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\begin{document}

\title{Insufficient synthesis and acylation modification of anthocyanins causes
photoinactivation of the oxygen-evolving complex in Zostera marina}



\author[1]{Meng Xin Wang}%
\author[1]{Yun Wen}%
\author[1]{Ming Yu Zhong}%
\author[1]{Wenhao Zhang}%
\author[1]{Chengying Luo}%
\author[1]{Di Zhang}%
\author[1]{Zimin Hu}%
\author[1]{Quan sheng Zhang}%
\affil[1]{Yantai University}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Zostera marina} among seagrass suffering from global decline is a
representative species in temperate regions in the Northern Hemisphere.
Given our recent findings, the decline of seagrasses may be associated
with the photosensitivity of the oxygen-evolving complex (OEC).
Therefore, understanding the mechanism of OEC photosensitivity is key to
understanding the continued decline in seagrasses. Herein, we explored
the screening-based photoprotection function in \emph{Z. marina} by
examining the inactivation spectrum of OEC and the differences in
photoresponse pathways following exposure to different spectrums. The
OEC inactivation was spectral-dependent. High-energy light significantly
reduced the PSII performance, OEC peripheral protein expression, and
photosynthetic O \textsubscript{2} release capacity. The increased
synthesis of carotenoids under blue light with severe OEC damage implied
its weak photoprotection property in \emph{Z. marina}. However,
anthocyanins key synthetic genes were lowly expressed with inefficient
accumulation under high-energy light. Furthermore, the acylation
modifications of anthocyanins, especially aromatic acylation
modifications were insufficient, leading to poor stability and light
absorption of anthocyanins. Based on the role of blue light receptors in
regulating the synthesis of anthocyanins in vascular plant, we
hypothesized that the absence of blue light receptor CRY2 in \emph{Z.
marina} causes the insufficient synthesis of anthocyanins and acyl
modifications, reducing the shielding against high-energy light,
subsequently causing OEC photoinactivation.%
\end{abstract}%



\sloppy


\textbf{Insufficient synthesis and acylation modification of
anthocyanins causes photoinactivation of the oxygen-evolving complex in
\emph{Zostera marina}}

Mengxin Wang\textsuperscript{1}, Yun Wen\textsuperscript{1}, Mingyu
Zhong\textsuperscript{1}, Wenhao Zhang\textsuperscript{1}, Chengying
Luo\textsuperscript{1}, Di Zhang\textsuperscript{1}, Zimin
Hu\textsuperscript{1}, Quansheng Zhang\textsuperscript{1*}

\textsuperscript{1}\emph{Ocean School, Yantai University, Yantai 264005,
PR China}

* Corresponding author. Tel.: +86 0535 6705305; fax: +86 0535 6706299.

\emph{E-mail address} : ytuqsz@hotmail.com (Q.S. Zhang).

\textbf{Abstract}

\emph{Zostera marina} among seagrass suffering from global decline is a
representative species in temperate regions in the Northern Hemisphere.
Given our recent findings, the decline of seagrasses may be associated
with the photosensitivity of the oxygen-evolving complex (OEC).
Therefore, understanding the mechanism of OEC photosensitivity is key to
understanding the continued decline in seagrasses. Herein, we explored
the screening-based photoprotection function in\emph{Z. marina} by
examining the inactivation spectrum of OEC and the differences in
photoresponse pathways following exposure to different spectrums. The
OEC inactivation was spectral-dependent. High-energy light significantly
reduced the PSII performance, OEC peripheral protein expression, and
photosynthetic O\textsubscript{2} release capacity. The increased
synthesis of carotenoids under blue light with severe OEC damage implied
its weak photoprotection property in \emph{Z. marina} . However,
anthocyanins key synthetic genes were lowly expressed with inefficient
accumulation under high-energy light. Furthermore, the acylation
modifications of anthocyanins, especially aromatic acylation
modifications were insufficient, leading to poor stability and light
absorption of anthocyanins. Based on the role of blue light receptors in
regulating the synthesis of anthocyanins in vascular plant, we
hypothesized that the absence of blue light receptor CRY2 in \emph{Z.
marina} causes the insufficient synthesis of anthocyanins and acyl
modifications, reducing the shielding against high-energy light,
subsequently causing OEC photoinactivation.

\textbf{Keywords} : Anthocyanin; Light screening; Photoreceptor;
Oxygen-evolving complex;\emph{Zostera marina} .

\textbf{Introduction}

Seagrasses are a foundation species in marine ecosystems, which provide
important ecosystem services by forming extensive seagrass beds
(Fourqurean et al., 2012; Cullen-Unsworth et al., 2018; Costa et al.,
2020). Despite their critical value, they are suffering a global
decline, becoming one of the most threatened ecosystems (Orth et al.,
2006). This decline has been attributed to global climate change and
habitat fragmentation, eutrophication, pollution, etc., as a direct
result of human activities (Burkholder et al., 2007; Duarte et al.,
2018; Hall-Spencer and Harvey, 2019; Nguyen et al., 2021). At the same
time, the inherent biological vulnerability of seagrasses has received
little attention.

