3.10 Protein biosynthesis of U. prolifera to 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. Transcriptomics data showed that genes that promote protein synthesis were down-regulated, e.g., DOT1 domain-containing protein (histone-lysine N-methyltransferase activity,0.44), ATP-dependent DNA helicase DDM1 (plays a role in formation, organization, stability and heritability of heterochromatin,0.39), WD repeat-containing protein 5 (contributes to histone modification,0.49), RuvB-like 2 (it has single-stranded DNA-stimulated ATPase and ATP-dependent DNA helicase (5' to 3') activity suggesting a role in nuclear processes e.g., recombination and transcription,0.44), werner syndrome ATP-dependent helicase homolog (multifunctional enzyme that has both magnesium and ATP-dependent DNA-helicase activity and 3'-5' exonuclease activity towards double-stranded DNA with a 5'-overhang, 0.35); same in RNA transcription e.g., AP2-like ethylene-responsive transcription factor AIL5 (transcriptional activator, 0.33), transcriptional activator Myb (transcriptional activator,0.35), ESF1 homolog (constitute a novel regulatory system for basal transcription, 0.39), transcription initiation factor TFIID subunit 5 (TAFs are components of the transcription factor IID (TFIID) complex that is essential for mediating regulation of RNA polymerase transcription,0.39), DNA-directed RNA polymerase I subunit 1 (DNA-dependent RNA polymerase catalyzes the transcription of DNA into RNA using the four ribonucleoside triphosphates as substrates, 0.49), RuvB-like protein 1 (proposed core component of the chromatin remodeling INO80 complex which is involved in transcriptional regulation, DNA replication and probably DNA repair,0.46), DUF2428 domain-containing protein (he posttranscriptional addition of methyl groups to specific residues in a tRNA molecule, 0.37), and genes that decay with RNA were up-regulated e.g., tetratricopeptide repeat protein SKI3 (involved in exosome-mediated RNA decay, 3.46);down-regulation of genes that promote protein degradation and inhibit protein synthesis, e.g., ribosome biogenesis protein BRX1 homolog (biogenesis of the 60S ribosomal subunit, 0.30), tRNA pseudouridine synthase A (transfer RNAs, 0.36), general transcription factor 3C polypeptide 5 (involved in RNA polymerase III-mediated transcription, 0.37), EEF1A lysine methyltransferase 1 (protein-lysine methyltransferase that selectively catalyzes the trimethylation of EEF1A at 'Lys-79', 0.41), protein SDA1 homolog (ribosomal large subunit biogenesis,0.47), 60S ribosomal export protein NMD3 (acts as an adapter for the xpo1-mediated export of the 60S ribosomal subunit,0.48), eukaryotic translation initiation factor 5 (formation of cytoplasmic translation initiation complex, 0.48), cytoplasmic tRNA 2-thiolation protein 2 (may act by forming a heterodimer with ctu1/atpbd3 that ligates sulfur from thiocarboxylated urm1 onto the uridine of tRNAs at wobble position, 0.49), protein-lysine methyltransferase METTL21D (protein-lysine N-methyltransferase that specifically trimethylates 'Lys-315' of VCP/p97, 0.50), hybrid signal transduction histidine kinase J (Acts as a receptor histidine kinase for a signal transduction pathway. This protein undergoes an ATP-dependent autophosphorylation at a conserved histidine residue in the kinase core, and a phosphoryl group is then transferred to a conserved aspartate residue in the receiver domain,0.40), Haloacid dehalogenase-like hydrolase domain-containing protein 3 (hydrolase,0.43), and nucleotide synthesis was inhibited, e.g., 5'-nucleotidase (hydrolyzes extracellular nucleotides into membrane permeable nucleosides, 0.47), pseudouridine-5'-phosphate glycosidase (Catalyzes the reversible cleavage of pseudouridine 5'-phosphate (PsiMP) to ribose 5-phosphate and uracil. Functions biologically in the cleavage direction, as part of a pseudouridine degradation pathway,0.48), cytosolic purine 5'-nucleotidase (may have a critical role in the maintenance of a constant composition of intracellular purine/pyrimidine nucleotides in cooperation with other nucleotidases. Preferentially hydrolyzes inosine 5'-monophosphate (IMP) and other purine nucleotides, 2.51).
