenna size corresponds to fewer
absorbed photons but also to a faster trapping rate (Croce and Van
Amerongen, 2014). Simultaneously, PSII assembly was activated to retain
PSII functionality. Because continuously impaired OEC led to a
relatively oxidized PQ pool (Makarova et al., 2007), the PGR5-dependent
PSI-CEF in \emph{Z. marina} was activated to supplement the PSI linear
electron transport. Activated PSI-CEF revealed by proteome data in the
present study was in agreement with chlorophyll fluorescence
physiological phenotype observed in our previous study (Yang et al.,
2017). The interactions between PsbO and HCF244, as well as PGR5 and
PsaN indicated the potential of the partially impaired OEC to enhance
PSII repair and activate the PGR5-dependent PSI-CEF.

Continuous impairment of OEC in this mechanism suggested electron
deficiency in the electron transport chain. Unchanged antioxidant levels
and unactivated alternative electron flows, such as chlororespiration,
Mehler reaction, malic acid synthesis and photorespiration, supported
this viewpoint. The low PSII excitation pressure caused by electron
deficiency made it difficult to form a strong trans-thylakoid proton
gradient (\selectlanguage{greek}Δ\selectlanguage{english}pH), and consequently, could not induce strong NPQ. This was
verified by the unchanged \selectlanguage{greek}Δ\selectlanguage{english}pH sensor PsbS protein, the low value of
NPQ\textsubscript{max} (Schubert et al., 2015), and slightly upregulated
ZEP and VDE. NPQ is usually dissected into at least three kinetic
components, in which qE, induced within 20-60 s, was the dominant NPQ
component (Horton et al., 1996; Nilkens et al., 2010). Further
examination of the dynamics of NPQ induction in\emph{Z. marina}
indicated the absence of the fast qE component, which accounted for the
slow development of NPQ. Moreover, the decrease of NPQ during the late
illumination indicated that the demand for dissipation decreased with
the increased OEC impairment. A low NPQ capacity has also been reported
in the seagrass \emph{Posidonia sinuosa} (Schubert et al., 2015).

The low levels of NPQ were conductive to the formation of the reduced
form of NADPH and ATP derived from photosynthetic electron transport,
which in turn contributed to carbon fixation. In C3 plants,
photosynthesis is typically restricted by the Rubisco capacity due to
its exceedingly low catalytic turnover rate and competition with
oxygenation reaction (Farquhar et al., 1980). The significantly enhanced
carboxylase activity of Rubisco and the upregulated NTRC (Nikkanen et
al., 2016) suggested an increased CO\textsubscript{2} fixation rate in
response to light exposure. Recoveries of activities of GAPDH and PRK,
the key enzymes of carbon fixation, were achieved by downregulated CP12
(Marri et al., 2009), suggesting the activation of carbon fixation
pathway. The well photosynthetic performance during photoinhibition was
also supported by the normal photosynthetic rate characterized by
O\textsubscript{2} evolution rate in \emph{Z. marina} .