Our recent studies indicated that oxygen-evolving complex (OEC)
of\emph{Zostera marina} and \emph{Phyllospadix iwatensis} , two
representative seagrasses in temperate regions, are prone to
photoinactivation under visible light (Zhao et al., 2021; Wang et al.,
2022). The OEC, located on the luminal side of the thylakoid lumen of
photosystem II (PSII), is the site for catalytic cleavage of water into
protons and molecular oxygen in photosynthesis (Gupta, 2020). Generally,
OEC photoinactivation is limited, thus, OEC stability and carbon
assimilation are effectively maintained by the regulations of ascorbic
acid and PSII and PSI cycle electron transport (Tan et al., 2020; Zhao
et al., 2021). However, during extreme light conditions, the percentage
of OEC inactivation can exceed 15\%, rendering the regulation mechanisms
inefficient in maintaining the photosynthetic performance, leading to
irreversible damage to the photosynthetic apparatus and a potential
decline in seagrass populations in the long term (Wang et al., 2022).
Therefore, to further understand the intrinsic causes of seagrass
population decline, understanding the mechanism of OEC inactivation is
crucial.

Normally, plants trigger a series of protective mechanisms to prevent
oxidative damage under stressful conditions (Takahashi and Badger,
2011). Screening-based photoprotection constitutes the first-line
defense of plants against potentially harmful solar radiation
(Solovchenko, 2010). The screening pigments accumulated in plants tissue
structures attenuate ultraviolet (UV) or the visible part of the
spectrum, reducing the excessive absorption of radiation by the
photosynthetic apparatus (Shick and Dunlap, 2002; Solovchenko and
Merzlyak, 2003). These screening pigments have been categorized into
four groups: mycosporine-like amino acids mainly in prokaryotes,
betalains, a nitrogenous compound limited to flowering plants,
carotenoids (Car), and phenolic compounds commom in all plants
(Solovchenko and Merzlyak, 2008). The screen function in the visible
region is majorly performed by Car and anthocyanins in phenolic
compounds (Hormaetxe et al., 2005; Landi et al., 2015). Carotenoids are
auxiliary pigments commonly found in photosynthetic autotrophs, which
transfer absorbed energy to chlorophyll a for photosynthesis and protect
the plant against photodamage through the xanthophyll cycle and light
screening (Frank et al., 1997; Baroli and Niyogi, 2000; Merzlyak and
Solovchenko, 2002). Under unfavorable conditions, the synthesis of
extrathylakoid, and extraplastidic Car is induced in microalgae to
protect the photosynthetic apparatus (Boussiba, 2000; Wang et al.,
2003). However, compared to microalgae, the screening function of Car in
higher is relatively less studied (Merzlyak and Solovchenko, 2002;
Hormaetxe et al., 2005; Merzlyak et al., 2005). Unlike Car, anthocyanins
in phenolic compounds are the most studied protective pigments against
stress (Chalker-Scott, 1999). Apart from their antioxidant activity,
anthocyanins have a good spectral absorption property for UV and the
blue-green component of visible light (Landi et al., 2015). Besides,
modifications to the anthocyanins enhance their stability and light
absorption capacity (Fan et al., 2008). Specifically, the resistance of
plant photosynthetic tissues to photodamage is increased with the
accumulation of anthocyanins (Landi et al., 2015; Zhu et al., 2018).

Herein, to establish the mechanism of OEC photoinactivation, \emph{Z.
marina,}whose complete genome has been sequenced, was used as the
research object (Olsen et al., 2016; Ma et al., 2021).\emph{Zostera
marina} is a completely submerged angiosperm in oceans, which evolved
from monocotyledons land plants and returned to the marine ecosystem
(Wissler et al., 2011). During its complex evolutionary process, the
omission of some photoreceptors occurred. Specifically, phytochromes
(PHYs) include PHYA and PHYB, lack PHYC-E, while only cryptochrome 1
(CRY1) is present in CRY, with CRY2 and CRY3 missing. In addition, UVR8
photoreceptor associated with UV is also absent (Olsen et al., 2016; Ma
et al., 2021). The Mn cluster of OEC is extremely unstable and prone to
photoactivated shedding (Hakala et al., 2005). However, the synthesis of
screening substances regulated by photoreceptors reduces the
photoactivated release of the Mn cluster through light screening (Landi
et al., 2015). Therefore, the absence of photoreceptors in \emph{Z.
marina}potentially restricts the synthesis of light-shielded compounds
that allow high-energy light to reach the chloroplasts easily, causing
OEC photoinactivation. To verify this hypothesis, \emph{Z. marina} was
treated with different light qualities to investigate (1) the spectral
dependence of OEC inactivation and (2) the differences in spectral of
photoresponse pathways.

\textbf{Materials and methods}

\textbf{Sample preparations and treatments}

\emph{Zostera marina} plants with intact rhizomes-systems and fresh
leaves were collected from the seagrass beds in Rongcheng (37\selectlanguage{ngerman}º 16'N,
122º 41'E), Weihai, Shandong province, China. Samples were pre-cultured
in an aquarium at 15 °C and a 10: 14 h light: dark cycle with a minimum
saturation light intensity of 70 photosynthetic photon flux density
(PPFD) for 3 days. Pre-cultured samples were dark-adapted overnight
prior to experimental treatment, following which leaves 2 cm above the
leaf sheath were sampled for experimentation. The inactivation spectrum
of OEC was determined by monitoring the prompt fluorescence kinetic on
leaves exposed to light at 380, 400, 420, 450, 530, 630, 660, 725, and
400-750 nm with a light intensity of 210PPFD for 15 min. Furthermore,
the PSII performance, OEC peripheral protein expression, photosynthetic
O\textsubscript{2} release capacity, and transcriptome differences were
measured on leaves exposed to white (WL, 400-750 nm), blue (BL, 420 nm)
and red light (RL, 660 nm) at 210 PPFD to investigate the effects of
photoreceptor absence.

