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.