Molecular Response of Ulva prolifera to Short-Term High Light Stress Revealed by a Multi-Omics Approach

Simple Summary High light stress is one of the main factors affecting the normal growth of Ulva prolifera. The response mechanism of U. prolifera to 12 h of high light stress was explored by the multi-omics method. We found that short-term high light could inhibit the assimilation process of U. prolifera, destroy the cellular structure, and inhibit respiration. Moreover, it was raised by the genes associated with photosynthetic pigment synthesis, optical system I, and electronic transport, and may be able to make up the ATP defects by circulating electronic transport. At the same time, it reduced NADPH production by attenuating photosystem II synthesis. The carbon fixed approach was also transformed from the C3 pathway to the C4 pathway. Revealing the response mechanism of U. prolifera to high light can provide a more theoretical basis for studying the outbreak of green tide of U. prolifera in the Yellow Sea. Abstract The main algal species of Ulva prolifera green tide in the coastal areas of China are four species, but after reaching the coast of Qingdao, U. prolifera becomes the dominant species, where the light intensity is one of the most important influencing factors. In order to explore the effects of short-term high light stress on the internal molecular level of cells and its coping mechanism, the transcriptome, proteome, metabolome, and lipid data of U. prolifera were collected. The algae were cultivated in high light environment conditions (400 μmol·m−2·s−1) for 12 h and measured, and the data with greater relative difference (p < 0.05) were selected, then analyzed with the KEGG pathway. The results showed that the high light stress inhibited the assimilation of U. prolifera, destroyed the cell structure, and arrested its growth and development. Cells entered the emergency defense state, the TCA cycle was weakened, and the energy consumption processes such as DNA activation, RNA transcription, protein synthesis and degradation, and lipid alienation were inhibited. A gradual increase in the proportion of the C4 pathway was recorded. This study showed that U. prolifera can reduce the reactive oxygen species produced by high light stress, inhibit respiration, and reduce the generation of NADPH. At the same time, the C3 pathway began to change to the C4 pathway which consumed more energy. Moreover, this research provides the basis for the study of algae coping with high light stress.


Introduction
From 2007 to 2020, large-scale green tide disasters occurred in the Yellow Sea in China [1], which severely affected the ecosystem and the lives of coastal residents in the sea area. Green tides are ecological anomaly caused by a sharp increase in green algae's biomass, and U. prolifera has always been a species in the outbreak of green tide algae in the Yellow Sea [2]. U. prolifera (Ulvales, Chlorophyta [1,3]) has strong resistance to the U. prolifera samples were collected from Qingdao waters (120 • 19 E, 36 • 04 N) in July 2008, and gametophytes' pure-line progeny were obtained through sterile subculturing in the laboratory. In this experiment, samples of U. prolifera gametophytes were subcultured in VSE medium at 20 • C, a light intensity of 120 µmol·m −2 ·s −1 , and light period/dark period = 12:12 h. After 15 days of cultivation, the algae with healthy growth and similar morphologies were taken. The experiment was divided into the high light intensity treatment (400 µmol·m −2 ·s −1 ) and the control group (120 µmol·m −2 ·s −1 ), with other conditions unchanged. After the two groups were cultured for 12 h, the algae were taken out immediately. After liquid nitrogen treatment and ultra-low-temperature freeze-drying, omics tests were performed separately. The experiment set up biological replicates, where the transcriptome and proteome had three replicates per group, and the metabolome and lipidome had six per group. Each of the above replicates contained ten fronds.

Transcriptomics Procedure
In the transcriptome experiment, the total RNA from U. prolifera samples was accurately quantified after extracting. mRNA capture and fragmentation were performed. After the first strand was synthesized, double-strand cDNA synthesis was performed. Subsequently, the library was amplified with quality testing, and the obtained cDNA library was subjected to high-throughput sequencing on Illumina Hiseq TM (Illumina Inc., San Diego, CA, USA). Then, fast quality control quality evaluation was performed on the original sequencing data, and the quality was cut by Trimmomatic to obtain relatively accurate and valid data [14]. Finally, gene annotation, RNA-seq sequencing evaluation, gene-structure analysis, expression-level analysis, expression-variation analysis, and gene-enrichment analysis were carried out [15].

Quantitative PCR Assay
The total RNA of U. prolifera was extracted from each group followed by reversetranscription into cDNA using the Fast-King cDNA first strand synthesis kit (Tiangen, Beijing, China). Then nine genes selected from the transcriptome were applied for qRT-PCR, where 18S rRNA was taken as the internal reference [12]. The target gene primers were designed using NCBI database online tool "Primer-BLAST" (Table S1), and Tiangen's Talent fluorescence quantitative detection kit (SYBR Green) was used for the qPCR experiment, with the formulate as follows: 2 × Talent qPCR PreMix 12.5 µL, positive and negative primers 0.75 µL, cDNA template 1 µL, RNAase-free ddH 2 O 10µL. The reaction system was placed in the FTC-2000 PCR instrument, with the program setting as follows: 3 min pre-denaturation at 95 • C, 40 times of recycles including 95 • C for 30 s, 60 • C for 30 s, and 72 • C for 30 s. All samples had four repeats, and gene differential expression was calculated by 2 −∆∆CT [16].