\textbf{Chlorophyll a fluorescence measurements}

For a prompt characterization of the OEC activity, the kinetic of prompt
fluorescence was monitored using a multi-function plant efficiency
analyzer 2 (M-PEA-2; Hansatech, Norfolk, UK). The chlorophyll
fluorescence parameters were calculated as previously described by
Strasser et al. (2010). The normalized fluorescence rise kinetics of
OJIP were calculated using the formula: \selectlanguage{greek}Δ\selectlanguage{english}\emph{V} t = \selectlanguage{greek}Δ\selectlanguage{english} {[}(\emph{F}
\textsubscript{t}\emph{- F} \textsubscript{O})/ (\emph{F}
\textsubscript{m} \emph{- F} \textsubscript{O}){]}; the maximal quantum
yield of the PSII by \emph{F} \textsubscript{v}/\emph{F}
\textsubscript{m} = (\emph{F} \textsubscript{m} \emph{- F}
\textsubscript{o})/ \emph{F} \textsubscript{m}; the degree of damage on
the donor side of PSII by \emph{W} \textsubscript{K} = (\emph{F}
\textsubscript{K}\emph{-} \emph{F} \textsubscript{O})/ (\emph{F}
\textsubscript{J} \emph{- F} \textsubscript{O}), and the active fraction
of OEC centers by OEC\textsubscript{centers} = {[}1\emph{-} (\emph{V}
\textsubscript{K}/\emph{V}
\textsubscript{J}){]}\textsubscript{treatment}/ {[}1 \emph{-}(\emph{V}
\textsubscript{K}/\emph{V} \textsubscript{J}){]}\textsubscript{control}.
The subscript ``control'' and ``treatment'' indicated that the
corresponding parameters were measured on the dark-adapted, and
light-stressed treatment samples, respectively. Each measurement was
conducted in triplicate.

\textbf{Western blotting analysis}

The peripheral protein expression was used to characterize the stability
of OEC. Chloroplasts were separated from the leaf samples using the
Plants Leaf Chloroplast Rude Divide Kit (GenMED Scientifics Inc,
Arlington, MA, USA). The chlorophyll contents were measured as
previously described by Porra et al. (1989). To compare quantitative
differences, control samples with 1.25, 2.5, and 5 \selectlanguage{greek}µ\selectlanguage{english}g chlorophyll
corresponding to 25, 50, and 100\% of the control sample, respectively,
were loaded on SDS-PAGE gel and separated with solubilized materials
from treatment leaves containing 5 \selectlanguage{greek}µ\selectlanguage{english}g chlorophyll. Next, a Western blot
assay with antibodies against PsbO, PsbP and PsbQ (Agrisera, V\selectlanguage{ngerman}ännäa,
Sweden) was performed following the protocol described by Fristedt et
al. (2009). RuBisCo large subunit (RbcL) were used as equal loading
controls. The chemiluminescent bands on the blots were quantified using
Image Lab software (Bio-Rad) on a Gel Doc XR+ system (Bio-Rad, Hercules,
CA, USA). The sample densities were normalized to the RbcL density. Each
measurement was conducted in triplicate.

\textbf{Oxygen evolution rate measurements}

The rate of photosynthetic O\textsubscript{2} evolution was determined
using a liquid-phase oxygen electrode system (Chlorolab2+; Hansatech,
Hercules, UK) to evaluate the overall photosynthetic performance. Leaf
fragments (\textasciitilde{} 30 mg) were placed in the reaction chamber
containing 2 mL of seawater at 15°C. The net photosynthetic rate (Pn)
and the respiration rate (R) of the samples were measured within 3 min
of white light irradiation (70 PPFD) and 2 min of the dark adaptation,
respectively. The rate of O\textsubscript{2} evolution (P) was
calculated as P = R + Pn and expressed as a percentage before the onset
of light stress. Each measurement was conducted in triplicate.

