Acclimation of intertidal macroalgae Ulva prolifera to UVB radiation: the important role of alternative oxidase.

BACKGROUND: Solar radiation is primarily composed of ultraviolet radiation (UVR, 200 - 400 nm) and photosynthetically active radiation (PAR, 400 - 700 nm). Ultraviolet-B (UVB) radiation accounts for only a small proportion of sunlight, and it is the primary cause of plant photodamage. The use of chlorofluorocarbons (CFCs) as refrigerants caused serious ozone depletion in the 1980s, and this had led to an increase in UVB. Although CFC emissions have significantly decreased in recent years, UVB radiation still remains at a high intensity. UVB radiation increase is an important factor that influences plant physiological processes. Ulva prolifera, a type of macroalga found in the intertidal zone, is intermittently exposed to UVB. Alternative oxidase (AOX) plays an important role in plants under stresses. This research examines the changes in AOX activity and the relationships among AOX, photosynthesis, and reactive oxygen species (ROS) homeostasis in U. prolifera under changes in UVB and photosynthetically active radiation (PAR). RESULTS: UVB was the main component of solar radiation impacting the typical intertidal green macroalgae U. prolifera. AOX was found to be important during the process of photosynthesis optimization of U. prolifera due to a synergistic effect with non-photochemical quenching (NPQ) under UVB radiation. AOX and glycolate oxidase (GO) worked together to achieve NADPH homeostasis to achieve photosynthesis optimization under changes in PAR + UVB. The synergism of AOX with superoxide dismutase (SOD) and catalase (CAT) was important during the process of ROS homeostasis under PAR + UVB. CONCLUSIONS: AOX plays an important role in the process of photosynthesis optimization and ROS homeostasis in U. prolifera under UVB radiation. This study provides further insights into the response of intertidal macroalgae to solar light changes.

A novel flavobacterial phage abundant during green tide, representing a new viral family, Zblingviridae.

Flavobacteriia are the dominant and active bacteria during algal blooms and play an important role in polysaccharide degradation. However, little is known about phages infecting Flavobacteriia, especially during green tide. In this study, a novel virus, vB_TgeS_JQ, infecting Flavobacteriia was isolated from the surface water of the Golden Beach of Qingdao, China. Transmission electron microscopy demonstrated that vB_TgeS_JQ had the morphology of siphovirus. The experiments showed that it was stable from -20°C to 45°C and pH 5 to pH 8, with latent and burst periods both lasting for 20 min. Genomic analysis showed that the phage vB_TgeS_JQ contained a 40,712-bp dsDNA genome with a GC content of 30.70%, encoding 74 open-reading frames. Four putative auxiliary metabolic genes were identified, encoding electron transfer-flavoprotein dehydrogenase, calcineurin-like phosphoesterase, phosphoribosyl-ATP pyrophosphohydrolase, and TOPRIM nucleotidyl hydrolase. The abundance of phage vB_TgeS_JQ was higher during Ulva prolifera (U. prolifera) blooms compared with other marine environments. The phylogenetic and comparative genomic analyses revealed that vB_TgeS_JQ exhibited significant differences from all other phage isolates in the databases and therefore was classified as an undiscovered viral family, named Zblingviridae. In summary, this study expands the knowledge about the genomic, phylogenetic diversity and distribution of flavobacterial phages (flavophages), especially their roles during U. prolifera blooms. IMPORTANCE: The phage vB_TgeS_JQ was the first flavobacterial phage isolated during green tide, representing a new family in Caudoviricetes and named Zblingviridae. The abundance of phage vB_TgeS_JQ was higher during the Ulva prolifera blooms. This study provides insights into the genomic, phylogenetic diversity, and distribution of flavophages, especially their roles during U. prolifera blooms.

Structure-Function Covariation of Phycospheric Microorganisms Associated with the Typical Cross-Regional Harmful Macroalgal Bloom.

