Rosetta gen. nov. (Chlorophyta): Resolving the identity of red snow algal rosettes.

Thick-walled rosette-like snow algae were long thought to be a life stage of various other species of snow algae. Rosette-like cells have not been cultured, but by manually isolating cells from 38 field samples in southern British Columbia, we assigned a variety of rosette morphologies to DNA sequence. Phylogenetic analysis of Rubisco large-subunit (rbcL) gene, ribosomal internal transcribed spacer 2 (ITS2) rRNA region, and 18S rRNA gene revealed that the rosette-like cells form a new clade within the phylogroup Chloromonadinia. Based on these data, we designate a new genus, Rosetta, which comprises five novel species: R. castellata, R. floranivea, R. stellaria, R. rubriterra, and R. papavera. In a survey of 762 snow samples from British Columbia, we observed R. floranivea exclusively on snow overlying high-elevation glaciers, whereas R. castellata was observed at lower elevations, near the tree line. The other three species were rarely observed. Spherical red cells enveloped in a thin translucent sac were conspecific with Rosetta, possibly a developmental stage. These results highlight the unexplored diversity among snow algae and emphasize the utility of single-cell isolation to advance the centuries-old problem of disentangling life stages and cryptic species.

Meta-analysis of transcriptomic profiles in Dunaliella tertiolecta reveals molecular pathway responses to different abiotic stresses.

Microalgae are photosynthetic organisms and a potential source of sustainable metabolite production. However, different stress conditions might affect the production of various metabolites. In this study, a meta-analysis of RNA-seq experiments in Dunaliella tertiolecta was evaluated to compare metabolite biosynthesis pathways in response to abiotic stress conditions such as high light, nitrogen deficiency and high salinity. Results showed downregulation of light reaction, photorespiration, tetrapyrrole and lipid-related pathways occurred under salt stress. Nitrogen deficiency mostly induced the microalgal responses of light reaction and photorespiration metabolism. Phosphoenol pyruvate carboxylase, phosphoglucose isomerase, bisphosphoglycerate mutase and glucose-6-phosphate-1-dehydrogenase (involved in central carbon metabolism) were commonly upregulated under salt, light and nitrogen stresses. Interestingly, the results indicated that the meta-genes (modules of genes strongly correlated) were located in a hub of stress-specific protein-protein interaction (PPI) network. Module enrichment of meta-genes PPI networks highlighted the cross-talk between photosynthesis, fatty acids, starch and sucrose metabolism under multiple stress conditions. Moreover, it was observed that the coordinated expression of the tetrapyrrole intermediated with meta-genes was involved in starch biosynthesis. Our results also showed that the pathways of vitamin B6 metabolism, methane metabolism, ribosome biogenesis and folate biosynthesis responded specifically to different stress factors. Since the results of this study revealed the main pathways underlying the abiotic stress, they might be applied in optimised metabolite production by the microalga Dunaliella in future studies. PRISMA check list was also included in the study.

Novel insights in cryptic diversity of snow and glacier ice algae communities combining 18S rRNA gene and ITS2 amplicon sequencing.

Melting snow and glacier surfaces host microalgal blooms in polar and mountainous regions. The aim of this study was to determine the dominant taxa at the species level in the European Arctic and the Alps. A standardized protocol for amplicon metabarcoding using the 18S rRNA gene and ITS2 markers was developed. This is important because previous biodiversity studies have been hampered by the dominance of closely related algal taxa in snow and ice. Due to the limited resolution of partial 18S rRNA Illumina sequences, the hypervariable ITS2 region was used to further discriminate between the genotypes. Our results show that red snow was caused by the cosmopolitan Sanguina nivaloides (Chlamydomonadales, Chlorophyta) and two as of yet undescribed Sanguina species. Arctic orange snow was dominated by S. aurantia, which was not found in the Alps. On glaciers, at least three Ancylonema species (Zygnematales, Streptophyta) dominated. Golden-brown blooms consisted of Hydrurus spp. (Hydrurales, Stramenophiles) and these were mainly an Arctic phenomenon. For chrysophytes, only the 18S rRNA gene but not ITS2 sequences were amplified, showcasing how delicate the selection of eukaryotic 'universal' primers for community studies is and that primer specificity will affect diversity results dramatically. We propose our approach as a 'best practice'.

