Extensive intragenomic variations of the 18S rDNA V4 region in the toxigenic diatom species Pseudo-nitzschia multistriata revealed through high-throughput sequencing.

Metabarcoding analysis is an effective technique for monitoring the domoic acid-producing Pseudo-nitzschia species in marine environments, uncovering high-levels of molecular diversity. However, such efforts may result in the overinterpretation of Pseudo-nitzschia species diversity, as molecular diversity not only encompasses interspecies and intraspecies diversities but also exhibits extensive intragenomic variations (IGVs). In this study, we analyzed the V4 region of the 18S rDNA of 30 strains of Pseudo-nitzschia multistriata collected from the coasts of China. The results showed that each P. multistriata strain harbored about a hundred of unique 18S rDNA V4 sequence varieties, of which each represented by a unique amplicon sequence variant (ASV). This study demonstrated the extensive degree of IGVs in P. multistriata strains, suggesting that IGVs may also present in other Pseudo-nitzschia species and other phytoplankton species. Understanding the scope and levels of IGVs is crucial for accurately interpreting the results of metabarcoding analysis.

Diversity and ecological function of urease-producing bacteria in the cultivation environment of Gracilariopsis lemaneiformis.

Urease-producing bacteria (UPB) provide inorganic nitrogen for primary producers by hydrolyzing urea, and play an important role in marine nitrogen cycle. However, there is still an incomplete understanding of UPB and their ecological functions in the cultivation environment of the red macroalgae Gracilariopsis lemaneiformis. This study comprehensively analyzed the diversity of culturable UPB and explored their effects on urea uptake by G. lemaneiformis. A total of 34 isolates belonging to four main bacterial phyla i.e. (Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria) were identified through 16S rRNA sequencing and were screened for UPB by urea agar chromogenic medium assay and ureC gene cloning. Our data revealed that only 8 strains contained urease. All of these UPB exhibited different urease activities, which were determined by the Berthelot reaction colorimetry assay. Additionally, the UPB strain (G13) isolated from G. lemaneiformis with higher urease activity was selected for co-culture with G. lemaneiformis to explore its role in promoting or inhibiting nitrogen uptake by macroalgae. The results showed a significant increase in urea consumption in the culture medium and the total cellular nitrogen in G. lemaneiformis in the UPB-co culture group compared to the sterile group. This suggests that the selected UPB strain positively influences nitrogen uptake by G. lemaneiformis. Similarly, isotopic assays revealed that the δ15N content of G. lemaneiformis was significantly higher in the UPB-co culture than in the control group, where δ15N-urea was the only nitrogen source in the culture medium. This indicates that the UPB helped G. lemaneiformis to absorb more nitrogen from urea. Moreover, the highest content of δ15N was found in G. lemaneiformis with epiphytic bacteria compared to sterilized (i.e. control), showing that epiphytic bacteria, along with UPB, have a compound effect in helping G. lemaneiformis absorb more nitrogen from urea. Taken together, these results provide unique insight into the ecological role of UPB and suggest that urease from macroalgae environment-associated bacteria might be an important player in marine nitrogen cycling.

Comparison and improvement of RNA extraction methods in Sargassum (Phaeophyta).

Sargassum (Sargassaceae) is widely distributed globally and plays an important role in regulating climate change, but the landscape of genomes and transcripts is less known. High-quality nucleic acids are the basis for molecular biology experiments such as high-throughput sequencing. Although extensive studies have documented methods of RNA extraction, these methods are not very applicable to Sargassum, which contains high levels of polysaccharides and polyphenols. To find a suitable method to improve the quality of RNA extracted, we compared and modified several popular RNA extraction methods and screened one practical method with three specific Sargassum spp. The results showed that three CTAB methods (denoted as Methods 1, 2, and 3) and the RNAprep Pure Plant Kit (denoted as Method 4) could, with slight modifications, effectively isolate RNA from Sargassum species, except for Method 4 used with S. fusiforme. By performing further screening, we determined Method 4 was the best choice for S. hemiphyllum and S. henslowianum, as revealed by RNA yields, RNA Integrity Number (RIN), extraction time, and unigene mapped ratio. For S. fusiforme, Methods 1, 2, and 3 showed no obvious differences among the yields, quality, or time to perform. In addition, one other method was tested, but we found the quality of the RNA extracted by TRIzol reagent methods (denoted as Method 5) performed the worst when compared with the above four methods. Therefore, our study provides four suitable methods for RNA extraction in Sargassum and is essential for future genetic exploration of Sargassum.