Unravelling the structure-function variation of phycospheric microorganisms and its ecological correlation with harmful macroalgal blooms (HMBs) is a challenging research topic that remains unclear in the natural dynamic process of HMBs. During the world's largest green tide bloom, causative macroalgae Ulva prolifera experienced dramatic changes in growth state and environmental conditions, providing ideal scenarios for this investment. Here, we assess the phycospheric physicochemical characteristics, the algal host's biology, the phycospheric bacterial constitutive patterns, and the functional potential during the U. prolifera green tide. Our results indicated that (i) variation in the phycosphere nutrient structure was closely related to the growth state of U. prolifera; (ii) stochastic processes govern phycospheric bacterial assembly, and the contribution of deterministic processes to assembly varied among phycospheric seawater bacteria and epiphytic bacteria; (iii) phycospheric seawater bacteria and epiphytic bacteria exhibited significant heterogeneity variation patterns in community composition, structure, and metabolic potential; and (iv) phycospheric bacteria with carbon or nitrogen metabolic functions potentially influenced the nutrient utilization of U. prolifera. Furthermore, the keystone genera play a decisive role in the structure-function covariation of phycospheric bacterial communities. Our study reveals complex interactions and linkages among environment-algae-bacterial communities which existed in the macroalgal phycosphere and highlights the fact that phycospheric microorganisms are closely related to the fate of the HMBs represented by the green tide. IMPORTANCE Harmful macroalgal blooms represented by green tides have become a worldwide marine ecological problem. Unraveling the structure-function variation of phycospheric microorganisms and their ecological correlation with HMBs is challenging. This issue is still unclear in the natural dynamics of HMBs. Here, we revealed the complex interactions and linkages among environment-algae-bacterial communities in the phycosphere of the green macroalgae Ulva prolifera, which causes the world's largest green tides. Our study provides new ideas to increase our understanding of the variation patterns of macroalgal phycospheric bacterial communities and the formation mechanisms and ecological effects of green tides and highlights the importance of phycospheric microorganisms as a robust tool to help understand the fate of HMBs.

Green Tides Significantly Alter the Molecular Composition and Properties of Coastal DOC and Perform Dissolved Carbon Sequestration.

Despite green tides (or macroalgal blooms) having multiple negative effects, it is thought that they have a positive effect on carbon sequestration, although this aspect is rarely studied. Here, during the world's largest green tide (caused by Ulva prolifera) in the Yellow Sea, the concentration of dissolved organic carbon (DOC) increased by 20-37% in intensive macroalgal areas, and thousands of new molecular formulas rich in CHNO and CHOS were introduced. The DOC molecular species derived from U. prolifera constituted ∼18% of the total DOC molecular species in the seawater of bloom area, indicating the profound effect that green tides have on shaping coastal DOC. In addition, 46% of the macroalgae-derived DOC was labile DOC (LDOC), which had only a short residence time due to rapid microbial utilization. The remaining 54% was recalcitrant DOC (RDOC) rich in humic-like substances, polycyclic aromatics, and highly aromatic compounds that resisted microbial degradation and therefore have the potential to play a role in long-term carbon sequestration. Notably, source analysis showed that in addition to the microbial carbon pump, macroalgae are also an important source of RDOC. The number of RDOC molecular species contributed by macroalgae even exceed (77 vs 23%) that contributed by microorganisms.

Legacy Effects of Late Macroalgal Blooms on Dissolved Inorganic Carbon Pool through Alkalinity Enhancement in Coastal Ocean.

Taking the world's largest green tide caused by the macroalga Ulva prolifera in the South Yellow Sea as a natural case, it is studied here if macroalgae can perform inorganic carbon sequestration in the ocean. Massive macroalgae released large amounts of organic carbon, most of which were transformed by microorganisms into dissolved inorganic carbon (DIC). Nearshore field investigations showed that, along with seawater deoxygenation and acidification, both DIC and total alkalinity (TAlk) increased significantly (both >50%) in the areas covered by dense U. prolifera at the late-bloom stage. Offshore mapping cruises revealed that DIC and TAlk were relatively higher at the late-bloom stage than at the before-bloom stage. Laboratory cultivation of U. prolifera at the late-bloom stage further manifested a significant enhancement effect on DIC and TAlk in seawater. Sulfate reduction and/or denitrification likely dominated the production of TAlk. Notably, half of the generated DIC and almost all the TAlk could persist in seawater under varying conditions, from hypoxia to normoxia and from air-water CO2 disequilibrium to re-equilibrium. The enhancement of TAlk allowed more DIC to remain in the seawater rather than escape into the atmosphere, thus having the long-term legacy effect of increasing DIC pool in the ocean.

Particulate Organic Carbon Released during Macroalgal Growth Has Significant Carbon Sequestration Potential in the Ocean.

