Molecular identification of Halomonas AZ07 and its multifunctional enzymatic activities to degrade Pyropia yezoensis under high-temperature condition.

BACKGROUND: Pyropia yezoensis a commercially important red seaweed species, is susceptible to various microorganisms infections, among which bacterial infections are the most prominent ones. Pyropia yezoensis is often affected by harmful bacterial communities under high temperatures that can lead to its degradation and economic losses. The current study aimed to explore Pyropia yezoensis-associated microbiota and further identify potential isolates, which can degrade Pyropia yezoensis under high-temperature conditions. METHODS AND RESULTS: The 16S rRNA gene sequencing was used to identify the agarolytic bacterial species. The results showed that Chromohalobacter sp. strain AZ6, Pseudoalteromonas sp. strain AZ, Psychrobacter sp. strain AZ3, Vibrio sp. strain AZ, and Halomonas sp. strain AZ07 exhibited algicidal properties as these strains were more abundant at high temperature (25 °C). Among the five isolated strains, the potential isolate Halomonas sp. strain AZ07 showed high production of agarolytic enzymes, including lipase, protease, cellulase, and amylase. This study confirmed that the isolated strain could produce these four different enzymes. The strain Halomonas AZ07 was co-treated with Pyropia yezoensis cells under two different temperature environments, including 13 °C and 25 °C. The degradation of Pyropia yezoensis occurred at the optimum temperature of 25 °C and effectively degraded their cell wall, proteins, lipids, and carbohydrates. CONCLUSION: The successful cultivation of Pyropia yezoensis in coastal farm environments is dependent on specific temperature and environmental factors, and lower temperatures have been observed to be particularly beneficial for the survival and growth of Pyropia yezoensis. The temperature below 13 °C was confirmed to be the best niche for the symbiotic relationship of microbiota associated with Pyropia yezoensis for its growth, development, and production.

Preparation and Vasodilation Mechanism of Angiotensin-I-Converting Enzyme Inhibitory Peptide from Ulva prolifera Protein.

Ulva prolifera, a type of green algae that can be consumed, was utilized in the production of an angiotensin-I converting enzyme (ACE) inhibitory peptide. The protein from the algae was isolated and subsequently hydrolyzed using a neutral protease. The resulting hydrolysate underwent several processes including Sephadex-G100 filtration chromatography, ultrafiltration, HPLC-Q-TOF-MS analysis, ADMET screening, UV spectrum detection test, molecular docking, and molecular dynamic simulation. Then, the ACE inhibitory peptide named KAF (IC50, 0.63 ± 0.26 µM) was identified. The effectiveness of this peptide in inhibiting ACE can be primarily attributed to two conventional hydrogen bonds. Additionally, it could activate endothelial nitric oxide synthase (eNOS) activity to promote the generation of nitric oxide (NO). Additionally, KAF primarily increased the intracellular calcium (Ca2+) level by acting on L-type Ca2+ channel (LTCC) and the ryanodine receptor (RyR) in the endoplasmic reticulum, and completed the activation of eNOS under the mediation of protein kinase B (Akt) signaling pathway. Our study has confirmed that KAF has the potential to be processed into pharmaceutical candidate functions on vasoconstriction.

Isolation, Characterization and Bioactive Properties of Alkali-Extracted Polysaccharides from Enteromorpha prolifera.