Proteomics Procedure
The samples were ground by liquid nitrogen and precipitated by TCA/acetone, then an appropriate amount of SDT lysate was added, respectively. The samples were bathed in boiling water for 15 min, then treated with ultrasonic treatment and centrifuged at 12,000× g. After the supernatant was collected, the protein was quantified by the BCA method [17], and the filtrate was collected by the FASP enzymatic hydrolysis method [18]. The peptides were desalted by the C18 Cartridge, then lyophilized and redissolved with 40 µL 0.1% formic acid solution. The peptides were quantified (OD 280 ). High performance liquid chromatography was used to separate each sample using the HPLC liquid phase system easy NLC with nanositre flow rate. After chromatographic separation, Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) was used for mass spectrometry analysis. The mass charge ratio of polypeptides and fragments was collected as follows: after each full scan, 20 fragments were collected (MS2 scan). MS2 activation type was HCD, isolation window was 2 m/z, secondary mass spectral resolution was 17,500 at 200 m/z, normalized collision energy was 30 eV, and underfill was 0.1%.

Metabolomics Procedure
The sample was quantitatively weighed for liquid nitrogen grinding, dissolved in methanol acetonitrile aqueous solution (v/v, 2:2:1), centrifuged at 14,000× g at 4 • C for 20 min, and then the supernatant was taken. The supernatant was then redissolved in acetonitrile aqueous solution (acetonitrile: water =1:1, v/v) for mass spectrometry. The supernatant was taken for sample analysis after centrifugation at 14,000× g at 4 • C for 15 min. The samples were separated on an Agilent 1290 Infinity LC ultra-performance liquid chromatography (UHPLC) HILIC column. The samples were separated by UHPLC and analyzed by Triple TOF 6600 mass spectrometers (AB SCIEX, Boston, MA, USA). The obtained original data were converted into the MZML format by Proteo Wizard (Palo Alto, CA, USA), and then the XCMS program was used for peak alignment, retention time correction, and peak area extraction. Accurate mass number matching (<25 PPM) Biology 2022, 11, 1563 4 of 22 and secondary spectral matching were used for metabolite structure identification, and a database built by the laboratory was retrieved. The integrity of the data extracted by XCMS was checked. The metabolites with missing values of more than 50% in the group were removed and did participate in the subsequent analysis. The extreme values were deleted, and the total peak area was normalized for the data to ensure the parallelism of comparison between the samples and metabolites. After being processed, the data were input into the software SIMCA-P 14.1 (Umetrics, Umea, Sweden) for pattern recognition. After the data were preprocessed by pareto-scaling, multi-dimensional statistical analysis was conducted, including unsupervised principal component analysis (PCA), partial least squares discriminant (PLS-DA) and orthogonal partial least-squares discriminant (OPLS-DA) analysis. One-dimensional statistical analysis included student's t-test and multiple of variation analysis, and volcano maps were drawn by R software (R Foundation for Statistical Computing, Vienna, Austria).

Lipidomics Procedure
After centrifugation at low temperature and high speed, the upper organic phase was taken, and the ammonia gas was blown dry. Isopropanol solution was added for resolution during mass spectrometry analysis. The samples were centrifuged for 15 min at 14,000× g under 10 • C in the vortex, and the supernatant was taken for sample analysis. The samples were separated by Nexera UHPLC LC-30A ultra performance liquid chromatography (Shimadzu Technologies, Kyoto, Japan). Electrospray ionization (ESI) positive and negative ion modes were used for detection, respectively. The samples were separated by UHPLC and analyzed by mass spectrometry with Q exactive plus mass spectrometer (Thermo Scientific, New York, NY, USA). Peak and lipid identification (secondary identification), peak extraction, peak alignment, and quantification were performed by lipaid search software version 4.1 (Thermo Scientific, New York, NY, USA). In the extracted data, lipid molecules of RSD > 30% were deleted. For the data extracted by lipaid search, lipid molecules with missing values > 50% in the group were deleted, and the total peak area was normalized for the data. SimCA-P 14.1 (Umetics, Umea, Sweden) was used for pattern recognition. After the data were preprocessed by Pareto-scaling, multi-dimensional statistical analysis was conducted, including unsupervised PCA, PLS-DA, and OPLS-DA analysis. One-dimensional statistical analysis included student's t-test and multiple of variation analysis, and R software drew volcano maps, hierarchical clustering analysis, and correlation analysis.

Basic Data of Transcriptome Analysis
Trimmomatic processed the raw data obtained by high-throughput sequencing to obtain the clean data. The average read length of each sample was more than 142 bp, with the total read length more than 39 Mb, the base amount more than 5.5 Gb, the GC ratio greater than 59%, and the Q30 ratio between 96.29 and 96.48%. It indicated good sequencing quality (Table S2). Trinity was used to assemble the clean data into transcripts with denovo assembly and remove redundancy. By taking the longest transcript in each transcript cluster as the unigene, 28,362 unigenes were obtained, with an average length of 1406 bp., wherein the longest sequence length was 26,903 bp (Table S3). After comparison, 1579 unigenes sequences were annotated in the databases of NR, KEGG, Swiss-prot, and KOG, and the numbers of annotated genes were 8502, 1801, 7670, and 5851, respectively ( Figure S1). Compared with the control group, there were 100 genes whose expression quantities were extremely significantly upregulated (|Log 2 Fold Change (FC)| > 2, and p-value < 0.05), and 167 genes were downregulated for U. prolifera under high light intensity.

Target Gene Verification Results
The selected nine genes were verified by real-time fluorescence quantitative PCR, and the data were analyzed. As shown in Figure 1, under the condition of 12 h high light intensity, the expression trends of nine genes were similar with the transcriptomics results, indicating that the transcriptome data were relatively reliable.
value < 0.05), and 167 genes were down-regulated for U. prolifera under high light intensity.