Substantial amounts of particulate organic carbon (POC) are released during macroalgal growth; however, the fate of these POCs and their carbon sequestration effects remain unclear. Here, field investigations found that Ulva prolifera caused a significant increase of POC in seawater below the surface during a macroalgal bloom. However, laboratory simulations revealed that 77.6% of these POC was easily degraded by microorganisms in a short period of time, concurrently resulting in the production of dissolved organic carbon (DOC) from POC transformation. Over a period of 3 months, the bioavailable components of macroalgae-released POC and POC-transformed DOC were degraded, leaving 39.6% of the antibiodegradable substances composed of biorecalcitrant POC and biorecalcitrant DOC. However, although the biorecalcitrant POC was rich in humic-like components resisting biodegradation, the biorecalcitrant POC exhibited greater sensitivity to photodegradation than biorecalcitrant DOC. The photodegradation removal rate of biorecalcitrant POC (14.1%) was more than 10 times that of biorecalcitrant DOC (1.2%). Ultimately, a substantial portion (36.3%) of the POC released by growing macroalgae could potentially perform long-term carbon sequestration after conversion to recalcitrant POC and recalcitrant DOC, and these inert carbons derived from macroalgal POC have been previously ignored and should also be included in macroalgal carbon sequestration accounting.

Green Tides in the Yellow Sea Promoted the Proliferation of Pelagophyte Aureococcus anophagefferens.

Harmful algal blooms formed by fast-growing, ephemeral macroalgae have expanded worldwide, yet there is limited knowledge of their potential ecological consequences. Here, we select intense green tides formed by Ulva prolifera in the Yellow Sea, China, to examine the ecological consequences of these blooms. Using 28-isofucosterol in the surface sediment as a biomarker of green algae, we identified the settlement region of massive floating green algae in the area southeast of the Shandong Peninsula in the southern Yellow Sea. The responses of the phytoplankton assemblage from the deep chlorophyll-a maximum layer were then resolved using high-throughput sequencing. We found striking changes in the phytoplankton community in the settlement region after an intensive green tide in 2016, characterized by a remarkable increase in the abundance of the pelagophyte Aureococcus anophagefferens, the causative species of ecosystem disruptive brown tides. Our study strongly suggests that the occurrence of massive macroalgal blooms may promote blooms of specific groups of microalgae through alteration of the marine environment.

Responses of the Macroalga Ulva prolifera Müller to Ocean Acidification Revealed by Complementary NMR- and MS-Based Omics Approaches.

Ocean acidification (OA) is a dramatic perturbation of seawater environments due to increasing anthropogenic emissions of CO2. Several studies indicated that OA frequently induces marine biota stress and a reduction of biodiversity. Here, we adopted the macroalga Ulva prolifera as a model and applied a complementary multi-omics approach to investigate the metabolic profiles under normal and acidified conditions. Our results show that U. prolifera grows at higher rates in acidified environments. Consistently, we observed lower sucrose and phosphocreatine concentrations in response to a higher demand of energy for growth and a higher availability of essential amino acids, likely related to increased protein biosynthesis. In addition, pathways leading to signaling and deterrent compounds appeared perturbed. Finally, a remarkable shift was observed here for the first time in the fatty acid composition of triglycerides, with a decrease in the relative abundance of PUFAs towards an appreciable increase of palmitic acid, thus suggesting a remodeling in lipid biosynthesis. Overall, our studies revealed modulation of several biosynthetic pathways under OA conditions in which, besides the possible effects on the marine ecosystem, the metabolic changes of the alga should be taken into account considering its potential nutraceutical applications.

Balancing Damage via Non-Photochemical Quenching, Phenolic Compounds and Photorespiration in Ulva prolifera Induced by Low-Dose and Short-Term UV-B Radiation.

The Yellow Sea green tide (YSGT) is the world's largest transregional macroalgal blooms, and the causative species Ulva prolifera (U. prolifera) suffers from ultraviolet-b radiation (UVBR) during the floating migration process. Previous study confirmed that U. prolifera displayed a wide variety of physiological responses characterized as acclimation to UVBR, while the response mechanisms against low-dose and short-term radiation (LDSTR) are not clear. A study with photosynthetically active radiation (PAR) and UVBR was designed: normal light (NL: 72 µmol photons m-2 s-1), NL+0.3 (UVBR: 0.3 W·m-2), and NL+1.6 (UVBR: 1.6 W·m-2). The results showed that high-dose UVBR inhibited photosynthesis in thalli, especially under long-term exposure, while a variety of physiological responses were observed under LDSTR. The inhibition of photosynthesis appeared to be ameliorated by the algae under LDSTR. Further analysis showed that U. prolifera achieved balancing damage by means of non-photochemical quenching (NPQ), accumulation of phenolic compounds coupled with the ASA-GSH cycle involved in the antioxidant process and enhanced photorespiratory metabolism under LDSTR. This study provides new insights into the balancing damage mechanisms of U. prolifera under LDSTR, enabling the thalli to adapt to the light conditions during the long duration and distance involved in floating migration.