Unraveling the Diverse Profile of N-Acyl Homoserine Lactone Signals and Their Role in the Regulation of Biofilm Formation in Porphyra haitanensis-Associated Pseudoalteromonas galatheae.

N-acyl homoserine lactones (AHLs) are small, diffusible chemical signal molecules that serve as social interaction tools for bacteria, enabling them to synchronize their collective actions in a density-dependent manner through quorum sensing (QS). The QS activity from epiphytic bacteria of the red macroalgae Porphyra haitanensis, along with its involvement in biofilm formation and regulation, remains unexplored in prior scientific inquiries. Therefore, this study explores the AHL signal molecules produced by epiphytic bacteria. The bacterium isolated from the surface of P. haitanensis was identified as Pseudoalteromonas galatheae by 16s rRNA gene sequencing and screened for AHLs using two AHL reporter strains, Agrobacterium tumefaciens A136 and Chromobacterium violaceum CV026. The crystal violet assay was used for the biofilm-forming phenotype. The inferences revealed that P. galatheae produces four different types of AHL molecules, i.e., C4-HSL, C8-HSL, C18-HSL, and 3-oxo-C16-HSL, and it was observed that its biofilm formation phenotype is regulated by QS molecules. This is the first study providing insights into the QS activity, diverse AHL profile, and regulatory mechanisms that govern the biofilm formation phenotype of P. galatheae. These findings offer valuable insights for future investigations exploring the role of AHL producing epiphytes and biofilms in the life cycle of P. haitanensis.

Carotenoid Differences and Genetic Diversity in Populations of Sargassum hemiphyllum and Sargassum fusiforme.

Sargassum hemiphyllum and Sargassum fusiforme are important benthic seaweeds that grow along the southeastern coast of China. The content of carotenoids in each population was detected by ultra-high performance liquid chromatography (UHPLC). The research results will enrich the theoretical basis and data support concerning the influencing factors of carotenoids in Sargassum. The inter-simple sequence repeat (ISSR) technique was used to study the genetic diversity of four S. hemiphyllum and two S. fusiforme populations, and the results provide a reference for the artificial cultivation of Sargassum. The total carotenoid content of Sargassum ranged from 161.79 ± 4.22 to 269.47 ± 6.15 µg/g. Among the carotenoids, ß-carotene and fucoxanthin accounted for 80%, and levels in S. hemiphyllum were generally higher than those in S. fusiforme. The carotenoid contents of S. hemiphyllum from different areas were significantly different (P < 0.05), and the total carotenoids content decreased toward the southern region. The average heterozygosity H ranged from 0.29 to 0.49, and the Shannon diversity index I ranged from 0.44 to 0.69. The polymorphic loci, genetic diversity, and other indicators of S. hemiphyllum populations were higher than those of S. fusiforme, and the diversity of cultivated populations was not significantly lower. The results showed that the genetic variation of Sargassum is limited, and thus, more sexual reproduction can be attempted in breeding. Considering morphological indicators, genetic diversity indexes, and carotenoid content, S. hemiphyllum appears to have a higher commercial development value.

Golgi fucosyltransferase 1 reveals its important role in α-1,4-fucose modification of N-glycan in CRISPR/Cas9 diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum (Pt) is a critical microbial cell factory to produce a wide spectrum of marketable products including recombinant biopharmaceutical N-glycoproteins. N-glycosylation modification of proteins is important for their activity, stability, and half-life, especially some special modifications, such as fucose-modification by fucosyltransferase (FucT). Three PtFucTs were annotated in the genome of P. tricornutum, PtFucT1 was located on the medial/trans-Golgi apparatus and PtFucT2-3 in the plastid stroma. Algal growth, biomass and photosynthesis efficiency were significantly inhibited in a knockout mutant of PtFucT1 (PtFucT1-KO). PtFucT1 played a role in non-core fucose modification of N-glycans. The knockout of PtFucT1 might affect the activity of PtGnTI in the complex and change the complex N-glycan to mannose type N-glycan. The study provided critical information for understanding the mechanism of protein N-glycosylation modification and using microalgae as an alternative ecofriendly cell factory to produce biopharmaceuticals.

Rapid identification of salmonella serovars by using Raman spectroscopy and machine learning algorithm.