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 (may be involved in pre-mRNA splicing, 0.37), eukaryotic translation initiation factor 5A-2 (the precise role of eIF-5A in protein biosynthesis is not known but it functions by promoting the formation of the first peptide bond,0.60), DNA binding helix-turn helix protein (it has the activity of transcription coactivators and participates in the transcription process,0.24), glutamate--cysteine ligase (synthesis of glutathione is involved in interpretation,0.30).photosystem I (PSI) biogenesis, 0.59), polynucleotide-3'-phosphatase ZDP (nick-sensing 3'-phosphoesterase involved in a base excision repair pathway required for active DNA demethylation, 0.46). And the expression of proteins involved in photosynthesis were up-regulated, e.g., uroporphyrinogen decarboxylase (involved in the synthesis of chlorophyll and porphyrin,3.83).
The combined metabolome and lipidome data showed that the expression of promoting amino acid metabolism was up-regulated, e.g., L-glutamate (1.90), L-methionine (3.06), L-glutamate (1.74).
In summary, after 12 h of intense light stress, the expression of protein synthesis related genes showed an overall trend of down-regulation in Ulva algae, including DNA activation, RNA transcription and protein folding and so on, meanwhile, it also inhibited nucleotide production. It showed that 12 h of high light stress is the turning point of U. prolifera tolerant to high light.
3.11 Signal transduction and growth of U. prolifera to high light stress
Accord to omics data, some important genes related to signal transduction and growth altered significantly after 12 h of high light stress. Transcriptomics data indicated that the expression of genes related to the second molecular were down-regulated, e.g., adenylate cyclase (plays essential roles in regulation of cellular metabolism by catalyzing the synthesis of a second messenger, 0.47), protein RRC1 (required for phytochrome B (phyB) signal transduction, 0.44). So did those in ion transport, e.g., potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 2 (modulated by intracellular chloride ions and pH, acidic pH shifts the activation to more negative voltages, 0.46), sodium/calcium exchanger 3 (mediates the electrogenic exchange of Ca2+ against Na+ ions across the cell membrane, and thereby contributes to the regulation of cytoplasmic Ca2+ levels and Ca2+-dependent cellular processes, 0.35), potassium voltage-gated channel subfamily H member 5 (channel properties may be modulated by cAMP and subunit assembly, 0.37), protein detoxification (enables the active transport of a solute across a membrane by a mechanism whereby two or more species are transported in opposite directions in a tightly coupled process not directly linked to a form of energy other than chemiosmotic energy, 0.48).
The genes expression of growth were down-regulated, e.g., GPCR-type G protein 2 (abscisic acid receptor. The GDP-bound form exhibits greater abscisic acid binding than the GTP-bound form. Required for seedling growth and fertility,0.49), and the genes that inhibit cell growth were up-regulated, e.g., ABC transporter G family member 31 (Together with ABCG25, export abscisic acid (ABA) from the endosperm to deliver it to the embryo via ABCG30 and ABCG40-mediated import to suppress radicle extension and subsequent embryonic growth, 2.33).
Proteomics data showed that the expression of proteins involved in signal transduction was down-regulated, e.g., inositol monophosphatase (participates in the phosphatidylinositol signaling pathway,0.57), Calcium-dependent protein kinase 22 (may play a role in calcium as a second messenger signal transduction pathway,0.07), Transmembrane transport protein expression is up-regulated, e.g., Vesicle-fusing ATPase (involved in vesicle-mediated transport pathways,1.61),ATP-energized ABC transporter (participates in transmembrane transport mechanisms,2.63). But at the same time, the expression of related proteins mediating mitochondrial protein transport was down-regulated e.g., mitochondrial import inner membrane translocase subunit Tim9 (mitochondrial intermembrane chaperone that participates in the import and insertion of multi-pass transmembrane proteins into the mitochondrial inner membrane,0.46). The expression of ion channel-related proteins was down-regulated, e.g., UPF0187 protein At3g61320 (participates in the formation of anion channels,1.58).