Target Gene Verification Results
The selected nine genes were verified by real-time fluorescence quantitative PCR, and the data were analyzed. As shown in Figure 1, under the condition of 12 h high light intensity, the expression trends of nine genes were similar with the transcriptomics results, indicating that the transcriptome data were relatively reliable. Figure 1. The nine significantly differential expressed genes in U. prolifera under high light intensity (400 µmol·m −2 ·s −1 ) were verified by qRT-PCR.

Basic Data of Proteome Analysis
According to the obtained mass spectrum, the Andromeda engine integrated by Max Quant was used for identification. The filtering was completed with PSM-level FDR ≤ 1%, and filtering was performed with protein-level FDR ≤ 1%. There were 18,100 identified peptide fragments and 2226 identified proteins. The unique peptide fragment is the protein's characteristic sequence. In this experiment, there were 309 unique peptide segments with a quantity of two ( Figure S2). The obtained proteins were mostly distributed between 10-50 kDa, of which 20-30 kDa had the most distribution ( Figure S3). Max Quant was used for the quantitative analysis of each group with Welch's t-test. It showed that the two groups contained 62 different proteins (|FC| ≥ 1.5, and p < 0.05), of which 21 were up-regulated, and 41 were down-regulated.

Basic Data of Metabolome Analysis
The chromatographic peak's response intensity and retention time in the positive and negative ion mode of the QC samples in the metabolome overlapped. SIMCA-P software was used for PCA analysis to demonstrate that the parallel samples of each group were closely clustered together, which showed that the experiment had good repeatability. In the positive ion mode, 3790 ion peaks were obtained; in the negative ion mode, 3606 ion peaks were obtained. PLS-DA measured the strength of influence and interpretation of metabolites' expression patterns on the classification of samples in each group by calculating variable importance for the projection (VIP). The PLS-DA model's evaluation parameters R 2 Y = 0.997 (for positive ions) and 0.971 (for negative ions) after seven interactive verification cycles. OPLS-DA was modified based on PLS-DA to filter out noises unrelated to classified information, which improved the model's analysis and effectiveness. In this model, R 2 Y = 1 (for positive ions) and 0.999 (for negative ions). In the above two models, R 2 Y was close to one, which explained the samples' metabolic differences in the two groups. On this basis, 29 significantly different metabolites (VIP > 1 and p < 0.05) were identified through statistical analysis and screening, wherein 24 were up-regulated, and 5 were down-regulated.

Basic Data of Lipidome Analysis
The response intensity and retention time in the UHPLC-Obitrap MS BPC of QC samples in the lipidome showed that the experiment had good repeatability. The PLS-DA and OPLS-DA models' evaluation parameters R 2 Y were equal to 0.968 and 0.993, respectively, which explained the metabolic differences between the samples in the two groups. In this study, 558 lipid molecules were identified with 21 subclasses, mainly involving triglyceride (TG), ceramidesglycerol 1 (CerG1), diacylglycerol (DG), DGDG, diacylglycerol monoacylglycerol (DGMG), MGDG, monogalactosyl monoacylglycerol (MGMG), phosphatidylglycerol (PG), and sulfoquinovosyl diacylglycerol (SQDG) ( Figure S4). There were five significantly different metabolites (VIP > 1, and p < 0.05), among which one was up-regulated, and four were down-regulated (Table S4).

Photosynthesis of U. prolifera in the Conditions of High Light Stress
Through multi-omics joint analysis, it was found that some important genes related to the process of photosynthesis in U. prolifera changed significantly after 12 h of high light intensity (Table 1). Transcriptomics data showed that genes that promote chlorophyll and carotenoid synthesis were up-regulated, e.g., glutamate-1-semialdehyde 2,1-aminomutase, ABC transporter C family member 3, and photosystem II CP43 reaction center protein.
On the other hand, the expression of pheophytinase decreased. The genes involved in promoting the synthesis of photosystem I and electron transport were up-regulated, e.g., photosystem I P700 chlorophyll apoprotein A1, cytochrome b6-f complex subunit 4, and ATP synthase subunit. Meanwhile, the PSII complex-related gene expression was downregulated, e.g., photosystem II protein D2 (psbD) and aldedh domain-containing protein.
The gene expression associated with the dark reaction process in photosynthesis, e.g., carbonic anhydrase was down-regulated. The expression of genes associated with phosphoenolpyruvate synthesis was up-regulated and metabolism was down-regulated, e.g., pyruvate, phosphate dikinase, phosphoenolpyruvate/phosphate translocator 1 and phosphoenolpyruvate carboxylase 1. a FC: 1 ≤ |Log 2 FC| < 2 indicated a significant difference of gene expression (q-value (modified p value after multiple hypothesis tests) < 0.05.), while |FC | ≥ 2 indicated extremely remarkable differences of gene expression. n = 3 biologically independent experiments. b FC ≥ 1.5 indicated that protein increased significantly (p-value < 0.05), while FC ≤ 0.6 indicated a significant decrease in protein. n = 3 biologically independent experiments. c FC > 1 indicated that protein increased, while FC < 1 indicated a decrease in protein. p-value < 0.05 means significant difference, p-value < 0.01 means extremely remarkable difference. n = 6 biologically independent experiments. "+" means cation of metabolite, while "-" means anion of metabolite. In all selected metabolites above, the VIP score > 1.0. d FC > 1 indicated that protein increased, while FC < 1 indicated a decrease in protein. p-value < 0.05 means significant difference, p-value < 0.01 means an extremely remarkable difference. n = 6 biologically independent experiments. In all selected lipids above, the VIP score > 1.0. The description of "a, b, c, d" in the follow tables is the same.
Proteomics data showed that the expression of proteins related to the biosynthesis of chlorophyll was up-regulated, e.g., uroporphyrinogen, heat shock protein 90-5, chloroplastic, and ABC transporter C family member 2 and the expression of C3 pathway-related proteins was inhibited, e.g., pyridoxal 5'-phosphate synthase subunit PDX1.
The combined metabolome and lipidome data showed that the content of photosynthetic membrane involved in photosynthesis increased, e.g., DGDG. However, MGDG as an intermediate for DGDG production decreased, indicating that 12-h high light stress promoted the synthesis of the photosynthetic membranes of U. prolifera. In summary, 12 h of intense high light stress promoted the synthesis of chlorophyll and carotenoid, PSI, and electron transport subunit, and complemented ATP deficiency by coupling with cyclic electron transport. Meanwhile, it weakened PSII synthesis and acyclic photophosphorylation, reduced NADPH generation, and inhibited carbohydrate synthesis in a dark reaction. Meanwhile, a shift from the C3 to the C4 pathway started by the promotion of phosphoenolpyruvate synthesis, while inhibiting phosphoenolpyruvate transport and consumption. Furthermore, high light induced a large amount of DGDG synthesis on the photosynthetic membrane while consuming the substrate MGDG. Those might be supplemented by MGMG. It was suggested that the 12 h time point was the turning point of U. prolifera tolerant to high light.