A widespread and escalating public health problem worldwide is foodborne illness, and foodborne Salmonella infection is one of the most common causes of human illness.For the three most pathogenic Salmonella serotypes, Raman spectroscopy was employed to acquire spectral data.As machine learning offers high efficiency and accuracy, we have chosen the convolutional neural network(CNN), which is suitable for solving multi-classification problems, to do in-depth mining and analysis of Raman spectral data.To optimize the instrument parameters, we compared three laser wavelengths: 532, 638, and 785 nm.Ultimately, the 532 nm wavelength was chosen as the most effective for detecting Salmonella.A pre-processing step is necessary to remove interference from the background noise of the Raman spectrum.Our study compared the effects of five spectral preprocessing methods, Savitzky-Golay smoothing (SG), Multivariate Scatter Correction (MSC), Standard Normal Variate (SNV), and Hilbert Transform (HT), on the predictive power of CNN models.Accuracy(ACC), Precision, Recall, and F1-score 4 machine learning evaluation indicators are used to evaluate the model performance under different preprocessing methods.In the results, SG combined with SNV was found to be the most accurate spectral pre-processing method for predicting Salmonella serotypes using Raman spectroscopy, achieving an accuracy of 98.7% for the training set and over 98.5% for the test set in CNN model.Pre-processing spectral data using this method yields higher accuracy than other methods.As a conclusion, the results of this study demonstrate that Raman spectroscopy when used in conjunction with a convolutional neural network model enables the rapid identification of three Salmonella serotypes at the single-cell level, and that the model has a great deal of potential for distinguishing between different serotypes of pathogenic bacteria and closely related bacterial species.This is vital to preventing outbreaks of foodborne illness and the spread of foodborne pathogens.

Different photosynthetic responses of haploid and diploid Emiliania huxleyi (Prymnesiophyceae) to high light and ultraviolet radiation.

Solar radiation varies quantitatively and qualitatively while penetrating through the seawater column and thus is one of the most important environmental factors shaping the vertical distribution pattern of phytoplankton. The haploid and diploid life-cycle phases of coccolithophores might have different vertical distribution preferences. Therefore, the two phases respond differently to high solar photosynthetically active radiation (PAR, 400-700 nm) and ultraviolet radiation (UVR, 280-400 nm). To test this, the haploid and diploid Emiliania huxleyi were exposed to oversaturating irradiance. In the presence of PAR alone, the effective quantum yield was reduced by 10% more due to the higher damage rate of photosystem II in haploid cells than in diploid cells. The addition of UVR resulted in further inhibition of the quantum yield for both haploid and diploid cells in the first 25 min, partly because of the increased damage of photosystem II. Intriguingly, this UVR-induced inhibition of the haploid cells completely recovered half an hour later. This recovery was confirmed by the comparable maximum quantum yields, maximum relative electron transport rates and yields of the haploid cells treated with PAR and PAR + UVR. Our data indicated that photosynthesis of the haploid phase was more sensitive to high visible light than the diploid phase but resistant to UVR-induced inhibition, reflecting the ecological niches to which this species adapts.

Endoplasmic reticulum-quality control pathway and endoplasmic reticulum-associated degradation mechanism regulate the N-glycoproteins and N-glycan structures in the diatom Phaeodactylum tricornutum.

Tunicamycin inhibits the first step of protein N-glycosylation modification. However, the physiological, transcriptomic, and N-glycomic effects of tunicamycin on important marine diatom Phaeodactylum tricornutum are still unknown. In this study, comprehensive approaches were used to study the effects of tunicamycin stress. The results showed that cell growth and photosynthesis were significantly inhibited in P. tricornutum under the tunicamycin stress. The soluble protein content was significantly decreased, while the soluble sugar and neutral lipid were dramatically increased to orchestrate the balance of carbon and nitrogen metabolisms. The stress of 0.3 µg ml-1 tunicamycin resulted in the differential expression of ERQC and ERAD related genes. The upregulation of genes involved in ERQC pathway, the activation of anti-oxidases and the differential expression of genes related with ERAD mechanism might be important for maintaining homeostasis in cell. The identification of N-glycans, especially complex-type N-glycan structures enriched the N-glycan database of diatom P. tricornutum and provided important information for studying the function of N-glycosylation modification on proteins. As a whole, our study proposed working models of ERQC and ERAD will provide a solid foundation for further in-depth study of the related mechanism and the diatom expression system.

Low-intensity transcranial ultrasound stimulation facilitates hand motor function and cortical excitability: A crossover, randomized, double blind study.