Four new purified polysaccharides (PAP) were isolated and purified from the Enteromorpha prolifera by alkali extraction, and further characterization was investigated. Their average molecular weights of PAP-1, PAP-2, PAP-3, and PAP-4 were estimated as 3.44 × 104, 6.42 × 104, 1.20 × 105, and 4.82 × 104 Da, respectively. The results from monosaccharide analysis indicated that PAP-1, PAP-2, PAP-3 were acidic polysaccharides and PAP-4 was a neutral polysaccharide. PAP-1 and PAP-2 mainly consist of galacturonic acid, while PAP-3 and PAP-4 mainly contained rhamnose. Congo red test showed that no triple helical structure was detected in the four polysaccharides. The antioxidant activities were investigated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), Superoxide, and 2, 2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical assay. In vitro antitumor activities were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PAP-1 exhibited relatively stronger antioxidant activities among the four polysaccharides in a dose-dependent manner. At a concentration of 1.00 mg/mL, the antioxidant activities of PAP-1 on the DPPH radical scavenging rate, superoxide anion radical scavenging rate, and ABTS radical rate at 1.00 mg/mL were 56.40%, 54.27%, and 42.07%, respectively. They also showed no significant inhibitory activity against MGC-803, HepG2, T24, and Bel-7402 cells. These investigations of polysaccharides provide a scientific basis for the use of E. prolifera as an ingredient in functional foods and medicines.

Differential gene expression for carotenoid biosynthesis in a green alga Ulva prolifera based on transcriptome analysis.

BACKGROUND: Carotenoids are widely distributed in plants and algae, and their biosynthesis has attracted widespread interest. Carotenoid-related research has mostly focused on model species, and there is a lack of data on the carotenoid biosynthetic pathway in U. prolifera that is the main species leading to green tide, a harmful plague of floating green algae. RESULTS: The carotenoid content of U. prolifera samples, that is the main species leading to green tide, a harmful plague of floating green algae at different temperatures revealed that its terpenoid was highest in the samples subjected to high temperature at 28 °C (H), followed by the samples subjected to low temperature at 12 °C (L). Its terpenoid was lowest in the samples subjected to medium temperature at 20 °C (M). We conducted transcriptome sequencing (148.5 million raw reads and 49,676 unigenes in total) of samples that were subjected to different temperatures to study the carotenoid biosynthesis of U. prolifera. There were 1125-3164 significant differentially expressed genes between L, M and H incubation temperatures, of which 11-672 genes were upregulated and 453-3102 genes were downregulated. A total of 3164 genes were significantly differentially expressed between H and M, of which 62 genes were upregulated and 3102 genes were downregulated. A total of 2669 significant differentially expressed genes were observed between L and H, of which 11 genes were upregulated and 2658 genes were downregulated. A total of 13 genes were identified to be involved in carotenoid biosynthesis in U. prolifera, and the expression levels of the majority were highest at H and lowest at M of incubation temperature. Both the carotenoid concentrations and the expression of the analysed genes were lowest in the normal temperature group, while low temperature and high temperature seemed to activate the biosynthesis of carotenoids in U. prolifera. CONCLUSIONS: In this study, transcriptome sequencing provided critical information for understanding the accumulation of carotenoids and will serve as an important reference for the study of other metabolic pathways in U. prolifera.

Biomass and terpenoids produced by mutant strains of Arthrospira under low temperature and light conditions.

The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14 °C, 43.75 µmol photons m-2 s-1 and performed breed conservation at 2.5 °C, 8.75 µmol photons m-2 s-1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16 °C and 43.75 µmol photons m-2 s-1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057 ± 80 mg l-1, 68.7 ± 2.5 mg l-1 day-1, 1839 ± 44 mg l-1, 60.6 ± 1.8 mg l-1 day-1, and 2113 ± 64 mg l-1, 77.7 ± 2.5 mg l-1 day-1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10 °C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28 °C, achieving 1140 mg l-1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-D-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.

Cloning and Functional Analysis of a Zeaxanthin Epoxidase Gene in Ulva prolifera.