Molecular cloning and transcriptional regulation of two γ-carbonic anhydrase genes in the green macroalga Ulva prolifera.

Ulva prolifera O.F. Müller (Ulvophyceae, Chlorophyta) is well known as a typical green-tide forming macroalga which has caused the world's largest macroalgal blooms in the Yellow Sea of China. In this study, two full-length γ-carbonic anhydrase (γ-CA) genes (UpγCA1 and UpγCA2) were cloned from U. prolifera. UpγCA1 has three conserved histidine residues, which act as an active site for binding a zinc metal ion. In UpγCA2, two of the three histidine residues were replaced by serine and arginine, respectively. The two γ-CA genes are clustered together with other γ-CAs in Chlorophyta with strong support value (100% bootstrap) in maximum likelihood (ML) phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analysis showed that stressful environmental conditions markedly inhibited transcription levels of these two γ-CA genes. Low pH value (pH 7.5) significantly increased transcription level of UpγCA2 not UpγCA1 at 12 h, whereas high pH value (pH 8.5) significantly inhibited the transcription of these two γ-CA genes at 6 h. These findings enhanced our understanding on transcriptional regulation of γ-CA genes in response to environmental factors in U. prolifera.

Effects of temperature on the nitrate reductase activity and growth of Ulva prolifera.

To better understand the effect of temperature on the growth and nitrate reductase activity (NRA) of Ulva prolifera and their relationships, the effects of five different temperatures (10, 15, 20, 25, and 30°C) were investigated in a laboratory setup. In this study, an optimization in vitro analysis method for Ulva prolifera NRA was developed. Under different treatments, the NRA, nitrate concentration, pH, the intracellular nitrate and nitrite concentrations, and the POC/PON were evaluated. The results of the in vitro analysis method showed it was optimal for the NRA assay when the extraction time was 6 min, enzymatic reaction time 30 min, volume of phenazine methosulfate (PMS) solution 50 µL, NADH concentration 0.36 mM, and KNO3 concentration 10 mM. The maximal NRA (NRAmax ) appeared on the 2nd day in the 10, 15, and 20°C (low-temperature) groups and on the 1st day in the 25 and 30°C (high-temperature) groups. The algal growth ended earlier at a high temperature, ending after 5 d at 30 and 25°C and 7 d at 20°C and 9 d at 15°C, and the alga at 10°C had been growing during the incubation period. Ulva prolifera cultivated in a range of 10-20°C had a long growth cycle and the NRA decreased with increasing temperature when exceeded 15°C, a positive correlation between algal growth and NRA was observed. This study supports NRA is a suitable proxy of the effects of temperature changes on the ability of Ulva prolifera to uptake and metabolize nitrogen nutrients.

Slow zeaxanthin accumulation and the enhancement of CP26 collectively contribute to an atypical non-photochemical quenching in macroalga Ulva prolifera under high light.

Non-photochemical quenching (NPQ) is an important photoprotective mechanism in plants, which dissipates excess energy and further protects the photosynthetic apparatus under high light stress. NPQ can be dissected into a number of components: qE, qZ, and qI. In general, NPQ is catalyzed by two independent mechanisms, with the faster-activated quenching catalyzed by the monomeric light-harvesting complex (LHCII) proteins and the slowly activated quenching catalyzed by LHCII trimers, both processes depending on zeaxanthin but to different extent. Here, we studied the NPQ of the intertidal green macroalga, Ulva prolifera, and found that the NPQ of U. prolifera lack the faster-activated quenching, and showed much greater sensitivity to dithiothreitol (DTT) than to dicyclohexylcarbodiimide (DCCD). Further results suggested that the monomeric LHC proteins in U. prolifera included only CP29 and CP26, but lacked CP24, unlike Arabidopsis thaliana and the moss Physcomitrella patens. Moreover, the expression levels of CP26 increased significantly following exposure to high light, but the concentrations of the two important photoprotective proteins (PsbS and light-harvesting complex stress-related [LhcSR]) did not change upon the same conditions. Analysis of the xanthophyll cycle pigments showed that, upon exposure to high light, zeaxanthin synthesis in U. prolifera was gradual and much slower than that in P. patens, and could effectively be inhibited by DTT. Based on these results, we speculate the enhancement of CP26 and slow zeaxanthin accumulation provide an atypical NPQ, making this green macroalga well adapted to the intertidal environments.