Energy Metabolism of U. prolifera in Conditions of High Light Stress
Through multi-omics joint analysis, it was found that after 12 h of intense light stress, some important genes related to energy metabolism in U.prolifera changed significantly ( Table 2). Transcriptomics data indicated that the expression of genes related to energy metabolism was down-regulated, such as adenylate kinase 5, acyl-CoA-binding domaincontaining protein 5, and pyruvate dehydrogenase E1 component subunit α-1. The expression of genes related to redox activity was also down-regulated, e.g., protein tas, cytochrome P450 4e3, and amino oxidase domain-containing. On the other hand, the transcriptome showed that the expression of genes that are involved in glycolysis was up-regulated, e.g., endoglucanase E-4 and endoglucanase 1 and both enzymes catalyzing the endohydrolysis of 1, 4-β-glucosidic linkages in cellulose, lichenin, and cereal β-D-glucans. Meanwhile, the gene expression of 4-α-glucanotransferase DPE2, which catalyzes starch to sucrose, was also up-regulated. The proteome showed that the expression of proteins related to energy metabolism was up-regulated, e.g., R-mandelonitrile lyase-like, NADH: ubiquinone oxidoreductase 30 kDa.
The metabolome showed that the content of metabolites related to TCA increased, e.g., L-malic acid, L-asparagine, and cyclohexylamine. Meanwhile, that of diethanolamine and ribitolalso increased. However, the content of sucrose decreased.
As a whole, it was found that after short-term high light stress, sucrose content and glycolysis gradually increased, while the TCA cycle gradually weakened, including the reduction in acetyl-CoA production and transport and reduction in proton production, respiratory terminal oxidase production, GTPase synthesis, and ATP production. Thus, the overall trend of energy metabolism was down-regulated which might make U. prolifera dormant.

Transcription and Translation of U. prolifera in Conditions of High Light Stress
Through multi-omics joint analysis, it was found that some important genes related to the process of protein synthesis and expression in Ulva changed significantly after 12 h of high light intensity (Table 3). Transcriptomics data showed that genes that activate DNA were down-regulated, e.g., DOT1 domain-containing protein, ATP-dependent DNA helicase DDM1, and RuvB-like 2; as well as those in RNA transcription, e.g., AP2-like ethylene-responsive transcription factor AIL5, transcriptional activator Myb, ESF1 homolog, and transcription initiation factor TFIID subunit 5. However, genes involved in exosome-mediated RNA decay were up-regulated, e.g., tetratricopeptide repeat protein SKI3. Furthermore, genes related to protein synthesis and degradation were down-regulated, e.g., ribosome biogenesis protein BRX1 homolog, tRNA pseudouridine synthase A, and general transcription factor 3C polypeptide 5. However, Carboxypeptidase inhibitor SmCI involved in the inhibition of pancreatic carboxypeptidase was up-regulated. Nucleotide synthesis was inhibited, e.g., 5'nucleotidase, pseudouridine-5'-phosphate glycosidase, and cytosolic purine 5'-nucleotidase.  Proteomics data showed that the expression of proteins related to protein synthesis was down-regulated, e.g., pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH3, eukaryotic translation initiation factor 5A-2, and DNA-binding helix-turn helix protein.
The combined metabolome data showed that the content of some amino acid increased, e.g., L-glutamate, L-methionine, and L-glutamate.
In summary, after 12 h of intense light stress, the expression of protein synthesisrelated genes showed an overall trend of down-regulation in Ulva algae, including DNA activation, RNA transcription, protein synthesis, and degradation. Meanwhile, it also inhibited nucleotide production. This showed that 12 h of high light stress is the turning point of U. prolifera tolerant to the condition of high light stress.