The xanthophyll cycle is a photoprotective mechanism in plants and algae, which protects the photosynthetic system from excess light damage under abiotic stress. Zeaxanthin is considered to play a pivotal role in this process. In this study, the relative content of xanthophylls was determined using HPLC-MS/MS in Ulva prolifera exposed to different salinities. The results showed that high-salt stress significantly increased the relative content of xanthophylls and led to the accumulation of zeaxanthin. It was speculated that the accumulated zeaxanthin may contribute to the response of U. prolifera to high-salt stress. Zeaxanthin epoxidase (ZEP) is a key enzyme in the xanthophyll cycle and is also involved in the synthesis of abscisic acid and carotenoids. In order to explore the biological function of ZEP, a ZEP gene was cloned and identified from U. prolifera. The CDS of UpZEP is 1122 bp and encodes 373 amino acids. Phylogenetic analysis showed that UpZEP clusters within a clade of green algae. The results of qRT-PCR showed that high-salt stress induced the expression of UpZEP. In addition, heterologous overexpression of the UpZEP gene in yeast and Chlamydomonas reinhardtii improved the salt tolerance of transgenic organisms. In conclusion, the UpZEP gene may be involved in the response of U. prolifera to high-salt stress and can improve the high-salt tolerance of transgenic organisms.

Purification identification and function analysis of ACE inhibitory peptide from Ulva prolifera protein.

In the present study, Ulva prolifera, an edible alga, was used to prepare angiotensin-I converting enzyme (ACE) inhibitory peptide. The algae protein was isolated and later hydrolyzed by five commercial enzymes (alcalase, papain, pepsin, trypsin, neutral protease), either individually or in combination. Hydrolysate, with the highest in vitro ACE inhibitory activity, was processed using the Sephadex-G100, ultrafiltration, HPLC-Q-TOF-MS, ADMET screening and molecular docking, respectively. The ACE inhibitory peptide DIGGL with a IC50 value of 10.32 ± 0.96 µM was then identified. The peptide against ACE by a non-competitive mode and mainly attributable to the three Conventional Hydrogen Bonds. It could activate Endothelial nitric oxide synthase activity in NO generation and reduce Endothelin-1 secretion induced by Angiotensin II in Human umbilical vein endothelial cells. Meanwhile, DIGGL could promote mice splenocytes proliferation, which was also effective when co-incubated with Con A or LPS, respectively. Besides, the anti-ACE peptide could remain active during the digestion of gastrointestinal proteases (pepsin-trypsin) in vitro.

Morphological and photosynthetic variations in the process of spermatia formation from vegetative cells in Porphyra yezoensis Ueda (Bangiales, Rhodophyta) and their responses to desiccation.

Porphyra yezoensis has a macroscopic foliage gametophyte phase with only a single cell layer, and is ideally suited for the study of the sexual differentiation process, from the vegetative cell to the spermatia. Firstly, we compared variations in the responses of the vegetative and male sectors to desiccation. Later, cell tracking experiments were carried out during the formation of spermatia from vegetative cells. The two sectors showed similar tolerance to desiccation, and the formation of spermatia from vegetative cells was independent of the degree of desiccation. Both light and scanning electron microscopy (SEM) observations of the differentiation process showed that the formation of spermatia could be divided into six phases: the one-cell, two-cell, four-cell, eight-cell, pre-release and spermatia phases. Photomicrographs of Fluorescent Brightener staining showed that the released spermatia had no cell walls. Photosynthetic data showed that there was a significant rise in Y(II) in the four-cell phase, indicating an increase in photosynthetic efficiency of PSII during this phase. We propose that this photosynthetic rise may be substantial and provide the increased energy needed for the formation and release of spermatia in P. yezoensis.

An Arabidopsis gene encoding a C2H2-domain protein with alternatively spliced transcripts is essential for endosperm development.