\textbf{RNA extraction, library construction and transcriptome
sequencing}

To establish the influence of light quality on the gene expression
levels, a transcript analysis was performed. Total RNA of\emph{Z.
marina} samples exposed to BL, WL and RL for 3 h was extracted using the
TRlzol Reagent (Invitrogrn). Next, the genomic DNA in the extracted RNA
was digested using DNase I (TaKaRa Shuzo, Kyoto, Japan). The quality and
concentration of the RNA were detected by agarose gel electrophoresis
and NanoDrop ND-2000 spectrophotometer (NanoDropTechnologies),
respectively. The high-quality RNA samples (OD260/OD280 \selectlanguage{english}[?] 1.8,
OD260/OD230 \selectlanguage{english}[?] 2.0) were selected and used to construct the sequencing
library. To prepare the RNA-seq transcriptome library, mRNA was isolated
from the total RNA using oligo(dT) coupled to magnetic beads. Next, a
SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, USA)
with random hexamer primers (Illumina) was used to synthesize an
end-repaired, and phosphorylated double-stranded cDNA, following the
manufacturer's protocol. The mRNA library was sequenced using the
Illumina HiSeq xten after quantification with TBS380, generating 150bp
paired-end reads. Full RNA-seq transcriptome data were submitted to the
National Center for Biotechnology Information database under the
accession numbers: SRR18740075, SRR18740076, SRR18740077, SRR18740078,
SRR18740079, SRR18740080, SRR18740081, SRR18740082, SRR18740083,
SRR18740084, SRR18740085, SRR18740086.

\textbf{Bioinformatics analysis}

The\emph{Z. marina} genome v.3.1 (Ma et al., 2021) was used as the
reference genome for bioinformatics analysis. Clean reads were mapped to
the reference genome using Bowtie2 v2.4.1. The gene expression levels
expressed as fragments per kilobase of exon model per million mapped
fragments (FPKM) were calculated using Expectation-Maximization (RSEM)
v1.3.1. Differentially expressed genes (DEGs) between treatments were
analyzed using the DESeq2 R package. To improve the accuracy of
identifying, we investigated the significant DEGs (fold change
\textgreater{}= 2.00 and\emph{P} -value \textless{} 0.05). Next, the
gene annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG)
analysis were performed to identify the biological pathways regulated by
the significant DEGs. The biological pathways with \emph{P} -value
\textless{} 0.05 were considered significantly enriched. The HeatMap
function in Tbtools was used for the visual analysis of the DEGs.

\textbf{Totalcarotenoids andanthocyanins content analysis}

The total carotenoid content was measured using a Plant Carotenoid
Content Assay Kit (BC4330, Solarbio, Beijing, China), based on the
spectral absorption properties of carotenoids, chlorophyll a and
chlorophyll b. The total anthocyanins content was measured as previously
described by Neff and Chory (1998) with some modifications. The samples
were dried at -60degC for 48 h using a freeze dryer (Alpha1-2LDplus,
Martin Christ, Osterode, Germany) and then ground into powder. Next,
approximately 0.01g per sample was put into a 2 mL Eppendorf tube, and 1
mL of methanol acidified with 1\% HCL was added. The tubes were
incubated overnight in dark at 4 \selectlanguage{english} until the green color of the leaf
tissue disappeared. Anthocyanins were separated from chlorophylls by
back-extraction with 400 \selectlanguage{greek}µ\selectlanguage{english}L chloroform and 400 \selectlanguage{greek}µ\selectlanguage{english}L ddH\textsubscript{2}O,
then centrifuging the tube for 5 min at 6000 r/min and measuring the
absorbance of the supernatant using a TU-1810 spectrophotometer (Beijing
Purkinje General Instrument Co., Ltd., China). The content of
anthocyanins was calculated as
OD\textsubscript{530nm}-0.25OD\textsubscript{657nm} to account for any
interference from chlorophylls. Finally, the total anthocyanin content
was calculated as cyanidin-3-glucoside using 29600 (E 1 cm 1\%) as the
extinction coefficient and 449.2 as the molecular weight.

\textbf{Liquid chromatography with tandem mass spectrometry}

The differences in anthocyanin acylation modification were detected by
liquid chromatography with tandem mass spectrometry (LC-MS/MS). The
lyophilisation of samples and analysis of extracts were all performed by
MetWare Biotechnology Ltd. (Wuhan, China). The detailed protocol is
presented in Methods S1.

\textbf{Data analysis}

All statistical analyses were performed with the SPSS 22.0 statistical
package (IBM Corp, Armonk, NY, USA). All parameters were analyzed using
one-way ANOVA and post hoc comparisons were performed using the tukey
trend test.

\textbf{Results}

\textbf{OEC inactivation spectrum}

The standardized OJIP curves showed K points at 0.3 ms after a short
period of stress at different light qualities (Fig. 1A), implying that
OEC damage on the donor side of PSII was triggered at all light
qualities. To further understand the photoinactivation of OEC,
calculation of fluorescence at point K in a standardized manner revealed
that\emph{W} \textsubscript{K} was gradually increased with an increase
in wavelength, with the greatest increase following UV and BL exposure
(Fig. 1B).

\textbf{3.2} \textbf{Physiological and biochemical responses to light
quality}

The amplitude of the standardized OJIP curve \selectlanguage{greek}Δ\selectlanguage{english}\emph{V} t was different
across the three light qualities (Fig. 2A), implying that light quality
significantly affected PSII performance. In addition, the decrease in
\emph{F} v/\emph{F} m was light quality dependent, with the greatest
decrease observed upon exposure to BL (Fig. 2B). Furthermore, a
significant decrease in OEC stability was observed under BL and was
characterized by a substantial reduction in OEC peripheral protection
proteins, including PsbO, PsbP, and PsbQ (Fig. 2C). Overall, BL induced
the inactivation of a most active OEC, evidenced by the changes in the
physiological parameter, OEC\textsubscript{centers} (Fig. 2D).