\textsuperscript{1}O\textsubscript{2} is usually formed by a
photosensitization reaction in which an oxygen molecule reacts
with\textsuperscript{3}P680 (Posp\selectlanguage{ngerman}í\selectlanguage{polish}š\selectlanguage{english}il, 2016). The sensitizer
molecule\textsuperscript{3}P680, produced
by\textsuperscript{3}{[}P680\textsuperscript{+}Phe\textsuperscript{-}{]}
which is generated either via spin conversion of primary radical pair
P680\textsuperscript{+}Phe\textsuperscript{-} or via recombination of
P680\textsuperscript{+}Q\textsubscript{A}\textsuperscript{-}, is
triggered by the over-reduction of Q\textsubscript{A} (Vass et al.,
1992; Hakala et al., 2005; Ohnishi et al., 2005; Vass, 2011). The
reduction degree of Q\textsubscript{A} was low in \emph{Z. marina} ,
which was evidenced by a slight decrease of 1 - \emph{V}
\textsubscript{J}. Therefore, the conditions for
\textsuperscript{1}O\textsubscript{2} production were not appropriate.
In fact, light exposure neither caused an upregulation of PsbH and GPX,
nor an increase of\textsuperscript{1}O\textsubscript{2}levels. The
strong interaction between PsbO and PsbH indicated that there was no
need for PsbH to stabilize the combination of excess \selectlanguage{greek}β-\selectlanguage{english}carotene and PSII
(Hall et al., 2016) to eliminate\textsuperscript{1}O\textsubscript{2}.
Moreover, the inhibition of alternative electron donation in our study
resulted in a significant damage of PSII RC described by the net losses
of D1 and CP43 proteins, indicating that the photodamage of PSII was
attributed to the long-lived P680\textsuperscript{+}rather
than\textsuperscript{1}O\textsubscript{2}. Thus, the long-lived
P680\textsuperscript{+} resulted from the interrupted electron supply
was usually considered as the damage source in the PSII donor-side
photoinhibition, which has been suggested by Vass (2012) and Tyystj\selectlanguage{ngerman}ärvi
(2008). Noticeably, a distinct low descending amplitude of\emph{F}
\textsubscript{v}\emph{/F} \textsubscript{m}was observed during light
exposure, which could be explained by most PSII RC remaining open to
contribute to the depletion of the pool of high-potential
P680\textsuperscript{+} via facilitating the direct charge recombination
of the P680\textsuperscript{+}Q\textsubscript{A}\selectlanguage{english}\textsuperscript{-}.
Because the competition
between\textsuperscript{3}{[}P680\textsuperscript{+}Phe\selectlanguage{english}\textsuperscript{-}{]}
formation and the direct recombination of the
P680\textsuperscript{+}Q\textsubscript{A}\selectlanguage{english}\textsuperscript{-} was
controlled by PSII excitation pressure (Vass and Cser, 2009), the low
reduction degree of Q\textsubscript{A}in \emph{Z. marina} prevented the
formation
of\textsuperscript{3}{[}P680\textsuperscript{+}Phe\selectlanguage{english}\textsuperscript{-}{]}.

The existence of alternative PSII electron donors has been reported in
some species (Thompson and Brudvig, 1988; Toth et al., 2009, 2011).
Based on in vivo data, the increased AsA level and the upregulated GLDH
content involved in AsA synthesis following light exposure provided a
clue of AsA as alternative electron donor. Similarly, in contrast to 62
\selectlanguage{greek}μ\selectlanguage{english}M DMBQ enhancing PSII-CEF, 125 \selectlanguage{greek}μ\selectlanguage{english}M DMBQ uncoupling PSII-CEF induced the
lower PSII electron donation capacity and PSII photochemical activity,
providing a clue of PSII-CEF as another alternative donation pathway.
Furthermore, a factorial design experiment with different combinations
of AsA and PSII-CEF inhibitions demonstrated that the suppression of
alternative electron donation damaged the PSII component, including a
decrease in\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m}, an
increase in\emph{W} \textsubscript{K}, as well as net losses of PSII RC
proteins and OEC peripheral proteins. With the duration of light
exposure, the inhibition effect became more significant, of which, the
most severe damage of PSII was caused by the dual inhibition of AsA and
PSII-CEF. Based on these results, we suggested that both AsA and
PSII-CEF were important photoprotective mechanisms, which provided
electrons to remit the oxidative stress from long-lived
P680\textsuperscript{+} during light exposure.

In conclusion, the water-splitting dysfunction caused by OEC
photoinactivation interrupted the electron supply from water to the
oxidized primary donor P680\textsuperscript{+}, resulting in the damage
to the PSII RC of\emph{Z. marina} . At least PSII-CEF and the
alternative donor AsA exerted photoprotective roles in the depletion of
P680\textsuperscript{+} by donating electrons to PSII (Fig. 7). In
contrast to acceptor-side photoinhibition caused by electron excess,
continuous impairment of OEC resulted in electron deficiency in the
electron transport chain during the PSII photoinhibition. For the
efficient use of the limited electrons, NPQ was inefficient and the
alternative electron flows associated with energy dissipation, such as
chlororespiration, Mehler reaction, malic acid synthesis and
photorespiration, were not significantly activated to decrease
unnecessary consumption (Fig. 7). The extremely sensitivity of OEC to
visible light was presumably a result of insufficient polyphenol levels
caused by a lack of blue light photoreceptors in a habitat that is rich
in blue light (Peng and Moriguchi, 2013; Jiang et al., 2016).