The endosperm is an essential part of the seed which sustains embryo development and contains storage reserves. Endosperm development involves a series of nuclear divisions in the absence of cytokinesis. In this study, it is shown that a mutant Arabidopsis plant carrying the disrupted At4g24900 gene exhibits severe defects during seed development. At4g24900 encodes a nuclear-localized C2H2-containing protein and the transcripts of this gene are alternatively spliced, consequently producing at least nine differentially spliced isoforms. The heterozygous T-DNA insertion mutation in this gene resulted in abortion of 25% of seeds, and the homozygous mutant allele displayed embryo lethality. Differential interfernce contrast (DIC) analyses revealed that the mutant has endosperm nuclei with more than one nucleolus and an embryo arrested at the globular to heart stage transition. Because this mutant exhibits a titan-like phenotype, it was designated ttl. The TTL gene is preferentially expressed in tissues with quickly dividing cells as revealed in P(TTL)::GUS (ß-glucuronidase) transgenic plants. It is proposed that TTL is likely to function as a key regulator of endosperm nuclear division.

Comparative Analysis of MicroRNAs between Sporophyte and Gametophyte of Porphyra yezoensis.

Porphyra yezoensis Ueda is an intertidal marine red algae that has received increasing attention as a model organism owing to its important role in biological research and the agronomic industry. The two generations of Porphyra yezoensis, the sporophyte and the gametophyte, have the same genome but show great differences in many aspects, including structural features, habitat, and gene expression. To identify miRNAs and their probable roles in P. yezoensis development, we constructed and sequenced libraries of small RNA from P. yezoensis sporophytes and gametophytes. The sequencing data were analyzed, and 14 miRNAs were identified, with only one common to these two samples. Our results show that P. yezoensis has a complex small RNA processing system containing novel miRNAs that have no identifiable homolog in other organisms. These miRNAs might have important regulatory roles in development of the different generations of P. yezoensis.

Social Environment and Healthy Investment Behavior: Joint Influence of Culture and Institution on China.

The influence of the social environment on healthy investment behavior is a vital research topic. This paper focuses on foreign direct investment (FDI) as an important part of its broad impact in improving the level of capital circulation and diversifying the non-systemic risk of a single country portfolio. Using data from 35 countries on direct investment in China, we find that the impact of the social environment on healthy investment behavior is mainly reflected in investors' resistance to cultural distance and their benefit compensation across institutional distance. In addition, their joint influence is still negative, dominated by cultural distance, which can still verify that institutional distance mitigates the negative effect of cultural distance on FDI. Therefore, in order to promote international healthy investment behavior, it is feasible to improve both the mitigation effect of the institution in the short term and promote the level of cultural exchange in the long term, according to the research results of this paper.

PSI-driven cyclic electron flow allows intertidal macro-algae Ulva sp. (Chlorophyta) to survive in desiccated conditions.

Ulva sp. (Chlorophyta) is a representative species of the intertidal macro-algae responsible for the green tides that occurred along the shores of Qingdao in 2008 and had detrimental effects on the preparation for the 2008 Beijing Olympic Games sailing competition. In view of its significance, we have investigated the photosynthetic performance of the photosystems and the changes in photosynthetic electron transport that occur during desiccation and rehydration of Ulva sp. The PSII activity in Ulva sp. declined gradually during the course of desiccation, which was reflected by the decreased maximum quantum yield and effective quantum yield, whereas the PSI activity fluctuated significantly. In contrast, the electron transport rates of PSII approached zero at severe levels of desiccation, but the electron transport of PSI, which still operated, could be suppressed effectively by a specific inhibitor. Furthermore, the electron transport of PSI during rehydration of desiccated thalli was recovered faster than that of PSII. All these results implied that the linear electron flow was abolished in desiccated Ulva sp., whereas the cyclic PSI activity was significantly elevated, was still active at severe levels of desiccation and could be restored faster than PSII activity. Based on these results, we concluded the PSI-driven cyclic electron flow might provide desiccation tolerance and additional flexibility for the cell physiology of Ulva sp. under desiccation conditions, which might be one of the most important factors that make Ulva sp. well suited to experience daily cycles of desiccation at low tide and rehydration at high tide.

Comparison of chlorophyll and photosynthesis parameters of floating and attached Ulva prolifera.