\textbf{3.3} \textbf{Photosynthetic O\textsubscript{2}evolution}

The complete measurement process of the O\textsubscript{2} evolution
rate was shown in Fig. 3A. The rate of O\textsubscript{2} evolution
(overall photosynthetic performance) was gradually decreased with
exposure time, with the greatest decrease occurring following BL
exposure (3B). Moreover, the O\textsubscript{2} content change curves
after 3 h of irradiation revealed that Pn and R were significantly
decreased following BL exposure, implying the severe impairment of the
overall leaf function (Fig. 3C).

\textbf{3.4 Overview oftranscriptome analysis}

A total of 129.42 GB of high-quality sequencing data with Q30 exceeding
92.53\% were obtained after filtering the raw sequencing data (Table 1).
Clean reads were mapped to the reference genome at a mapping rate higher
than 92.13\% (Table 2), implying a high level of gene expression in all
treatment groups. The principal component analysis revealed that the
four treatment groups were distributed in the different regions of the
three-dimensional space, hence, they could be distinguished based on the
light quality (Fig. 4).

\textbf{3.5 Functional enrichment analysis}

The KEGG analysis revealed that ``Photosynthesis - antenna proteins'',
``MAPK signaling pathway - plant'', ``Glycine, serine and threonine
metabolism'', ``Plant hormone signal transduction'' and some other
pathways were significantly enriched following exposure to BL, WL, and
RL (Fig. 5A-C). However, ``Carotenoid biosynthesis'' pathway was only
enriched in BL, while ``Flavonoid biosynthesis'' pathway was minimally
enriched in BL compared to WL and RL (Fig. 5A-C). Furthermore,
``Anthocyanin biosynthesis'' pathway was only enriched in RL, although
it was not significant (Fig. S1).

\textbf{3.6 Expression of related synthetic genes and carotenoids
content}

Following BL exposure, nine DEGs were mapped to the ''Carotenoid
biosynthesis'' pathway. The genes encoding Car synthesis,
including\emph{PSY} , were significantly upregulated following BL
exposure, implying that BL effectively promoted Car accumulation (Fig.
6A). Indeed, Car content was significantly increased with BL exposure,
consistent with the signaling pathway enrichment analysis and gene
expression.

\textbf{3.7 Expression of related synthetic genes and anthocyanin
content}

The DEGs associated with anthocyanins synthesis identified by pathway
enrichment assignments were less upregulated following BL exposure than
WL and RL (Fig. 7A). Specially, the genes encoding phenylalanine
ammonia-lyase (PAL), a rate-limiting enzyme during polyphenol synthesis,
and chalcone synthase (CHS), a key enzyme during flavonoid synthesis,
were lowly expressed under BL exposure (Fig. 7A), implying a limited
ability to induce anthocyanin synthesis. Furthermore, the changes in
anthocyanins content during the exposure period confirmed the
inefficiency of BL-induced anthocyanin synthesis. Although there was a
gradual increase in anthocyanins content with exposure time across the
three light qualities, it was consistently the lowest under BL exposure
(Fig. 7B).

\textbf{3.8} \textbf{Acylated anthocyanins}

A total of ten anthocyanins modified by acylation, including seven
aromatic acyl-substituted, were detected in\emph{Z. marina} following
light exposure. The lowest level of aromatic acyl-substituted
anthocyanins, particularly the
delphinidin-3-O-(6-O-p-coumaroyl)-glucoside and
pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside, which may have a good
light absorption ability due to the aromatic acyl-modification, was
detected following BL exposure (Table 3).

\textbf{Discussion}

In the present work, the OEC dysfunction \emph{in vivo} was spectral
dependent, with a progressive increase in impairment with increasing
light wavelength. The effects of light quality on OEC function and
stability, overall photosynthetic performance, and gene expression at
three typical light qualities, including composite WL and two critical
lights in the visible range (BL and RL), were characterized to
understand the spectral dependence of \emph{Z. marina} . Based on
chlorophyll fluorescence, protein expression, and photosynthetic
O\textsubscript{2} evolution analyses, BL is the key light quality
inducing OEC inactivation and reducing the photosynthetic rate.
Transcriptome analysis further revealed that compounds with
light-screening functions are involved in the \emph{Z. marina} responses
to light. Specifically, pathways associated with Car and phenolic
compounds synthesis following light exposure are enriched. Considering
the UV wavelengths are mostly attenuated in marine ecosystems (Olsen et
al., 2016), this study focused on the light-shielding substances (Car
and anthocyanins) with absorption peaks in the visible region.
Interestingly, ''Carotenoid biosynthesis'' and ''Anthocyanin
biosynthesis'' pathways were enriched in BL and RL, respectively,
implying varying light-screening capacity in response to different light
qualities.

