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).