Metabolomics data showed that a few metabolites involved in signal transduction were up-regulated, such as L-glutamate (1.90), adenosine (1.86), succinate (1.67), while isoleucyl-glutamate (0.71) was down-regulated.
In summary, after 12 h of high light, Ulva algae showed downward-regulated overall expression of signal transduction process-related genes, while the expression of intermediate metabolites were up-regulated. It was speculated that the signal transduction pathway of U. proifera was rapidly inhibited during 12 h irradiation, and downward-regulated trend after that time. Meanwhile, the expression of growth inhibition related genes was up-regulated, the expression of growth promotion related genes was down-regulated and, resulting in the growth and development of U. prolifera was inhibited.
3.12 Cell division, gametogenesis and apoptosis of U. prolifera to 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. Transcriptomics data indicated that the expression of genes involved in cell division were down-regulated, e.g., DNA mismatch repair protein MSH4 (promotes homologous recombination through facilitating chiasma formation during prophase I, 0.28), DNA replication licensing factor MCM5 (essential to undergo a single round of replication initiation and elongation per cell cycle in eukaryotic cells, 0.40), single mybhistone 3 (binds preferentially double-stranded telomeric repeats, but may also bind to the single telomeric strand, 0.40), RING finger and CHY zinc finger domain-containing protein 1 (contributes to the regulation of the cell cycle progression,0.41), chromosome transmission fidelity protein 18 homolog (involved in sister chromatid cohesion and fidelity of chromosome transmission, 0.41), origin of replication complex subunit 1A (component of the origin recognition complex that binds origins of replication, 0.42), chromosome transmission fidelity protein 18 (essential for the fidelity of chromosome transmission and also required for the DNA replication block checkpoint, 0.43), ATP-dependent DNA helicase DDX11 (cooperates also with TIMELESS factor during DNA replication to regulate proper sister chromatid cohesion and mitotic chromosome segregation, 0.43), cyclin-dependent kinase-like 4 (cyclin-dependent catalysis activity, 0.44), DNA replication licensing factor MCM6 (component of the MCM2-7 complex that may function as a DNA helicase and which is essential to undergo a single round of replication initiation and elongation per cell cycle in eukaryotic cells, 0.45), WD repeat-containing protein WRAP73 (act as regulator of spindle anchoring at the mitotic centrosome, 0.45), bloom syndrome protein homolog (participates in DNA replication and repair, 0.46), centrosomal protein of 135 kDa (involved in centriole biogenesis, 0.46), POC1 centriolar protein homolog A (may play an important role in centriole assembly and/or stability and ciliogenesis, 0.36), mitotic spindle checkpoint protein MAD2 (required for the execution of the mitotic checkpoint, 0.49), regulator of telomere elongation helicase 1 homolog (acts as an anti-recombinase to counteract toxic recombination and limit crossover during meiosis, 0.39), lys-63-specific deubiquitinaseBRCC36 (required for normal mitotic spindle assembly and microtubule attachment to kinetochores, 0.49), heat shock-like 85 kDa protein (molecular chaperone that promotes the maturation, structural maintenance and proper regulation of specific target proteins involved for instance in cell cycle control and signal transduction, 0.50); and some genes promoting the cell division were up-regulated, e.g., histone acetyltransferase MCC1 (histone acetyltransferase that probably regulates acetylation status of histone H3 during meiosis and may influence recombination and chromosome segregation, 2.87), protein chromatin remodeling 24 (an essential component of the spindle assembly checkpoint, chromatin remodeling factor that regulate homologous recombination and non-homologous recombination, 2.73), DNA excision repair protein ERCC-6-like (contributes to the mitotic checkpoint by recruiting MAD2 to kinetochores and monitoring tension on centromeric chromatin, 2.78), serine/threonine-protein kinase mos (suppresses the mitotic cell cycle in oocytes, forcing them to undergo meiosis II to produce haploid gametes, 2.04).
