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).
3.8 Acylated
anthocyanins
A
total of ten anthocyanins modified by acylation, including seven
aromatic acyl-substituted, were detected inZ. 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).
Discussion
In
the present work, the OEC dysfunction 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 Z. marina .
Based on chlorophyll fluorescence,
protein expression, and photosynthetic O2 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 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 inZ. 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 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).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 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 inZ. marina (Olsen et al.,
2016), implying they do not have a significant effect on 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 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 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 Z. marina requires further investigations.
Conclusion
The
absence of CRY2 photoreceptor inZ. 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 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.