Signal Transduction, Ion Transport, and Cytoskeleton Synthesis of U. prolifera in Conditions of High Light Stress
According to omics data, some important genes related to signal transduction and growth altered significantly after 12 h of high light stress (Table 4). Transcriptomics data indicated that the expression of genes related to signal transduction were down-regulated, e.g., adenylate cyclase and protein RRC1. Moreover, ABC transporter G family member 31 that suppresses radicle extension and subsequent embryonic growth was up-regulated, while GPCR-type G protein 2 that is required for seedling growth and fertility was downregulated. On the other side, the expression of genes related to ion transport was also down-regulated, e.g., potassium/sodium hyperpolarization-activated cyclic nucleotidegated channel 2, sodium/calcium exchanger 3, and sodium-and chloride-dependent GABA transporter 2. Many genes on cytoskeleton synthesis were down-regulated, e.g., kinesinlike protein KIN-4A, tubulin glycylase 3A, and protein tilB homolog, while several of them were up-regulated, e.g., tubulin polyglutamylase TTLL4, dynein assembly factor 5, and flagellar associated protein.
Proteomics data showed that the expression of proteins involved in signal transduction was down-regulated, e.g., inositol, and calcium-dependent protein kinase 22. The expression of related proteins mediating mitochondrial protein transport was downregulated, e.g., mitochondrial import inner membrane translocase subunit Tim9. However, UPF0187 protein At3g61320 which participates in the formation of anion channels was up-regulated, as was the transmembrane transport proteins, e.g., vesicle-fusing ATPase and ATP-energized ABC transporter.
Metabolomics data showed that a few metabolites involved in signal transduction were up-regulated, such as L-glutamate, adenosine, and succinate, while isoleucyl-glutamate was down-regulated.
In summary, after 12 h of high light stress, U. prolifera showed a decrease in signal transduction generation, inhibition of growth-related gene expression, weakened ion transport and microfibril and microtubule synthesis, and overall inhibition of cilium synthesis. Therefore, cytoskeleton synthesis was generally inhibited, and cell growth was limited.

Cell Division, Gametogenesis, and Apoptosis of U. prolifera in Conditions of High Light Stress
According to multiple omics analysis, it was found that after 12 h of high light stress on U. prolifera, some important genes in the process of cell division, gametogenesis, and apoptosis in U. prolifera had significant changes (Table 5). Transcriptomics data indicated that the expression of many genes involved in cell division was down-regulated, e.g., DNA mismatch repair protein MSH4, DNA replication licensing factor MCM5, single mybhistone 3, and heat shock-like 85 kDa, and some genes promoting the cell division were up-regulated, e.g., histone acetyltransferase MCC1, protein chromatin remodeling 24, and serine/threonine-protein kinase mos.
Transcriptomics data indicated that the expression of genes involved in gametogenesis were down-regulated, e.g., thioredoxin domain-containing protein 3 homolog, 26 S proteasome non-ATPase regulatory subunit 12 homolog, and cilia-and flagella-associated protein 91, while C-factor which is necessary for spore differentiation was up-regulated. Moreover, some genes are expressed to inhibit apoptosis, e.g., serine/threonine-protein kinase, dnaJ homolog subfamily A member 1, and WD repeat-containing protein 35. However, some genes are expressed to promote apoptosis, e.g., metacaspase-1.
Proteomics data showed that the expression of proteins that promote cell division was down-regulated, e.g., SNF1-related protein kinase regulatory subunit gamma and R-mandelonitrile lyase, while dnaJ protein homolog 2 which plays a continuous role in plant development was up-regulated.
According to the omics data above, it was found that after 12 h of high light, the gene expression related to cell division and gametogenesis showed an overall downward trend in U. prolifera. At the same time, the expression of apoptosis-related genes changed, which means the reproductive development of U. prolifera was inhibited by high light stress conditions. It was speculated that 12 h of high light intensity is the turning point of U. prolfera cell division and reproduction.

Resistance of U. prolifera in Conditions of High Light Stress
According to multiple omics analysis, it was found that after 12 h of high light stress on U. prolifera, some important genes related to resistance in U. prolifera had significant changes ( Table 6). The transcriptome showed that many genes were expressed to increase resistance to stress. Some were involved in disease, e.g., disease resistance protein RGA4, disease resistance protein TAO1, and TMV resistance protein N; some were involved in extradition, e.g., Broad substrate specificity ATP-binding cassette transporter ABCG2 and neurotrypsin. Others were related to salt tolerance, e.g., mannitol dehydrogenase; DNA damage tolerance, e.g., disease resistance protein RPP5. However, a few genes were expressed to decrease resistance. They were related to cellular defense responses, e.g., DEAD-box ATP-dependent RNA helicase 50, 17.6 kDa class I heat shock protein 3, and activator of 90 kDa heat shock protein ATPase homolog 1; or DNA damage repair, e.g., deoxy ribodipyrimidine photo-lyase; or photoprotection, e.g., carotene biosynthesis-related protein CBR (other specific categories are shown in Table 6). Proteomics data showed that the expression of proteins involved in antioxidant response was down-regulated, e.g., glutathione S-transferase, ascorbate peroxidase, and peroxidase, as was glutamate-cysteine ligase which is involved in detoxification. How-ever, 10 kDa chaperonin which promotes the refolding and proper assembly of unfolded polypeptides generated under stress conditions was up-regulated.
According to the metabolome data, most of the metabolites on anti-stress were upregulated, including γ-L-glutamyl-L-glutamic acid, L-glutamate, and D-proline. However, there were also metabolites that were down-regulated, e.g., galactinol, L-pyroglutamic acid, and L-glutamine.
According to omics data above, it was found that after 12 h of high light stress, some aspects of resistance were enhanced, including disease, extrudation, salt tolerance, DNA damage tolerance, stress adaptation, lipoxygenases activity, riboflavin, degradation, calcium depend kinase activity, salicylic acid synthesis, and cell recognition and adhesion; others were down-regulated, including cellular defense responses, DNA damage repair, photoprotection, antioxidant and innate immunity. It seemed that in various ways, U. prolifera employ mechanisms to cope with light stress conditions.