To clarify the differences in photoprotection functions, the expression
of relevant synthetic genes and the content of Car and anthocyanins were
analyzed. Although the content of Car was significantly increased
following BL exposure, the level of OEC inactivation in BL was still
more severe than WL and RL, implying that Car were not the major
photoprotector in\emph{Z. marina} . On the other hand, anthocyanin is
another substance playing an important light-shielding role in the
visible region (Landi et al., 2015), which demonstrated a synthetic
regulation consistent with photosynthetic properties. The key genes
associated with anthocyanin synthesis as well as the anthocyanin content
were upregulated and increased following light exposure, respectively.
Furthermore, the degree of anthocyanin accumulation varied with light
quality, implying photoreceptors regulated the process. However, unlike
the significant induction of anthocyanin accumulation by exposure to BL
in most plants (Kondo et al., 2014; Zoratti et al., 2014; Tao et al.,
2018; Zhang et al., 2018), anthocyanin synthesis in \emph{Z. marina} was
inefficient. Plant responses to BL are mainly regulated by CRY1, and
CRY2 receptors, which regulate the synthesis of secondary metabolites,
such as anthocyanins (Ahmad et al., 1995), apart from mediating the
hypocotyl elongation and flowering (Jenkins et al., 2001).\emph{Zostera
marina} has a special evolutionary process, achieving the most severe
habitat shift among the flowering plants, and experiencing the extensive
losses of photoreceptors, including CRY2 (Olsen et al., 2016).
Therefore, the inefficiency of BL-induced anthocyanin synthesis in
\emph{Z. marina} may be due to the absence of the CRY2 photoreceptor.
This is consistent with the findings on Arabidopsis mutants, where the
absence of the CRY2 receptor reduced anthocyanin accumulation (Li et
al., 2013). At the same time, the massive synthesis of anthocyanins
under RL compared to BL and WL, can be attributed to the presence of
PHYA and PHYB receptors. PHYA, and PHYB are major photoreceptors under
RL, which can control the expression of the genes regulating anthocyanin
synthesis (Yanovsky et al., 1998). Besides, PHYC, PHYD, and PHYE
receptors associated with the photoperiodic regulation of flowering in
most plants were absent in\emph{Z. marina} (Olsen et al., 2016),
implying they do not have a significant effect on \emph{Z. marina}
flowering that is more temperature controlled.

More than 500 anthocyanins with specific chemical structures synthesized
by plants have been identified. Their main differences are in the degree
of hydroxylation of the anthocyanin chromophore and the modifications
added to the backbone (Andersen and Markham, 2006; Kovinich et al.,
2014). Modification in the acyl groups, especially aromatic acyl,
increases the stability and light absorption ability of anthocyanins
compared to non-acylated modifications (Stintzing and Carle, 2004; Luo
et al., 2007; Yonekura-Sakakibara et al., 2008). Coumaric acid, caffeic
acid, sinapic acid, ferulic acid, and p-hydroxybenzoic acid are the main
aromatic acids involved in aromatic acyl substitution
(Yonekura-Sakakibara et al., 2009). Besides, modifications on multiple
aromatic acyl groups improve the anthocyanin properties (Kovinich et
al., 2015), thus playing an important role in photoprotection. However,
unlike the multiple acylation modifications (i.e., polyacylation) of
anthocyanins in wild-type Arabidopsis (Luo et al., 2007;
Yonekura-Sakakibara et al., 2012), only one acylation substitution
occurs in \emph{Z. marina} anthocyanins. Furthermore, the light
shielding efficiency in BL could be worse than WL and RL, due to the
lower content of aromatic acyl-substituted anthocyanins in BL,
especially delphinidin-3-O-(6-O-p-coumaroyl)-glucoside and
pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside.

The acylation modification of anthocyanins is catalyzed by anthocyanin
acyltransferases, whose activity is mainly regulated by phosphorylation
and gene transcription (Fan et al., 2008). As a signal transducer,
photoreceptors can induce the binding of transcription factor MYB to
acyltransferase promoter, facilitating gene expression (Rinaldo et al.,
2015). Our transcriptome data revealed differences in the expression of
the transcription factor MYB and acyltransferases under different light
qualities (Fig. S2), implying that the absence of photoreceptors in
\emph{Z. marina} may affects the acylation modification of anthocyanins
through gene expression. Since anthocyanins modification is not
regulated by all MYB transcription factors (Tamagnone et al., 1998; Du
et al., 2009), and anthocyanin acyltransferases are anthocyanin-specific
and acyl receptor-specific (Fan et al., 2008), the regulation of
anthocyanin acylation in \emph{Z. marina} requires further
investigations.

\textbf{Conclusion}

The absence of CRY2 photoreceptor in\emph{Z. marina} led to the
insufficient synthesis of anthocyanins and reduced the level of aromatic
acylated modification of anthocyanins. Thus, excessive
photosynthetically effective radiation, especially the high-energy
blue-green light enriched in marine ecosystems, was not effectively
shielded, leading to OEC inactivation. Irreversible photoinactivation of
OEC in harsh environments limits \emph{Z. marina} growth leading to its
population decline in the long run. Therefore, the absence of
photoreceptors is an important intrinsic factor for seagrass
degradation. The findings in this study provide a new insight for
seagrasses restoration by modifying the photoreceptors through the
gene-editing technology to enhance their resilience to environmental
changes, thus, slowing down their population decline.

\textbf{Acknowledgements}

This work was supported by the National Natural Science Foundation of
China (No. 41376154) and the Yantai Municipal Key Research and
Development Project (No. 2019XDHZ096).

\textbf{Conflict of Interest}

The authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a
potential conflict of interest.