Transcriptomics data indicated that the expression of genes involved in gametogenesis were down-regulated, e.g., thioredoxin domain-containing protein 3 homolog (may be required during the final stages of sperm tail maturation, 0.30), 26S proteasome non-ATPase regulatory subunit 12 homolog A (required for gametogenesis and sporophyte development acts redundantly with RPN5B, 0.50), cilia- and flagella-associated protein 91 (may play a role in spermatogenesis, 0.40), and some genes promoting the gametogenesis were up-regulated, e.g., C-.factor (necessary for cellular aggregation, for spore differentiation, and for gene expression that is initiated after 6 hour of starvation, 2.15).
Transcriptomics data indicated that the expression of genes related to apoptosis were down-regulated, e.g., serine/threonine-protein kinase atg1 (involved in autophagy, 0.26), dnaJ homolog subfamily A member 1 (functions as co-chaperone for HSPA1B and negatively regulates the translocation of BAX from the cytosol to mitochondria in response to cellular stress, thereby protecting cells against apoptosis, 0.47), nucleotide-binding oligomerization domain-containing protein 1 (forms an intracellular sensing system along with ARHGEF2 for the detection of microbial effectors during cell invasion by pathogens, 0.37), WD repeat-containing protein 35 (may promote CASP3 activation and TNF-stimulated apoptosis, 0.35), and some genes promoting the gametogenesis were up-regulated, e.g.,metacaspase-1 (acts as a positive regulator of cell death, 3.14), homocysteine methyltransferase (Catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation, methionine is precursors of plant endogenous hormones ethylene and polyamines synthesis, 2.16), and the expression of ascorbate peroxidase (may play a role in the protection of oocyte incorporated cells from rapid apoptotic degradation, 0.41) protecting cells and reducing apoptosis were down- regulated.
Proteomics data showed that the expression of proteins that promote cell division were down-regulated, e.g., SNF1-related protein kinase regulatory subunit gamma (plays redundant role with PV42a in regulating male gametogenesis and pollen tube guidance, 0.57), (R)-mandelonitrile lyase 2 (participates in the formation of propagules, 0.52). Upregulation of development-related proteins, e.g., dnaJ protein homolog 2 (plays a continuous role in plant development probably in the structural organization of compartments, 2.27).
According to omics data above, it was found that after short-term 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 was up-regulated, which means the reproductive development of U. prolifera was inhibited by high light. It was speculated that 12 h of high light was the turning point of U. prolfera cell division and reproduction.
3.13 Resistance of U. prolifera to 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 resistance in U. prolifera had significant changes. Transcriptome showed that the expression of some genes on resistance were up-regulated. Some were involved in disease, e.g., as disease resistance protein RGA4 (that triggers a defense system which restricts the pathogen growth, 2.04), disease resistance protein TAO1 (TIR-NB-LRR receptor-like protein that contributes to disease resistance induced by the Pseudomonas syringae type III effector AvrB. Acts additively with RPM1 to generate a full disease resistance response to P.syringae expressing this type III effector, 2.08), disease resistance protein RPP5 (May have additional roles in adaptation to various stress conditions and in DNA damage tolerance, 3.10)and TMV resistance protein N (that triggers a defense system including the hypersensitive response, which restricts the pathogen growth, 2.30/3.03); some in osmotic substance synthesis, e.g., broad substrate specificity ATP-binding cassette transporter ABCG2 (part of the ATP-binding cassette family that actively extrudes a wide variety of physiological compounds, dietary toxins and xenobiotics from cells, 2.22), neurotrypsin (exocytosis, 2.30/2.35/2.36/2.91), mannitol dehydrogenase (provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level, 2.66),thiamine thiazole synthase (involved in biosynthesis of the thiamine precursor thiazole, 2.51), protein VMS (involved in the endoplasmic reticulum associated degradation pathway, 2.9); some in antioxidant response, e.g., hydroperoxide isomerase ALOXE3 (non-heme iron-containing lipoxygenase which is atypical in that it displays a prominent hydroperoxide isomerase activity and a reduced lipoxygenases activity, 2.36), Riboflavin biosynthesis protein PYRR (Riboflavin is involved in the antioxidant and peroxidation processes of plants, thus affecting the production of reactive oxygen species during oxidative damage and subsequent allergic reactions,2.99), some in apoptosis signal transduction, e.g., calcium-dependent protein kinase 34 (activated by calcium. Autophosphorylation may play an important role in the regulation of the kinase activity, 2.03), and although protein-tyrosine-phosphatase MKP1 (protein-tyrosine-phosphatase that acts as a negative regulator of MPK6 and MPK3 signaling by dephosphorylating and repressing MPK6 and MPK3. May be involved in salt and genotoxic stress responses, 0.41) was down-regulated, it was catalytic factor that inhibits apoptosis, and the final result is still to weaken apoptosis. And V-type proton ATPase subunit E (V-ATPase is responsible for acidifying a variety of intracellular compartments in eukaryotic cells, 2.51) expression can accelerate the decomposition of cells.