Cell Membrane Synthesis and Repair of U. prolifera in Conditions of High Light Stress
According to the omics data, some important genes related to cell membrane synthesis and repair altered significantly after 12 h of high light stress (Table 7). Transcriptomics data indicated that the expression of genes related to cell membrane, cytoderm, and plasmodesmata synthesis was up-regulated, while lipid alienation was down-regulated, e.g., Enoyl-[acyl-carrier-protein] reductase [NADH], sterol sensor 5-transmembrane proteinand UDP-glucuronate 4-epimerase 1. Moreover, genes associated with tRNA synthesis were up-regulated, e.g., tRNA 2'-phosphotransferase and ribonuclease Z, mitochondrial. Proteomics data showed that the expression of proteins involved in lipid biosynthesis was up-regulated, e.g., CDP-diacylglycerol-serine O-phosphatidyl transferase 2 and adipocyte plasma membrane-associated protein.
In summary, after 12 h of intense light stress, genes were expressed to promote the formation of the cell membrane, cytoderm, and plasmodesmata, and reduce lipid dissimilation metabolism. Moreover, synthesis of tRNA was also promoted, which might be related to promotion cell repair.

High Light Intensity Conditions Affecting the Composition of Photosynthetic Membranes
When the external environment changes drastically, algae have evolved multiple mechanisms to avoid harm [19,20]. The algae cell membrane is a significant hydrophobic barrier separating it from the surrounding environment [21]. Therefore, maintaining or regulating the physical and biochemical properties of cell membranes is very important. Regarding the thylakoid-membrane glycerolipids for photosynthesis and photoprotection in chloroplasts, different light conditions will affect them [22,23]. Unsaturated fatty acids are also important components of biofilms [24]. They can increase the fluidity of the membrane, which is important for activating the enzymes on the membrane [25,26].This transcriptomics data (Table 7) showed that the transcript expressions of the sterol sensor 5-transmembrane proteins involved in sterol synthesis were up-regulated. Sterols are essential eukaryotic lipids that are required for a variety of physiological roles [27]. Under the condition of high light, the photosynthetic membrane of U. prolifera was damaged to a certain extent, which accelerated the repair process to ensure the normal photosynthesis of the body [28].
Metabolomics data (Table 7) showed that metabolite 4,7,10,13,16,19-docosahexaenoic acids involved in the synthesis of unsaturated fatty acids decreased; while α-linolenic acid and linoleic acid were up-regulated. The biosynthesis of these fatty acids was corelated because linoleic acid was the synthetic precursor of α-linolenic acid, and the latter was the synthetic precursor of docosahexaenoic acid. The product of fatty acid metabolism was the precursor of lipoic acid metabolism, which involved ferredoxin-thioredoxin reductase. The thioredoxin has been recognized as the key system for transmitting the light-induced reduction signal to the target proteins [29,30].
DGDG and MGDG are the main membrane lipids ( Table 7) that constitute the chloroplast photosynthetic membrane of higher plants, accounting for more than 80% of the chloroplast membrane lipids [31]. Among them, DGDG is one of the most important compounds constituting photosynthetic membranes and exists in almost all biofilms [32]. It accounts for more than 20% of total lipids and can replace other phospholipids under special circumstances [33]. Moreover, DGDG plays an important role in maintaining the oligomer structure of the photosystem II light-harvesting pigment-protein complex and regulating the photosystem II and the oxygen-evolution activity of its core complex [34,35]. The metabolomics results of this study showed that 12-h high light stress increased DGDG content and decreased MGDG content at the same time. In higher plants containing a large quantity of hexadecenoic acids, the biosynthesis of DGDG started from palmitic acid and became cis-9-octadecenoyl-CoA through acetylation, chain lengthening, and hydrogenation [36]. The latter reacted with 3phosphoglycerol to form lysophosphatidic acid [37] and then deacylated to form phosphatidic acid. Phosphatidic acid as a substrate could generate phosphatidylglycerol and DAG. DAG reacted with uridine diphosphate galactose to generate MGDG under the catalysis of MGDG synthase. The latter was combined with galactose-1-phosphate and finally generated DGDG under the catalysis of DGDG synthase [38]. In summary, it was speculated that high light induced a large amount of DGDG synthesis on the photosynthetic membranes and consumed the substrate MGDG, which could be supplemented by MGMG [39].