In mid-May 2008 a serious green tide caused mainly by floating Ulva prolifera (Müller) J. Agardh (Chlorophyta, Ulvales) thalli struck the coastal area of Qingdao, China. To understand the present physiological conditions of the floating alga, in this work both laboratory and field investigations were conducted on the floating U. prolifera thalli in comparison with the attached U. prolifera thalli collected from the area. The floating thalli of three distinctively different colors and attached thalli at three different stages of sporangium formation process were characterized under a microscope, while their photosynthetic parameters were determined with chlorophyll fluorescence technology. On the other hand, the sporangium formation status of the floating U. prolifera thalli was surveyed both in the laboratory and in the field. Comparisons showed that both of the paired morphological characteristics and the paired physiological parameters of the floating and attached U. prolifera thalli were consistent. Furthermore, some spores were confirmed in the field and some motile particles were found within the floating thalli. These results suggest that the floating U. prolifera thalli with different colors could be at different stages of sporangium formation. However, our results also showed that the floating alga thalli have only a limited reproductive potential. This might limit the duration and the further geographic expansion of the green algal bloom.

Correction: FtsHi4 Is Essential for Embryogenesis Due to Its Influence on Chloroplast Development in Arabidopsis.

[This corrects the article DOI: 10.1371/journal.pone.0099741.].

Overexpression of a cytosol-localized rhamnose biosynthesis protein encoded by Arabidopsis RHM1 gene increases rhamnose content in cell wall.

l-Rhamnose (Rha) is an important constituent of pectic polysaccharides, a major component of the cell walls of Arabidopsis, which is synthesized by three enzymes encoded by AtRHM1, AtRHM2/AtMUM4, and AtRHM3. Despite the finding that RHM1 is involved in root hair formation in Arabidopsis, experimental evidence is still lacking for the in vivo enzymatic activity and subcellular compartmentation of AtRHM1 protein. AtRHM1 displays high similarity to the other members of RHM family in Arabidopsis and in other plant species such as rice and grape. Expression studies with AtRHM1 promoter-GUS fusion gene showed that AtRHM1 was expressed almost ubiquitously, with stronger expression in roots and cotyledons of young seedlings and inflorescences. GFP::AtRHM1 fusion protein was found to be localized in the cytosol of cotyledon cells and of petiole cells of cotyledon, indicating that AtRHM1 is a cytosol-localized protein. The overexpression of AtRHM1 gene in Arabidopsis resulted in an increase of rhamnose content as much as 40% in the leaf cell wall compared to the wild type as well as an alteration in the contents of galactose and glucose. Fourier-transform infrared analyses revealed that surplus rhamnose upon AtRHM1 overexpression contributes to the construction of rhamnogalacturonan.

Buoyancy potential of dominant green macroalgal species in the Yellow Sea's green tides, China.

Large-scale green tides caused by Ulva prolifera, occurred for 12 consecutive years in the Yellow Sea of China. To resolve the abrupt shift in species composition between attached and floating macroalgal assemblages, field experiments were conducted from May to July 2017 to quantify the net buoyancy force and compare the floating potential of the common green macroalgae from the red algal seaweed Pyropia yezoensis rafts. At the same time, U. prolifera from different sampling locations were tested to study variable buoyancy of this species and the associated influencing factors. Our results illustrated a stronger positive buoyant force and a proportionally greater buoyancy capacity of U. prolifera, compared to the other co-occurring species. Buoyancy is a dynamic trait and is closely correlated with light intensity, morphology and physiological status. The positive buoyancy of U. prolifera is an important factor that helps explain its predominance in the Yellow Sea's large-scale green tides.

Reproduction diversity of Enteromorpha prolifera.