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Table 1. Summary of the raw sequencing data\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Sample\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Raw reds\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Raw bases\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Clean reads\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Clean bases\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Error rate (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
Q30 (\%)\strut
\end{minipage} & \begin{minipage}[t]{0.12\columnwidth}\raggedright\strut
GC (\%)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Control 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
51696844\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7806223444\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
51248218\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7654100841\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0254\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.7\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.89\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Control 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
43195248\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6522482448\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
42884144\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6396532081\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0247\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
94.34\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.91\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Control 3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
53426210\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8067357710\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
52988854\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7916574395\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0256\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.52\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.55\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Blue 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
47966530\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7242946030\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
47571454\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7087546538\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0259\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.25\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.55\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Blue 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
46431768\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7011196968\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
46022620\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6853807472\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0259\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.15\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.34\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Blue 3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
46629886\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7041112786\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
46209142\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6869230840\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.026\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.07\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.54\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
White 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
47783712\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7215340512\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
47304796\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7048592380\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0266\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
92.53\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.5\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
White 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
48806370\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7369761870\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
48448086\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7234007292\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0253\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.81\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.84\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
White 3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
42486188\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6415414388\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
42179286\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6296228913\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0252\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.87\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.73\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Red 1\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
55690780\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8409307780\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
55279256\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8264478362\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0254\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
93.66\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.57\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Red 2\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
42774264\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6458913864\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
42383990\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
6336610478\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0263\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
92.85\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.53\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Red 3\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
53363724\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
8057922324\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
53060834\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
7781592173\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.0227\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
96.52\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
45.34\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Q30 refers to the percentage of bases with sequencing quality above
99.9\% in total bases.

Table 2. Summary of the transcriptome sequence alignment\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Sample & Total reads & Total mapped & Multiple mapped & Uniquely
mapped\tabularnewline
Control 1 & 51248218 & 47669935 (93.02\%) & 3540284 (6.91\%) & 44129651
(86.11\%)\tabularnewline
Control 2 & 42884144 & 39508764 (92.13\%) & 2835227 (6.61\%) & 36673537
(85.52\%)\tabularnewline
Control 3 & 52988854 & 49921524 (94.21\%) & 3307605 (6.24\%) & 46613919
(87.97\%)\tabularnewline
Blue 1 & 47571454 & 44874551 (94.33\%) & 2337446 (4.91\%) & 42537105
(89.42\%)\tabularnewline
Blue 2 & 46022620 & 43045726 (93.53\%) & 2256231 (4.9\%) & 40789495
(88.63\%)\tabularnewline
Blue 3 & 46209142 & 43708260 (94.59\%) & 2148207 (4.65\%) & 41560053
(89.94\%)\tabularnewline
White 1 & 47304796 & 44443715 (93.95\%) & 2421034 (5.12\%) & 42022681
(88.83\%)\tabularnewline
White 2 & 48448086 & 45367323 (93.64\%) & 2510172 (5.18\%) & 42857151
(88.46\%)\tabularnewline
White 3 & 42179286 & 39200137 (92.94\%) & 2207280 (5.23\%) & 36992857
(87.7\%)\tabularnewline
Red 1 & 55279256 & 51774839 (93.66\%) & 2963989 (5.36\%) & 48810850
(88.3\%)\tabularnewline
Red 2 & 42383990 & 39667109 (93.59\%) & 2402614 (5.67\%) & 37264495
(87.92\%)\tabularnewline
Red 3 & 53060834 & 50230425 (94.67\%) & 2573786 (4.85\%) & 47656639
(89.82\%)\tabularnewline
\bottomrule
\end{longtable}

Table 3. Content of acylated anthocyanins\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& & Anthocyanin content (\selectlanguage{greek}μ\selectlanguage{english}g/g) & Anthocyanin content (\selectlanguage{greek}μ\selectlanguage{english}g/g) &
Anthocyanin content (\selectlanguage{greek}μ\selectlanguage{english}g/g)\tabularnewline
& Compounds & BL & WL & RL\tabularnewline
aliphatic acyl-substituted &
Delphinidin-3-O-(6-O-malonyl-beta-D-glucoside) & 0.172\selectlanguage{ngerman}±0.02 &
0.164\selectlanguage{ngerman}±0.001 & 0.153\selectlanguage{ngerman}±0.03\tabularnewline
& Delphinidin-3-O-(6-O-malonyl)-glucoside-3'-glucoside & 0.004\selectlanguage{ngerman}±0.001 &
0.006\selectlanguage{ngerman}±0.001 & 0.006\selectlanguage{ngerman}±0.002\tabularnewline
& Pelargonidin-3-O-(6-O-malonyl-beta-D-glucoside) & 0.173\selectlanguage{ngerman}±0.03 &
0.033\selectlanguage{ngerman}±0.03 & 0.101\selectlanguage{ngerman}±0.09\tabularnewline
& Total amount & 0.349 & 0.203 & 0.260\tabularnewline
aromatic acyl-substituted & Cyanidin-3-(6-O-p-caffeoyl)-glucoside &
0.417\selectlanguage{ngerman}±0.02 & 0.400\selectlanguage{ngerman}±0.07 & 0.603\selectlanguage{ngerman}±0.23\tabularnewline
& Cyanidin-3-O-(6-O-p-coumaroyl)-glucoside & 0.015\selectlanguage{ngerman}±0.01 & 0.017\selectlanguage{ngerman}±0.01 &
0.013\selectlanguage{ngerman}±0.01\tabularnewline
& Delphinidin-3-O-(6-O-p-coumaroyl)-glucoside & 0 & 0.013\selectlanguage{ngerman}±0.002 &
0.015\selectlanguage{ngerman}±0.005\tabularnewline
& Malvidin-3-O-(6-O-p-coumaroyl)-glucoside & 0.013\selectlanguage{ngerman}±0.01 & 0.037\selectlanguage{ngerman}±0.01 &
0.228\selectlanguage{ngerman}±0.1\tabularnewline
& Malvidin-3-O-5-O-(6-O-coumaroyl)-diglucoside & 0.110\selectlanguage{ngerman}±0.004 &
0.166\selectlanguage{ngerman}±0.08 & 0.150\selectlanguage{ngerman}±0.008\tabularnewline
& Pelargonidin-3-O-(6-O-p-coumaroyl)-glucoside & 0 & 0.006\selectlanguage{ngerman}±0.005 &
0.003\selectlanguage{ngerman}±0.005\tabularnewline
& Peonidin-3-O-(6-O-p-coumaroyl)-glucoside & 0.02\selectlanguage{ngerman}±0.003 & 0.023\selectlanguage{ngerman}±0.005 &
0.048\selectlanguage{ngerman}±0.011\tabularnewline
& Total amount & 0.575 & 0.662 & 1.06\tabularnewline
\bottomrule
\end{longtable}