Changes in Photosynthetic Pigments Affected by High Light Stress Conditions
The content of chlorophyll can be induced by light. In this study, the expression of chlorophyll and carotenoid-related genes ( Table 1) was enhanced under 12 h high light stress (400 µmol·m −2 ·s −1 ). The Chl a and yield of U. prolifera cultured under weak light (62 µmol·m −2 ·s −1 ) for one day were twice that under high light conditions (324 µmol·m −2 ·s −1 ). However, within one week of culture, there was no difference in the Chl a yield of all samples [40]. The synthesis of Chl a in the red alga Corallina elongata can be induced by red light pulses [41] and regulated by light intensity. After 5 h of light treatment, the pigment reached a steady state. When the irradiance increased, chlorophyll synthesis also increased, indicating that this steady state was dynamic [42]. For floating U. prolifera, the surface and lower layers of the algal mat had different photosynthetic responses [43]. The surface algae mat dissipated excess energy through the quantum control of photosynthesis (energy quenching or redistribution between PSII/PSI) and reduced the photosynthetic system's damage. The lower algal mat increased Chl a and Chl b and reduced the ratio of Chl a/b to improve its ability to use light energy [44]. Therefore, U. prolifera has strong photosynthetic plasticity [45,46]. Due to the waves' interference, it quickly adapted to the frequent exchange between the surface and the lower environments through the change of pigment compositions, energy quenching, and energy redistribution between PSII/PSI [44].
Chlorophyll synthesis and catabolism were dynamically balanced, and the change in the ratio of Chls a/b under different physiological conditions was reflected in this experiment's transcriptome data. After 12 h of high light treatment, the expressions of glutamate-1-semialdehyde 2,1-aminomutase-related genes ( Table 1) were up-regulated to promote the production of Chl b [47,48]. The expression level of pheophorbide hydrolase-related genes, which was the key rate-limiting enzyme of the chlorophyll catabolism pathway, was downregulated. It slowed down the communication of Chl b to Chl a, and reduced the ratio of Chl a/b. However, Chl a increased, thereby improving its ability to use light energy as a whole. The contents of the photosystem's auxiliary proteins and pigments were regulated by light. Under 700 µmol·m −2 ·s −1 , the chlorophyll content in Ulva sp. decreased within a few minutes, while the carotenoids remained unchanged [49]. Moreover, under 800 µmol·m −2 ·s −1 , the upper layer of the meadow was involved in the gene up-regulation of light adaptation (rubisco, ferredoxin, and chlorophyll-binding protein) and light protection (antioxidant enzymes, genes related to lutein cycle, and tocopherol biosynthesis), indicating the activation of more defense mechanisms [50]. However, under the condition of 400 µmol·m −2 ·s −1 for 12 h, the antioxidant ability of U. prolifera decreased, but the expression of disease resistance, DNA tolerance, and other defense mechanisms was enhanced (Table 6). In addition to common photosensitive pigments such as carotenoids and chlorophyll, seven rhodopsin types, two leuco dyes, and one photoprotein were found in Chlamydomonas reinhardtii, wherein rhodopsin was a flavin-based photoreceptor sensitive to blue light [51]. Among the light-harvesting proteins, LHCX and LHCZ genes had a stronger up-regulation effect under 400 µmol·m −2 ·s −1 than that under 60 µmol·m −2 ·s −1 [52]. The similar proteins ElipL1, ElipL2, Cbrx, and OHP in U. linza were also upregulated by high light within 3 h under 2000 µmol·m −2 ·s −1 [53]. However, photolyase (Table 6), a blue-light receptor that could bind to folic acid and FAD, was down-regulated in this study, which showed that the blue light sensitivity of U. prolifera weakened after 12 h of high light. Photolyase can repair UV-induced DNA damage in a light-dependent manner and the plant's blue light photoreceptors, which mediate light-dependent regulation of seedling development [54], e.g., the germination of U. prolifera spores [55]. Moreover, blue light can improve the photosynthetic rate of Ulva sp. [56]. Thus, the rate of photosynthesis of U. prolifera decreased because its sensitivity to blue light was weakened. However, the study showed that 12 h of high light stress could promote the expression of DNA repair process of U. prolifera (Table 6). Therefore, it meant that U. prolifera actively reduced photosynthetic rate and growth rate in response to 12-h high light stress under the premise of protecting DNA.
U. prolifera has a mechanism to resist photoinhibition. When the required light energy exceeds the range that the photosynthetic system can withstand, the photosynthetic function declines and light inhibition occurs. Plants have multiple protection mechanisms in response to excessive light energy [57]. For example, Macrocystis pyrifera, Chondrus crispus, and Ulva lactuca promote self-shading by increasing biomass and reduce photoinhibition [58]. The dinoflagellate uses the flavin cycle for photoprotection through heat dissipation [59]. When the light intensity exceeded 400 µmol·m −2 ·s −1 , the electron flow reached saturation, with the increased excitation pressure and NPQ [60]. When Ulva fasciata was exposed to 1500 µmol·m −2 ·s −1 , protein D1 rapidly degraded, and its PES medium form was destroyed. The NPQ ability decreased to a steady state within 110 min, but it quickly recovered in low light [61]. When non-photochemical quenching did not work properly, U. fasciata maintained NPQ by keeping a small proportion of high fast-light PSII combinations. However, there was high-thermal activity with high light because the degradation of Cyt 6 f seriously hindered electrons' transmission, which led to NPQ [62]. The results of this study showed that U. prolifera attenuated PSII synthesis and acyclic photosynthetic phosphorylation, and enhanced respiration, and the carbon fixation mode changed from C3 pathway to C4 pathway. At the same time, RRC1 which is required for phytochrome B (phyB) signal transduction [63] was reduced and phyB is a major photoreceptor in plants [64].
Far-red light enhanced the circulating electron flow around PSI and induced the expression of LHCSR to trigger NPQ in U. prolifera. Lhcb1 and CP29 were adjusted up-regulated under FRL, which meant that the PSII antenna size increased [65]. NPQ induction could be related to individual proteins, for example, psbS content was positively correlated. The latter played an important role in reconstructing the PSII-CLHCII super complex and the energy balance regulation of the thylakoid membrane [66], Or it was regulated by zeaxanthin and triggered and controlled by the transthylakoid proton gradient (∆pH) under high light (1954 µmol·m −2 ·s −1 ). More importantly, it was regulated by the assemblage of the light-harvesting complex (LHC) family. Under high light conditions, the expression of LHCSR was even higher than that of PSBS [67]. However, U. prolifera's NPQ lacked a rapid activation mechanism under high light, and its monomeric LHC proteins only contained CP29 and CP26 instead of CP24. Furthermore, a significant increase in the expression level of CP26 did not change the concentrations of the photoprotective proteins psbS and lhcSR, with the gradual synthesis of zeaxanthin. The atypical NPQ made U. prolifera more suitable for the complex sea environment [62]. The transcriptome data in this experiment also showed that under high light (400 µmol·m −2 ·s −1 ) treatment for 12 h, U. prolifera was involved in light capture, PSI and PSII, and the expressions of genes related to photosynthetic pigment synthesis (Table 1). It was very similar to the atypical NPQ of U. prolifera.