Enteromorpha prolifera (Muell.) J. Agardh (Chlorophyta, Ulvophyceae), which is distributed widely in the inter-tidal zone of the ocean, is one of the most common fouling green algae. However, the present understandings of the life history of E. prolifera have been insufficient to explain their seasonal abundances. Thus it is essential to investigate how many reproductive strategies are likely to contribute to the successful colonization and flourishing of the green alga. In the present study the reproduction diversity of E. prolifera was observed and studied systematically by culturing chopped tissues. Our results showed that there are in total seven pathways of reproduction for E. prolifera including sexual, asexual and vegetative reproduction. It was indicated that the variety of the reproductive ways and the large quantity of reproductive cells produced and released during the reproductive season are the two key factors that facilitate colonization of E. prolifera. The reproduction of the alga E. prolifera mainly depends on asexual methods. The results presented here contribute to increasing our understanding about how the opportunistic macroalgae successfully maintain colonization and excessive growth.

Molecular cloning and expression analysis of two key genes, HDS and HDR, in the MEP pathway in Pyropia haitanensis.

The 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (HDS) gene and the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene are two important genes in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In this study, we reported the isolation and characterization of full-length HDS (MF101802) and HDR (MF159558) from Pyropia haitanensis. Characteristics of 3-D structures of the PhHDS and PhHDR proteins were analysed respectively. The results showed that the full-length cDNA of PhHDS, which is 1801 bp long, contained a 1455 bp open reading frame (ORF) encoding a putative 484 amino acid residue protein with a predicted molecular mass of 51.60 kDa. Meanwhile, the full-length cDNA of PhHDR was 1668 bp and contained a 1434 bp ORF encoding a putative 477 amino acid 2 residue protein with a predicted molecular mass of 51.49 kDa. The expression levels of the two genes were higher in conchocelis than that in leafy thallus. Additionally, the expression levels could be influenced by light, temperature and salinity and induced by methyl jasmonate (MJ) and salicylic acid (SA). This study contributed to our in-depth understanding of the roles of PhHDS and PhHDR in terpenoid biosynthesis in Pyropia haitanensis and the regulation of the two genes by external environments.

Recyclable and Intrinsically Anti-cyanobacterial Polyanionic Membranes.

Cyanobacteria blooms possess serious threats to water resources. Herein, we report the synthesis of polyanionic membranes (PA-M) by in situ photo-crosslinking of a sulfate-based anionic monomer followed by cation-exchange with metal cations, Fe3+ (PA-Fe), Cu2+ (PA-Cu), or Zn2+ (PA-Zn). The effect of cations on the anti-cyanobacterial activities against both Microcystis aeruginosa (M. aeruginosa) and Anabaena flos-aquae (A. flos-aquae) was investigated. All the prepared metal-containing membranes (PA-Fe, PA-Cu, PA-Zn) exhibit high anti-cyanobacterial activities and long-term anti-cyanobacterial stability, demonstrating that the synthesized PA-M membranes can be used as an effective and safe inhibitor to control cyanobacterial blooms.

[Mechanism of heavy metal ions on RNase activity from bovine pancreas].

This paper studied mechanism of Ce3+, Cd2+, Pb2+ on RNase activity from bovine pancreas. The results showed that the activity of RNase was enhanced under the treatment by Ce3+, Cd2+, Pb2+ at lower concentration (10-60 or 10-30 mumol.L-1), but was inhibited by Ce3+, Cd2+, Pb2+ at higher concentration (40 or 70 mumol.L-1 above), and the inhibition was in the order as Pb2+ > Cd2+ > Ce3+. The equilibrium dialysis demonstrates that RNase may have one Ca(2+)-binding site. The fluorescence titration showed that one molecule of RNase has one binding site for Ce3+, the association constant k for its low-affinity Ce(3+)-binding site is 1.22 x 10(8) L.mol-1. However, it can bind three Cd2+ or Pb2+ and the association causing constant k for its low-affinity Cd2+ or Pb(2+)-binding site is 1.8 x 10(8) L.mol-1, 2.01 x 10(8) L.mol-1, respectively, and caused the conformational changes of RNase.