On the other hand, transcriptome data revealed some important genes involved in the process of stress resistance were down-regulated significantly. Some were involved in cellular defense responses, e.g., DEAD-box ATP-dependent RNA helicase 50 (probably involved in resistance to biotic and abiotic stresses, 0.22), 17.6 kDa class I heat shock protein 3 (0.27), class I heat shock protein (response to reactive oxygen species, 0.48), activator of 90 kDa heat shock protein ATPase homolog 1 (activates the ATPase activity of HSP90AA1 leading to increase in its chaperone activity, 0.50); some in DNA damage repair, e.g., deoxy ribodipyrimidine photo-lyas (photolyase involved in the repair of UV radiation-induced DNA damage, 0.42); some in photoprotection, e.g., carotene biosynthesis-related protein CBR (it forms photoprotective complexes within the light-harvesting antennae, 0.47) and some in cell osmotic pressure-related gene expression, e.g., molybdenum cofactor sulfurase (sulfurates the molybdenum cofactor, 0.49); some in oxidoreductase activity, e.g., Fe2OG dioxygenase domain-containing protein (oxidoreductase activity, 0.36), amino oxidase domain-containing protein (oxidoreductase activity, 0.47); and some in innate immune response, e.g., NLR family CARD domain-containing protein 3 (attenuates signaling pathways activated by Toll-like receptors and the DNA sensor sting/tmem173 in response to pathogen-associated molecular patterns, such as intracellular poly (dA : dT), but not poly (I : C), or in response to DNA virus infection, including that of herpes simplex virus 1, 2.53 / 3.16).
Proteomics data showed that the expression of proteins involved in antioxidant was down-regulated, e.g., glutathione S-transferase (involved in the redox homeostasis, especially in scavenging of ROS under oxidative stresses,0.16),ascorbate peroxidase it is one of the important antioxidant enzymes in reactive oxygen metabolism of plants,0.58), peroxidase (oxidordeuctase,0.61), alkyl hydroperoxide reductase/thiol specific antioxidant/Mal allergen (RDOX enzyme activity, antioxidant activity, 0.44). The expression of proteins involved in detoxification was down-regulated, e.g., glutamate-cysteine ligase (synthesis of glutathione is involved in interpretation,0.30). The expression of osmotic regulation related proteins was up-regulated, e.g., Delta-1-pyrroline-5-carboxylate (co-expression of rice OsP5CS1 and OsP5CS2 genes in transgenic tobacco resulted in elevated proline biosynthesis and enhanced abiotic stress tolerance,1.57). Down-regulated expression of related proteins involved in regulating protein refolding, e.g., 10 kDa chaperonin (seems to function only as a co-chaperone, along with cpn 60, may facilitate the correct folding of imported proteins. May also prevent misfolding and promote the refolding and proper assembly of unfolded polypeptides generated under stress conditions in the mitochondrial matrix, 0.38).
According to the metabolome data, most of metabolites on anti-stress were up-regulated, including γ-L-glutamyl-L-glutamic acid (4.91), L-glutamate (1.90), D-proline (6.61), 1-aminocyclopropanecarboxylic acid (1.88), L-methionine (3.06), dimethyl sulfone (1.18), L-asparagine (2.39), (S)-2-aminobutyric acid (1.80), 2,3-dihydroxy-3-methylbutyric acid (3.73), ribitol (1.44), L-threonate (1.34), and D-lyxose (1.70). However, there were also metabolites down-regulated, e.g., galactinol (0.82), L-pyroglutamic acid (0.82), and L-glutamine (0.67).