Objective: Transcranial ultrasound stimulation (TUS) is a new form of non-invasive brain stimulation. Low-intensity TUS is considered highly safe. We aimed to investigate the effect of low-intensity TUS on hand reaction responses and cortical excitability in healthy adults. Methods: This study used a crossover, randomized, and double-blind design. A total of 20 healthy participants were recruited for the study. All the participants received TUS and sham stimulation on separate days in random order. The finger tapping test (tapping score by using a tablet) and motor evoked potential (MEP) were assessed before and after stimulation, and discomfort levels were assessed using a visual analog scale (VAS) score. Results: No significant differences in tapping score or MEP amplitude between the two experimental conditions were registered before stimulation. After stimulation, tapping scores were increased regardless of the specific treatment, and the real stimulation condition receiving TUS (90.4 ± 11.0 points) outperformed the sham stimulation condition (86.1 ± 8.4 points) (p = 0.002). The MEP latency of real TUS (21.85 ± 1.33 ms) was shorter than that of sham TUS (22.42 ± 1.43 ms) (p < 0.001). MEP amplitude of real TUS (132.18 ± 23.28 µV) was higher than that of sham TUS (114.74 ± 25.5 µV, p = 0.005). There was no significant difference in the discomfort score between the two conditions (p = 0.163). Conclusion: Transcranial ultrasound stimulation (TUS) can decrease the hand reaction response time and latency of the MEP, enhance the excitability of the motor cortex, and improve hand motor function in healthy individuals without obvious discomfort.

MYB gene family in the diatom Phaeodactylum tricornutum revealing their potential functions in the adaption to nitrogen deficiency and diurnal cycle.

The MYB transcription factor (TF) family is one of the largest and most important TF families, regulating the growth and response of microalgae to stress. However, the gene structure and characteristics of Phaeodactylum tricornutum MYB TFs, and their functions under nitrogen deficiency, have not been explored yet. To identify all P. tricornutum MYB (PtMYB) genes, the MYB gene family was analyzed at the genome-wide level in this study. A total ofm26 PtMYB genes were identified from the genome of P. tricornutum. These PtMYB genes were divided into 5 subfamilies: 5R-MYB, 4R-MYB, R2R3-MYB, R1R2R3-MYB, and MYB-related proteins. Phylogenetical motif and gene structure analyses of MYB genes indicated that the number and proportion of MYB TFs were species-specific, and MYB genes exhibited a lot of duplication events from microalgae to higher plants. Furthermore, the differentially expressed patterns of all 26 PtMYB TFs implied that PtMYB genes might have functional specificity under nitrogen deficiency. Homology analysis of MYB genes revealed that PtMYB3, PtMYB15, and PtMYB21 might play important roles in the regulation of the diurnal cycle and response to nitrogen stress in P. tricornutum. PtMYB3, PtMYB15, and PtMYB21 genes might be used as potential candidate genes for further studying the regulatory mechanisms of P. tricornutum under nitrogen deficiency. This work provides an important foundation for the future research of the potential functions of PtMYB genes and its diurnal regulatory mechanisms under nitrogen deficiency.

Global Profiling of N-Glycoproteins and N-Glycans in the Diatom Phaeodactylum tricornutum.

N-glycosylation is an important posttranslational modification in all eukaryotes, but little is known about the N-glycoproteins and N-glycans in microalgae. Here, N-glycoproteomic and N-glycomic approaches were used to unveil the N-glycoproteins and N-glycans in the model diatom Phaeodactylum tricornutum. In total, 863 different N-glycopeptides corresponding to 639 N-glycoproteins were identified from P. tricornutum. These N-glycoproteins participated in a variety of important metabolic pathways in P. tricornutum. Twelve proteins participating in the N-glycosylation pathway were identified as N-glycoproteins, indicating that the N-glycosylation of these proteins might be important for the protein N-glycosylation pathway. Subsequently, 69 N-glycans corresponding to 59 N-glycoproteins were identified and classified into high mannose and hybrid type N-glycans. High mannose type N-glycans contained four different classes, such as Man-5, Man-7, Man-9, and Man-10 with a terminal glucose residue. Hybrid type N-glycan harbored Man-4 with a terminal GlcNAc residue. The identification of N-glycosylation on nascent proteins expanded our understanding of this modification at a N-glycoproteomic scale, the analysis of N-glycan structures updated the N-glycan database in microalgae. The results obtained from this study facilitate the elucidation of the precise function of these N-glycoproteins and are beneficial for future designing the microalga to produce the functional humanized biopharmaceutical N-glycoproteins for the clinical therapeutics.