Algihabitans albus gen. nov., sp. nov., isolated from a culture of the green alga Ulva prolifera.

A bacterial strain, designated HHTR 118T, was isolated from a culture of the green alga Ulvaprolifera obtained from offshore seawater of Qingdao, Shandong Province, China. Cells of strain HHTR 118T were rod-shaped and motile with a single flagellum, and approximately 0.3-0.4 µm wide and 0.8-1.4 µm long. The strain was Gram-stain-negative, strictly aerobic, catalase-negative and oxidase-positive. Optimal growth was observed at 30 °C, at pH 8.0 and with 1 % (w/v) NaCl. Nitrate was not reduced. Sucrose, sodium citrate and l-leucine stimulated growth, but not lactose, fructose, xylose, d-mannose, glucose, raffinose, rhamnose, ornithine or lysine. The DNA G+C content of strain HHTR 118T calculated on the basis of the genome sequence was 64.9 mol% and the genome size is 4.6 Mbp. The major quinone was ubiquinone 10 and the predominant cellular fatty acids (>10 % of total fatty acids) were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c). The predominant polar lipids were phosphatidylglycerol, one unidentified phospholipid, two unidentified aminolipids and three unidentified polar lipids. Phylogenetic analysis, based on 16S rRNA gene sequences, demonstrated that strain HHTR 118T was affiliated with the family Rhodospirillaceae. On the basis of the 16S rRNA gene sequence data as well as physiological and biochemical characteristics, we concluded that strain HHTR 118T represents a novel species of a novel genus. We propose the name of Algihabitans albus gen. nov., sp. nov. for this novel species. The type strain of the novel species is strain HHTR 118T (=KCTC 62395T=MCCC 1K03486T).

High salt stress in the upper part of floating mats of Ulva prolifera, a species that causes green tides, enhances non-photochemical quenching.

Salt stress is a major abiotic stress factor that can induce many adverse effects on photosynthetic organisms. Plants and algae have developed several mechanisms that help them respond to adverse environments. Non-photochemical quenching (NPQ) is one of these mechanisms. The thalli of algae in the intertidal zone that are attached to rocks can be subjected to salt stress for a short period of time due to the rise and fall of the tide. Ulva prolifera causes green tides and can form floating mats when green tides occur and the upper part of the thalli is subjected to high salt stress for a long period of time. In this study, we compared the Ulva prolifera photosynthetic activities and NPQ kinetics when it is subjected to different salinities over various periods of time. Thalli exposed to a salinity of 90 for 4 d showed enhanced NPQ, and photosynthetic activities decreased from 60 min after exposure up to 4 d. This indicated that the induction of NPQ in Ulva prolifera under salt stress was closely related to the stressing extent and stressing time. The enhanced NPQ in the treated samples exposed for 4 d may explain why the upper layer of the floating mats formed by Ulva prolifera thalli were able to survive in the harsh environment. Further inhibitor experiments demonstrated that the enhanced NPQ was xanthophyll cycle and transthylakoid proton gradient-dependent. However, photosystem II subunit S and light-harvesting complex stress-related protein didn't over accumulate and may not be responsible for the enhanced NPQ.

Synergistic effects of HSE and LTR elements from hsp70 gene promoter of Ulva prolifera (Ulvophyceae, Chlorophyta) upon temperature induction1.