According to omics data above, it was found that after 12 h of high light, some gene expression on stress resistance were up-regulation trend.U. prolifera after 12 hours of strong light, cell recognition and adhesion and other aspects of stress resistance, while the antioxidant and innate immunity and other aspects of weakened. Different stress mechanisms of Enteromorpha prolifera have different coping mechanisms to 12 h’s strong light stress.
3.14 Cell membrane synthesis and repair of U. prolifera to high light stress
Accord to omics data, some important genes related to cell membrane synthesis and repair altered significantly after 12 h of high light stress. Transcriptomics data indicated that the expression of genes related to cell membrane and cytoderm synthesis were up-regulated and lipid alienation were down-regulated, e.g., Enoyl-[acyl-carrier-protein] reductase [NADH] (catalyzes the last reduction step in the de novo synthesis cycle of fatty acids. Involved in the elongation cycle of fatty acids which are used in lipid metabolism. Required for normal plant growth, 2.23), sterol sensor 5-transmembrane protein (involved in cholesterol biosynthesis and uptake, 3.27), UDP-glucuronate 4-epimerase 1 (involved in the synthesis of the negatively charged monosaccharide that forms the backbone of pectic cell wall components, 2.19), UDP-glucose 6-dehydrogenase 5 (involved in the biosynthesis of UDP-glucuronic acid (UDP-GlcA), providing nucleotide sugars for cell-wall polymers, 2.23), callose synthase 12 (structural component of plasmodesmatal canals, 2.62), Cycloartenol-C-24-methyltransferase 1 (catalyzes the methyl transfer from S-adenosyl-methionine to the C-24 of cycloartenol to form 24-methylene cycloartenol, 0.45), cycloeucalenol cycloisomerase (converts pentacyclic cyclopropyl sterols to tetracyclic sterols, 0.45); and genes associated with tRNA synthesis are up-regulated, e.g., Cycloartenol-C-24-methyltransferase 1 (catalyzes the methyl transfer from S-adenosyl-methionine to the C-24 of cycloartenol to form 24-methylene cycloartenol, 0.45), cycloeucalenol cycloisomerase (converts pentacyclic cyclopropyl sterols to tetracyclic sterols, 0.45). tRNA 2'-phosphotransferase (Catalyzes the last step of tRNA splicing, the transfer of the splice junction 2'-phosphate from ligated tRNA to NAD to produce ADP-ribose 1''-2'' cyclic phosphate, 7.56), Ribonuclease Z, mitochondrial (Probably involved in tRNA maturation, by removing a 3'-trailer from precursor tRNA, 2.04).
Proteomics data showed that the expression of proteins involved in lipid biosynthesis was up-regulated, e.g., CDP-diacylglycerol-serine O-phosphatidyl transferase 2 (lipid biosynthesis,1.97), adipocyte plasma membrane-associated protein (Cell membrane biosynthesis, 3.31).
The combined metabolome and lipidome data showed that the expression of certain metabolites was up-regulated during the process of biosynthetic fatty acid (e.g., cis-9-Palmitoleic acid (2.28), myristic acid (2.54), palmitic acid (2.11), α-linolenic acid (1.71), linoleic acid (1.53)), and unsaturated fatty acids (carbon-fixed-3-hydroxypropionic acid (1.65) in prokaryotes, DGDG (3.01) in lipidome). There were also down-regulated expressions, e.g., 4,7,10,13,16,19-docosahexaenoic acid (0.62) in metabolomics, and MGDG (0.69) and MGMG (0.60) in lipidome.
In summary, after 12 h of intense light stress, the expression of promoting synthesis of cell membranes, cell walls and plasmodesmata related genes showed an overall trend of up-regulation in Ulva algae, indicating that short-term high light promoted the repair of cell membrane system of Ulva. Meanwhile, the expression of genes related to lipid metabolism decreased. The results showed that high light stress for 12 h might lead to cell membrane damage of U. prolifera, and the synthesis process of cell membrane system was increased to protect the body.