Laminarin, a Major Polysaccharide in Stramenopiles.

During the processes of primary and secondary endosymbiosis, different microalgae evolved to synthesis different storage polysaccharides. In stramenopiles, the main storage polysaccharides are ß-1,3-glucan, or laminarin, in vacuoles. Currently, laminarin is gaining considerable attention due to its application in the food, cosmetic and pharmaceuticals industries, and also its importance in global biogeochemical cycles (especially in the ocean carbon cycle). In this review, the structures, composition, contents, and bioactivity of laminarin were summarized in different algae. It was shown that the general features of laminarin are species-dependence. Furthermore, the proposed biosynthesis and catabolism pathways of laminarin, functions of key genes, and diel regulation of laminarin were also depicted and comprehensively discussed for the first time. However, the complete pathways, functions of genes, and diel regulatory mechanisms of laminarin require more biomolecular studies. This review provides more useful information and identifies the knowledge gap regarding the future studies of laminarin and its applications.

Influence of phosphorus on the uptake and biotransformation of arsenic in Porphyra haitanensis at environmental relevant concentrations.

Edible seaweeds are rich in essential vitamins and minerals, which made them a popular food worldwide. Porphyra haitanensis is one of the most commonly consumed seaweeds with the known ability to accumulate a high level of total arsenic (As). A large number of articles have shown arsenic and phosphorus (P) interactions in microalgae due to the plant's inability to differentiate arsenate from phosphate. However, very limited information is available for edible seaweed at environmentally relevant concentrations. In this study, P. haitanensis was treated with arsenic as AsV (As1: 0.06 µM, As2: 0.4 µM, As3: 1.2 µM) and phosphorous (P1: 3.2 µM, P2: 13 µM) in a filtered seawater matrix under laboratory condition for six days. A better growth rate was found in seaweeds grown in P2 treatments. Moreover, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content measurements revealed that a higher P concentration prevent seaweeds from lipid peroxidation and oxidative stress. Transcriptome studies indicated the As replacement to P has the ability to target seaweed cell membrane composition, transmembrane transport, DNA and ATP binding. The inorganic As (iAs) had a concentration of 0.54 to 4.45 mg/kg in P. haitanensis on Day 6 with As1, As2, and As3 treatments under low P regime (P1), which exceeds the limits of iAs concentration (0.1-0.5 mg/kg) in National Food Safety Standard-Limits of Pollutants in Food (GB 2762-2017). High P regime (P2) not only reduced the total As but also iAs effectively, even in the highest As treatment (As3), the iAs concentration was less than 0.5 mg/kg on Day 6. These findings provide a good insight for seafood safety guarantees and are important for the management of coastal artificial seaweed farming.

Environmental factors shape the epiphytic bacterial communities of Gracilariopsis lemaneiformis.

Macroalgae host various symbionts on their surface, which play a critical role in their growth and development processes. However, there is still incomplete understanding of this epiphytic bacteria-host algae interactions. This study comprehensively analysed variation of the epiphytic bacterial communities (EBC) composition of red macroalga Gracilariopsis lemaneiformis at different geographic locations and environmental factors (i.e., nitrogen and phosphorus), which shape the EBC composition of G. lemaneiformis. The composition and structure of EBC were characterized using high throughput sequencing of the V3-V4 hypervariable region of the 16S rRNA gene. The results revealed that epiphytic bacteria varied significantly among three different geographic locations in China, i.e., Nan'ao Island (NA), Lianjiang County (LJ), and Nanri Island (NR). Redundancy analysis (RDA) showed that the relative abundance of Bacteroidetes, Firmicutes, Verrucomicrobia, and Epsilonbacteraeota at NR were strongly positively correlated with total nitrogen (TN), total phosphorus (TP), nitrate nitrogen (NO3-N), and dissolved inorganic nitrogen (DIN), but negatively correlated with nitrite nitrogen (NO2-N). The relative abundance of Cyanobacteria at NA and LJ were strongly positively correlated with NO2-N, but negatively correlated with TN, TP, NO3-N, and DIN. Besides, the Mantel test results indicated that the EBC composition was significantly correlated with these environmental factors, which was also confirmed by Spearman correlation analysis. Thus, environmental factors such as NO3-N and DIN play a key role in the community composition of epiphytic bacteria on G. lemaneiformis. This study provides important baseline knowledge on the community composition of epiphytic bacteria on G. lemaneiformis and shows correlation between different epiphytic bacteria and their surrounding environmental factors.