\textbf{Acknowledgments}

This work was financially supported by the National Natural Science
Foundation of China (No. 41376154). We wish to thank E. Tyystj\selectlanguage{ngerman}ärvi
(University of Turku) for constructive suggestions to the improvement of
this manuscript.

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\textbf{Figure Legends}

\textbf{Fig. 1 Preferential impairment of OEC induced by light.} (A)
Variations in the amplitude \emph{L} \textsubscript{1} of the kinetics
component calculated by fitting the DF decay kinetics to the time
function DF(\emph{t} ) = \emph{L} \textsubscript{1} \selectlanguage{ngerman}× exp(-t/\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{1}) + \emph{L} \textsubscript{2} \selectlanguage{ngerman}× exp(-t/\selectlanguage{greek}\emph{τ}
\selectlanguage{english}\textsubscript{2}) + \emph{L} \textsubscript{3}. The inset shows DF
decay kinetics at \emph{I} \textsubscript{1} in response to light
exposure. (B) Changes in the relative variable fluorescence at the
K-step (\emph{W} \textsubscript{k}) in response to light exposure. (C)
Variations in OEC peripheral proteins PsbO, PsbP and PsbQ during light
exposure. Values were \% of dark 100\% and normalized to RbcL amount.
The significantly different value (Tukey's tests, \emph{P} \textless{}
0.05) from 0 min or dark is marked with an asterisk (*). (D) Changes in
the normalized chlorophyll fluorescence intensity of OJIP transients
(\emph{F} \textsubscript{t}/\emph{F} \textsubscript{O}) in response to
light exposure and plotted on a logarithmic time scale. (E and F)
Photoinhibition measured by the decrease in\emph{F}
\textsubscript{v}/\emph{F} \textsubscript{m} in the presence of
lincomycin. (E) The decreases of\emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m} standardized based on 0 min fitted well with the
function\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m} = exp
(-K\textsubscript{PI} \selectlanguage{ngerman}× t) (all \emph{R} \textsuperscript{2} values
\textgreater{} 0.9). (F) Dependence of the K\textsubscript{PI} on photon
flux density (\emph{P} \textless{} 0.05). The means ± s.d. were
calculated from three independent samples. The means ± s.d. were
calculated from three independent samples. Each curve represents the
average of three replicates.

\textbf{Fig. 2 GO enrichment analysis and PPI network of DEPs.} (A) The
biological processes in GO enrichment analysis of DEPs. The DEPs are on
the left and the GO pathway names are on the right (\emph{P} \textless{}
0.05). (B) PPI network of DEPs. Red nodes indicate upregulated proteins
and blue nodes indicate downregulated proteins. The larger size of the
node represented the higher connectivity of the protein, which indicated
more interactions with other proteins. The width of the line represents
the capacity of the interaction between proteins.

\textbf{Fig. 3} \textbf{Photosynthetic activity and NPQ response to
light exposure.} (A) Changes in O\textsubscript{2} evolution rate and
Rubisco carboxylase activity in response to light exposure. The
significantly different value (Tukey's tests, \emph{P} \textless{} 0.05)
from 0 min is marked with an asterisk (*). (B) chlorophyll fluorescence
during NPQ formation. (C) The kinetics of NPQ induction fitted with the
function NPQ = A × exp (-x/\emph{t} ) + y\textsubscript{0}. The means ±
s.d. were calculated from three independent samples.

\textbf{Fig. 4} \textbf{The relevant parameters of PSII} \textbf{damage
response to light exposure.} (A) Variations in probabilities for an
electron moving further than Q\textsubscript{A}(1-\emph{V}
\textsubscript{J}) and chloroplast\textsuperscript{1}O\textsubscript{2}
contents after light exposure. The significantly different value from
Dark (Tukey's tests, \emph{P}\textless{} 0.05) is marked with an
asterisk (*). (B) Changes in OJIP transients in response to light
exposure and plotted on a logarithmic time scale. The means ± s.d. were
calculated from three independent samples. Each curve represents the
average of three replicates.

\textbf{Fig. 5} \textbf{AsA and PSII-CEF response to light exposure.}(A)
Variations in GLDH contents determined by densitometry and chloroplast
AsA levels in response to light exposure. The significantly different
value from 0 min (Tukey's tests, \emph{P} \textless{} 0.05) is marked
with an asterisk (*). (B) Changes in chlorophyll a fluorescence kinetics
as summarized by \selectlanguage{greek}Δ\selectlanguage{english}\emph{V} \textsubscript{t} and OJIP curves in response
to light exposure and different concentrations of DMBQ. The signals are
plotted on a logarithmic time scale. Each curve represents the average
of three replicates.