High Light Affecting the Signal Transduction Pathways of U. prolifera
Light-induced cAMP changes significantly increase stress-response proteins in Arabidopsis, so adenylate cyclase may act as a light sensor in higher plants [68]. The transcript expression quantity of cyaC, the primary subtype of adenylate cyclase in algae, is strongly affected by light, which is about 300 times stronger than that of the dark-treated control group [69]. Under subsaturated white light irradiation, the oxygen release of photosynthesis is correlated with cAMP change, showing that electron transfer can regulate the accumulation of cAMP in G. sesquipedale and U. rigida, that is, cAMP level is regulated by light intensity [70]. The transcriptome data here showed that the transcript expression of adenylate cyclase was down-regulated (Table 4). At the same time, metabolome data showed that the expression of related metabolites involved in signal channels was up-regulated (Table 4).
Light can change calcium-dependent protein kinases. In plants, the multi-gene family of CDPKs (calcium-dependent protein kinase) encodes structurally conserved singlemolecule calcium sensor/protein kinase, which plays essential roles in multiple signal transduction pathways. In this experiment, after 12 h of high-light stress for U. prolifera, the protein content of CDPKs was significantly down-regulated (Table 4), but the expressions of CDPKs-related transcripts were significantly up-regulated, indicating a large consumption of CDPKs and an increased demand. However, the transcript expressions of Calcium: Cation Antiporter, glucose 6-phosphoric acids/phosphates, and phosphoenolpyruvate/phosphate anti-transporter proteins, and K + -channel ERG-related proteins (including PAS/PAC sensor domain and K + -channel KCNQ) were significantly downregulated (Table 4), indicating that the demands for calcium and potassium-ion transport inside and outside the membrane decreased. Besides, changes in light intensity promoted the release of cations to the outside of the cell quickly. For example, within the first two minutes, light caused the release of sodium ions in Ulva lobata and Ulva expansa to be twice that of the group in the dark, with 86 Rb + and 85 Sr involved in the tracer released [71]. However, in addition to HCO 3− , other anions such as 36 Cl − , 35 SO 4 2− and [ 14 C] acetate were not affected by light [72].

Growth and Stress-Resistance of U. prolifera under High Light
Light is an essential factor in controlling the growth of algae, and light intensity affects the algae's growth and metabolism [73]. When the sunshine duration exceeds 12 h per day, compared with the light intensity of 10 µmol·m −2 ·s −1 , the chloroplast surface area of Ulva spp. under 100 µmol·m −2 ·s −1 increases with the increasing light duration [74]. Under low light intensity, the long light period will also promote spores in Ulva spp. The algae's growth rate in the light period (150 AE·m −2 ·s −1 ) is significantly higher than that in the dark period. It increases by about two times within 24 h because actin ACT expression is induced and inhibited by light and dark treatments [75]. However, in this experiment, under 400 µmol·m −2 ·s −1 , the expression transcript of SDA1 involved in regulating the actin skeleton was down-regulated ( Table 3), suggesting that cell growth was inhibited [76]. The enhancement of endopeptidase activity was considered the main reason for the decreased protein content during plant senescence [77], and the growth of U. prolifera under high light was also inhibited [78]. The up-regulated endopeptidase in this transcriptome data ( Table 5) was also involved in the overall down-regulation of cell cycle checkpoint kinase expression of cyclin-dependent kinase CDK1 (the main engine of mitosis) and impaired cell cycle regulation.
The growth and distribution of algae are affected by many environmental factors such as temperature, light, and chemicals in the water. Relevant studies have gradually discovered algae stress-related proteins that resist adversity stress [79,80]. In this study, dnaJ protein. (Table 5), which was up-regulated in the proteome and down-regulated in the transcriptome, used as co-chaperone for HSP70 [81], and involved in stress resistance [82].

Conclusions
Based on four omics data analysis, high light stress mainly affected the mutual conversion of pentose and glucuronic acids, fatty acid biosynthesis, steroid biosynthesis, photosynthesis, pyrimidine metabolism, and carbohydrate metabolism, and other metabolic pathways, and regulated the cell cycle in U. prolifera. DGDG and MGDG metabolites were regulated by influencing the ascorbic acid and alginate metabolism, fatty acid metabolism, and energy metabolism to control changes in the photosynthetic membranes of U. prolifera, thereby affecting its photosynthesis. The results provide a further study on the mechanism of U. prolifera's tolerance to high light stress and have laid a foundation for solving the reason of U. prolifera green tide.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biology11111563/s1, Figure S1: Gene annotation Venn diagram in transcriptome; Figure S2: Statistical table of the number of unique peptide segments in the proteome; Figure S3: Statistics of proteome data; Figure S4: Statistical of lipid subclasses and number; Table S1: Primers design of ten different genes; Table S2: QC data statistics of transcriptome; Table S3: Statistics of transcriptome assembly results; Table S4: Statistics of lipid molecules with significant differences.
Author Contributions: C.C. and P.H. conceived the idea. C.C., T.J. and K.G. designed the experiments. K.G. and T.J. performed the experiments. K.G., Y.L. and C.C. analyzed the data. K.G., Y.L. and C.C. wrote and finalized the manuscript. H.Z. and X.L. were responsible for the collection of information related to the research field of this article. All authors have read and agreed to the published version of the manuscript.