Structural and Magnetical Studies of Mixed-Valence Hexavanadate Hybrids: How Organic Ligands Affect the Magnetism of Polyoxometalates?

In this Communication, we illustrate the influence of organic ligands on magnetic structure and behavior by employing a mixed-valence Lindqvist-type hexavanadate as a research platform. Through covalently attaching to different halogen-containing organic ligands, the derived hybrid materials have different magnetism compared to their parent structure. Single-crystal X-ray analyses show that the introduction of organic ligands can modify the crystal packing manners of the derivatives, leading to further changes of the interaction between magnetic units. This work demonstrates that organic functionalization can remarkably affect the magnetism of polyoxometalates by adjusting the distance and location of the magnetic fractions.

Seaweed farms provide refugia from ocean acidification.

Seaweed farming has been proposed as a strategy for adaptation to ocean acidification, but evidence is largely lacking. Changes of pH and carbon system parameters in surface waters of three seaweed farms along a latitudinal range in China were compared, on the weeks preceding harvesting, with those of the surrounding seawaters. Results confirmed that seaweed farming is efficient in buffering acidification, with Saccharina japonica showing the highest capacity of 0.10 pH increase within the aquaculture area, followed by Gracilariopsis lemaneiformis (ΔpH = 0.04) and Porphyra haitanensis (ΔpH = 0.03). The ranges of pH variability within seaweed farms spanned 0.14-0.30 unit during the monitoring, showing intense fluctuations which may also help marine organisms adapt to enhanced pH temporal variations in the future ocean. Deficit in pCO2 in waters in seaweed farms relative to control waters averaged 58.7 ± 15.9 µatm, ranging from 27.3 to 113.9 µatm across farms. However, ΔpH did not significantly differ between day and night. Dissolved oxygen and Ωarag were also elevated in surface waters at all seaweed farms, which are benefit for the survival of calcifying organisms. Seaweed farming, which unlike natural seaweed forests, is scalable and is not dependent on suitable substrate or light availability, could serve as a low-cost adaptation strategy to ocean acidification and deoxygenation and provide important refugia from ocean acidification.

The complete plastid genome and phylogenetic analysis of Gracilaria chilensis.

Gracilaria chilensis is an economically important species of macroalgae. The plastid genome sequence of G. chilensis is 185,640 bp with a GC content of 29.34%. A total of 236 genes were determined, containing 203 protein-encoding genes, three rRNA genes, 30 tRNA genes, and one intron (with intronic ORF) inserted into the trnM gene. The gene content and structure of Gracilariaceae species were relatively well conserved. The phylogenetic analysis, based on the red algal plastid genomes, suggested that G. chilensis had a closer relationship with Gracilaria tenuistipitata var. liui in Gracilaria.

Glutamine Synthetase (GS): A Key Enzyme for Nitrogen Assimilation in The Macroalga Gracilariopsis lemaneiformis (Rhodophyta).

This study aimed to address the importance of glutamine synthetase II (GSII) during nitrogen assimilation in macroalga Gracilariopsis lemaneiformis. The cDNA full-length sequence of the three glGSII genes was revealed to have the 5' m7 G cap, 5'-untranslated region, open reading frame (ORF), 3'-untranslated region, and a 3' poly (A) tail. The three glGSIIs were classified into plastid glGS2 and cytosolic glGS1-1 and glGS1-2, having conserved GSII domains but different cDNA sequences. The complicated 5' end flanking region indicates complex function of glGS genes. glGS1 genes were significantly up-regulated under the different NH4+ : NO3- ratio (i.e., 40:10, 25:25, 10:40, and 0:50) except glGS2 which dramatically up-regulated under the low NH4+ : NO3- ratio (i.e., 10:40 and 0:50) during different cultivation times. These different expression patterns perhaps are due to the different biological roles of GS1 and GS2 in the gene family. Furthermore, hypothetical working model of nitrogen assimilation pathway exhibiting the role of glGS1 and glGS2 is proposed. Finally, glGS2 was expressed in Escherichia coli BL21 (DE3), and the optimal conditions for culture (15°C, overnight), purification (500 mM imidazole washing), and activity (pH 7.4, 37°C) were established. This study lays a very important foundation for exploring the role of GS in nitrogen assimilation in algae and plants.