Figure 1. (A) Changes in O-J phase of normalized chlorophyll a
fluorescence intensity in response to 15 min of different light
qualities exposure, respectively. (B) Action spectrum of OEC
photoinhibition. The inset shows the changes in \emph{W} k following WL
exposure. The means \selectlanguage{ngerman}± SD are calculated from three independent samples.

Figure 2. (A) Changes in normalized chlorophyll a fluorescence intensity
of \selectlanguage{greek}Δ\selectlanguage{english}\emph{V} \textsubscript{t} in response to 3 h of BL (blue circles),
WL (light gray triangles) and RL (red inverted triangles) exposure,
respectively. Each curve represents the average of three replicates. (B)
Variations in \emph{F} \textsubscript{v}/\emph{F} \textsubscript{m} in
response to BL, WL and RL exposure, respectively. (C) Changes in three
peripheral proteins of OEC are analyzed by Western blot. The
significantly different value from dark (Tukey's tests,
\emph{p}\textless{} 0.05) is marked with an asterisk (*). (D) Variations
in OEC\textsubscript{centers} in response to BL, WL and RL exposure,
respectively. The means \selectlanguage{ngerman}± SD are calculated from three independent
samples. Different letters indicate a significant difference
(\emph{p}\textless{} 0.05, one-way ANOVA).

Figure 3. (A) The typical O\textsubscript{2} content change curve in the
reaction chamber of the Liquid-Phase Oxygen Electrode System for
monitoring the O\textsubscript{2} evolution rate. (B) Time course of
changes in the O\textsubscript{2} evolution rate in response to 3 h of
BL, WL and RL exposure, respectively. The means ± SD are calculated from
three independent samples. (C) Changes in the oxygen content change
curve during the O\textsubscript{2} evolution rate monitoring after 3 h
of BL, WL and RL exposure, respectively. The rising and falling slope of
the curve under light and darkness represent the net photosynthetic rate
and respiration rate, respectively.

Figure 4. Principal component analysis (PCA) of identified genes. Light
blue squares represent the samples of control, whereas red circles,
green triangles, and dark blue inverted triangles indicate samples
treated by BL, RL, and WL, respectively. Different spot of each color
represents the biological replicates for this treatment.

Figure 5. (A-C) Enriched KEGG pathways among the DEGs in response to the
BL, WL, and RL treatments, respectively. Vertical axis shows the pathway
names, whereas the horizontal axis shows the enrichment factor. The size
of each spot reflects the number of DEGs in this pathway, and each spot
color corresponds to the different \emph{P} -value ranges. Pathways
with\emph{P} -values \textless{} 0.05 are significantly enriched.

Figure 6. (A) Expression profiles of the DEGs related to the carotenoid
biosynthesis pathway. (B) Time course of changes in the carotenoid
contents in response to 3 h of BL, WL and RL exposure. The means ± SD
are calculated from three independent samples. Different letters
indicate a significant difference (\emph{p} \textless{} 0.05, one-way
ANOVA).

Figure 7. (A) Expression profiles of the DEGs related to the
anthocyanins biosynthesis. (B) Time course of changes in the anthocyanin
contents in response to 3 h of BL, WL and RL exposure. The means ± SD
are calculated from three independent samples. Different letters
indicate a significant difference (\emph{p} \textless{} 0.05, one-way
ANOVA).

\textbf{Hosted file}

\verb`Figure.docx`  available at \url{https://authorea.com/users/342402/articles/567809-insufficient-synthesis-and-acylation-modification-of-anthocyanins-causes-photoinactivation-of-the-oxygen-evolving-complex-in-zostera-marina}

\selectlanguage{english}
\FloatBarrier
\end{document}

