Abstract
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 inZ. 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
O2 release
capacity.
The
increased synthesis of carotenoids under blue light with severe OEC
damage implied its weak
photoprotection property in 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 Z.
marina causes the insufficient synthesis of anthocyanins and acyl
modifications, reducing the shielding against high-energy light,
subsequently causing OEC photoinactivation.
Keywords : Anthocyanin; Light
screening;
Photoreceptor;
Oxygen-evolving complex;Zostera marina .
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) ofZostera marina and 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, Z. marina,whose complete genome has been sequenced, was used as the research
object (Olsen et al., 2016; Ma et al., 2021).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 Z. marinapotentially restricts the synthesis of light-shielded compounds that
allow high-energy light to reach the
chloroplasts easily, causing OEC
photoinactivation.
To verify this hypothesis, 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.
Materials and methods
Sample preparations and
treatments
Zostera
marina plants with intact rhizomes-systems and fresh leaves were
collected
from the seagrass beds in Rongcheng
(37º 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 O2 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.
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:
ΔV t = Δ
[(F t- F O)/
(F m - F O)];
the maximal quantum yield of the
PSII by F v/F m =
(F m -
F o)/ F m;
the degree of damage on the donor
side of PSII by W K = (F K- F O)/ (F J -
F O), and the
active fraction of OEC centers by OECcenters = [1- (V K/V J)]treatment/ [1 -(V K/V J)]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.
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
µg 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 µg chlorophyll.
Next, a Western blot assay with
antibodies against PsbO, PsbP and PsbQ (Agrisera, Vännäa, 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.
Oxygen evolution rate measurements
The
rate of photosynthetic
O2 evolution was determined using a
liquid-phase oxygen electrode
system (Chlorolab2+; Hansatech, Hercules, UK) to evaluate
the overall photosynthetic
performance. Leaf fragments
(~ 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
O2 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.
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 ofZ. 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 ≥ 1.8, OD260/OD230 ≥ 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.
Bioinformatics analysis
TheZ. 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 >= 2.00 andP -value < 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 P -value < 0.05 were considered
significantly enriched. The HeatMap
function in Tbtools was used for the visual analysis of the DEGs.
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 -60°C 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 ℃ until the green color
of the leaf tissue disappeared.
Anthocyanins were separated from
chlorophylls by back-extraction with 400
µL chloroform and 400 µL
ddH2O, 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
OD530nm-0.25OD657nm 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.
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.
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.
Results
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 thatW K was
gradually increased with an increase in wavelength, with the greatest
increase following UV and BL exposure (Fig. 1B).
3.2 Physiological
and biochemical responses to light quality
The
amplitude of the standardized OJIP curve ΔV t was different across
the three light qualities (Fig. 2A),
implying that light quality significantly affected PSII performance.
In
addition,
the
decrease in F v/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, OECcenters (Fig.
2D).
3.3 Photosynthetic
O2evolution
The
complete measurement process of the O2 evolution rate
was shown in Fig. 3A. The rate of O2 evolution
(overall photosynthetic
performance) was gradually decreased with exposure time, with the
greatest decrease occurring following BL exposure (3B). Moreover, the
O2 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).
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).