Besides heat stress, the 70 kDa heat shock proteins (HSP70s) have been shown to respond to cold stress. However, the involved cis-acting elements remain unknown. The hsp70 gene from the green macroalga Ulva prolifera (Uphsp70) has been cloned, from which one heat shock element HSE and one low-temperature-responsive element LTR were found in the promoter. Using the established transient expression system and quantitative GUS assay, a series of element deletion experiments were performed to determine the functions of HSE and LTR in response to temperature stress. The results showed that under cold stress, both HSE and LTR were indispensable, since deletion leads to complete loss of promoter activity. Under heat stress, although the HSE could respond independently, coexistence with LTR was essential for high induced activity of the Uphsp70 promoter. Therefore, synergistic effects exist between HSE and LTR elements in response to temperature stress in Ulva, and extensive bioinformatics analysis showed that the mechanism is widespread in algae and plants, since LTR coexists widely with HSE in the promoter region of hsp70. Our findings provide important supplements to the knowledge of algal and plant HSP70s response to temperature stress. We speculated that for algal domestication and artificial breeding, HSE and LTR elements might serve as potential molecular targets to temperature acclimation.

Ulvibacterium marinum gen. nov., sp. nov., a novel marine bacterium of the family Flavobacteriaceae, isolated from a culture of the green alga Ulva prolifera.

A Gram-stain negative, aerobic, rod-shaped, and non-motile bacterium, designated strain CCMM003T, was isolated from a culture of the green alga Ulva prolifera. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain CCMM003T belongs to the family Flavobacteriaceae and exhibits a close relationship to Pseudozobellia thermophila DSM 19858T (92.5%). Optimal growth occurred in the presence of 4% (w/v) NaCl, at pH 7.0 and 30 °C. The polar lipids of strain CCMM003T consisted of phosphatidylethanolamine and six unidentified lipids. The predominant isoprenoid quinone was MK-6. The major fatty acids were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH). The DNA G + C content of strain CCMM003T calculated on the basis of the genome sequence was 41.2 mol% and the genome size was 5.9 Mbp. On the basis of data from this polyphasic study, strain CCMM003T is considered to represent a novel genus and species of the family Flavobacteriaceae, for which the name Ulvibacterium marinum gen. nov., sp. nov. is proposed. The type strain is CCMM003T (= MCCC 1K03244T =KCTC 52639T).

Marinicaulis aureum sp. nov., isolated from a culture of the green alga Ulva prolifera, and emended description of the genus Marinicaulis.

A Gram-stain negative, facultatively anaerobic, rod-shaped motile bacterium with a single polar flagellum, designed strain HHTR114T, was isolated from a culture of the green alga Ulva prolifera obtained from offshore seawater at Qingdao, China. Optimum growth occurred in the presence of 2-3% (w/v) NaCl, at pH 7.0-8.0 and 30 °C. The major fatty acids (> 10% of total fatty acids) were C16:0 (24.7%), C18:1ω7c 11-methyl (24.3%) and summed feature 3 (C16:1ω6c and/or C16:1ω7c, 19.7%). The major polar lipids were phosphatidylethanolamine, glycolipid and four unidentified polar lipids. The DNA G + C content of strain HHTR114T calculated on the basis of the genome sequence was 58.2% and the genome size is 4.1 Mbp. The predominant isoprenoid quinone was Q-10. The estimated DNA-DNA hybridization values were 21.4% [18.6-24.4%] between strain HHTR114T and Marinicaulis flavus SY-3-19T. On the basis of polyphasic analysis, strain HHTR114T is considered to represent a novel species, for which the name Marinicaulis aureum sp. nov. is proposed. The type strain of the type species is HHTR114T (= KCTC 62394T = MCCC 1K03481T).

Comparative proteomic analysis of Ulva prolifera response to high temperature stress.