\textbf{Fig. 6 Photoprotection of AsA and PSII-CEF response to light
exposure.} (A) Time course of the changes in maximal photochemical yield
of the PSII (\emph{F} \textsubscript{v}/\emph{F} \textsubscript{m}) and
the relative variable fluorescence at the K-step (\emph{W}
\textsubscript{k}) in response to different inhibitors. The significant
effects of rotenone and DMBQ on\emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m} and\emph{W} \textsubscript{K} during HL exposure were
examined with repeated measures ANOVA (all \emph{p} values \textless{}
0.05). (B) The changes in PSII RC proteins D1, CP43 and OEC peripheral
proteins PsbO, PsbP, PsbQ after 3 h of treatment. Values were \% of dark
100\% and normalized to RbcL amount. The significantly different value
from 0 min (Tukey's tests, \emph{P} \textless{} 0.05) is marked with an
asterisk (*). Data are expressed as mean \selectlanguage{ngerman}± s.d. (\emph{n} = 3).

\textbf{Fig. 7} \textbf{Schematic model of donor-side photoinhibion
derived from the photoinactivated OEC.} The red arrows represent the
activated pathways in which the degree of activation are represented by
the width of line and the dark arrows represent the pathways that were
not significantly activated. Positions of photosynthetic complexes in
thylakoid membrane are based on published annotations (Eberhard and
Finazzi GWollman, 2008; Gururani et al., 2015; Li et al., 2018). PQ,
plastoquinone; Cytb6f, cytochrome b6f; PC, plastocyanin; Fd, ferredoxin;
FNR, ferredoxin NADP\textsuperscript{+} reductase; MDA,
monodehydroascorbate; Mal, malate; PTOX, ubiquinol oxidase; GLYK,
D-glycerate 3-kinase; MDH, malate dehydrogenase; OAA, oxaloacetic acid;
SOD, superoxide dismutase; APX, ascorbate peroxidase.

\textbf{Supporting Information}

\textbf{Fig. S1} Overview of the identified proteins. (a) Protein
profile statistical information. (b) The principal component analysis
(PCA) of identified proteins. (c) Volcano plots of proteins with
differential expression in Light vs. Dark group. Blue nodes indicate
significantly downregulated proteins (n = 38); red nodes represent
upregulated proteins (n = 89).

\textbf{Fig. S2} Normalized spectral power distribution of the 6000 K
LED lamp used in the experiments.

\textbf{Fig. S3} The effect of 50 \selectlanguage{greek}µ\selectlanguage{english}M rotenone on the initial rate of
P700+ re-reduction (Vre-red) following light exposure of 3 h. There was
no significant difference between Light and Light + Rotenone treatments
(Tukey's tests, \emph{P} \textgreater{} 0.05). Data are expressed as
mean \selectlanguage{ngerman}± s.d. (n = 3).

\textbf{Fig. S4} The effect of 50 \selectlanguage{greek}µ\selectlanguage{english}M rotenone and 125 \selectlanguage{greek}µ\selectlanguage{english}M DMBQ on\emph{F}
\textsubscript{v}/\emph{F} \textsubscript{m} under darkness for 190 min.
There was no significant difference of\emph{F}
\textsubscript{v}/\emph{F} \textsubscript{m} between the three
treatments (Tukey's tests, all \emph{p} values \textgreater{} 0.05).
Data are expressed as mean \selectlanguage{ngerman}± s.d. (n = 3).

\textbf{Fig. S5} The effect of 1.45\% (v/v) dimethyl sulfoxide (DMSO) on
OJIP transients and\emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m}following light exposure of 3 h. There was no
significant difference of\emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m} between Light and Light + DMSO treatments (Tukey's
tests, \emph{P} \textgreater{} 0.05). Data are expressed as mean ± s.d.
(n = 3).

\textbf{Table S1} List of 32 proteins identified by TMT in Z. marina
following light exposure.

\textbf{Table S2} Chlorophyll fluorescence parameters used in the study.

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