BACKGROUND: Ulva prolifera belongs to green macroalgae and is the dominant species of green tide. It is distributed worldwide and is therefore subject to high-temperature stress during the growth process. However, the adaptation mechanisms of the response of U. prolifera to high temperatures have not been clearly investigated yet. METHODS: In this study, isobaric tags for relative and absolute quantitation (iTRAQ) labelling was applied in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) to conduct comparative proteomic analysis of the response of U. prolifera to high-temperature stress and to elucidate the involvement of this response in adaptation mechanisms. Differentially expressed proteins (DEPs) of U. prolifera under high temperature (denote UpHT) compared with the control (UpC) were identified. Bioinformatic analyses including GO analysis, pathway analysis, and pathway enrichment analysis was performed to analyse the key metabolic pathways that underlie the thermal tolerance mechanism through protein networks. Quantitative real-time PCR and western blot were performed to validate selected proteins. RESULTS: In the present study, 1223 DEPs were identified under high temperature compared with the control, which included 790 up-regulated and 433 down-regulated proteins. The high-temperature stimulus mainly induced the expression of glutathione S-transferase, heat shock protein, ascorbate peroxidase, manganese superoxide dismutase, ubiquitin-related protein, lhcSR, rubisco activase, serine/threonine protein kinase 2, adenylate kinase, Ca2+-dependent protein kinase (CDPK), disease resistance protein EDS1, metacaspase type II, NDPK2a, 26S proteasome regulatory subunit, ubiquinone oxidoreductase, ATP synthase subunit, SnRK2s, and cytochrome P450. The down-regulated proteins were photosynthesis-related proteins, glutathione reductase, catalase-peroxidase, thioredoxin, thioredoxin peroxidase, PP2C, and carbon fixation-related proteins. Furthermore, biological index analysis indicated that protein content and SOD activity decreased; the value of Fv/Fm dropped to the lowest point after culture for 96 h. However, APX activity and MDA content increased under high temperature. CONCLUSION: The present study implied an increase in proteins that were associated with the stress response, oxidative phosphorylation, the cytokinin signal transduction pathway, the abscisic acid signal transduction pathway, and the glutathione metabolism pathway. Proteins that were associated with photosynthesis, carbon fixation in photosynthesis organisms, and the photosynthesis antenna protein pathway were decreased. These pathways played a pivotal role in high temperature regulation. These novel proteins provide a good starting point for further research into their functions using genetic or other approaches. These findings significantly improve the understanding of the molecular mechanisms involved in the tolerance of algae to high-temperature stress.

Cloning and characterization of nitrate reductase gene in Ulva prolifera (Ulvophyceae, Chlorophyta).

Ulva spp. dominates green tides around the world, which are occurring at an accelerated rate. The competitive nitrogen assimilation efficiency in Ulva is suggested to result in ecological success against other seaweeds. However, molecular characterization of genes involved in nitrogen assimilation has not been conducted. Here, we describe the identification of the nitrate reductase (NR) gene from a green seaweed Ulva prolifera, an alga which is responsible for the world's largest green tide in the Yellow Sea. Using rapid amplification of cDNA ends and genome walking, the NR gene from U. prolifera (UpNR) was cloned, which consisted of six introns and seven exons encoding 863 amino acids. According to sequence alignment, the NR in U. prolifera was shown to possess all five essential domains and 21 key invariant residues in plant NRs. The GC content of third codon position of UpNR (82.75%) was as high as those of green microalgae, and the intron number supported a potential loss issue from green microalga to land plant. Real-time quantitative PCR results showed that UpNR transcript level was induced by nitrate and repressed by ammonium, which could not be removed by addition of extra nitrate, indicating that U. prolifera preferred ammonium to nitrate. Urea would not repress NR transcription by itself, while it weakened the induction effect of nitrate, implying it possibly inhibited nitrate uptake rather than nitrate reduction. These results suggest the use of UpNR as a gene-sensor to probe the N assimilation process in green tides caused by Ulva.

Spatio-temporal distribution of micropropagules of green algae along the Jiangsu coast.

We conducted continuous monitoring at 13 stations along the Jiangsu coast to study the spatiotemporal distribution, population succession of micropropagules of green algae, and their impact on the outbreak of Southern Yellow Sea green tide. The study discovered that: 1) Green algae micropropagules had obvious temporal and spatial distribution and population changes along the Jiangsu coast. The monthly average abundance of micropropagules of green algae at station BH1, which was the high-value area, was 1230 inds/L. Station XS2 had the second-highest value area. Green algae micropropagules had an average monthly abundance of 836 inds/L. Between stations XS2 and BH1, the amount of green algae micropropagules steadily declined in comparison to other stations. The abundance was greatest from spring to early summer, and Ulva prolifera micropropagules predominated; 2) Compared with salinity, temperature had a more obvious effect on the micropropagules of green algae along the Jiangsu coast; 3) Green algae micropropagules on the Jiangsu coast could be a potential additional source on the outbreak of Southern Yellow Sea green tide. More data are needed to corroborate this conclusion. For the purpose of preventing and managing green tide, it is crucial to investigate the Southern Yellow Sea's potential supplementary source. This study analyzes the spatiotemporal distribution and population changes of green algae micropropagules along the Jiangsu coast, as well as their impact on green tide outbreaks, providing scientific data support for the prevention and control of green tides in the Southern Yellow Sea.