Hydrocytium expands the phylogenetic, morphological, and genomic diversity of the poorly known green algal order Chaetopeltidales.

PREMISE: Chaetopeltidales is a small, understudied order of the green algal class Chlorophyceae, that is slowly expanding with the occasional discoveries of novel algae. Here we demonstrate that hitherto unrecognized chaetopeltidaleans also exist among previously described but neglected and misclassified species. METHODS: Strain SAG 40.91 of Characium acuminatum, shown by previous preliminary evidence to have affinities with the orders Oedogoniales, Chaetophorales, and Chaetopeltidales (together constituting the OCC clade), was investigated with light and electron microscopy to characterize its morphology and ultrastructure. Sequence assemblies of the organellar and nuclear genomes were obtained and utilized in bioinformatic and phylogenetic analyses to address the phylogenetic position of the alga and its salient genomic features. RESULTS: The characterization of strain SAG 40.91 and a critical literature review led us to reinstate the forgotten genus Hydrocytium A.Braun 1855, with SAG 40.91 representing its type species, Hydrocytium acuminatum. Independent molecular markers converged on placing H. acuminatum as a deeply diverged lineage of the order Chaetopeltidales, formalized as the new family Hydrocytiaceae. Both chloroplast and mitochondrial genomes shared characteristics with other members of Chaetopeltidales and were bloated by repetitive sequences. Notably, the mitochondrial cox2a gene was transferred into the nuclear genome in the H. acuminatum lineage, independently of the same event in Volvocales. The nuclear genome data from H. acuminatum and from another chaetopeltidalean that was reported by others revealed endogenized viral sequences corresponding to novel members of the phylum Nucleocytoviricota. CONCLUSIONS: The resurrected genus Hydrocytium expands the known diversity of chaetopeltidalean algae and provides the first glimpse into their virosphere.

"DNA signaturing" database construction for Tetradesmus species identification and phylogenetic relationships of Scenedesmus-like green microalgae (Scenedesmaceae, Chlorophyta).

Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST.

The austral biflagellate Chloromonas rubroleosa (Chlorophyceae) is the closest relative of the unusual quadriflagellate genus Chlainomonas, both found in snow.

The quadriflagellate genus Chlainomonas frequently dominates red snow globally. It is unusual in several respects, with two separated pairs of flagella, apparent cell division via extrusion of cytoplasmic threads, and being nested phylogenetically within the biflagellate genus Chloromonas. Here, we showed that the austral species Chloromonas (Cr.) rubroleosa, originally described from Antarctic red snow, is a close biflagellate relative of Chlainomonas, challenging the monophyly of Chlainomonas as currently conceived. Sequences of the 18S rRNA gene robustly linked Cr. rubroleosa with near-identical environmental sequences from Antarctic red snow and Chlainomonas from North America, Japan, and Europe. Furthermore, the 18S rRNA and rbcL gene sequences of Cr. rubroleosa were almost identical to New Zealand and North American collections of Chlainomonas. Cr. rubroleosa and New Zealand Chlainomonas are separated by only a single-base substitution across the ITS1-5.8S-ITS2 rRNA loci (and according to ITS2, the North American collection is the next closest relative). This again raises the possibility that Chlainomonas is a life-cycle stage of vegetatively biflagellate organisms, although this remains confounded by the scarcity of biflagellates in field populations, the apparent cell division by quadriflagellates, and the absence of Chlainomonas-type cells in cultures of Cr. rubroleosa. The latter species is broadly similar to Chlainomonas, being poor at swimming, with similar pigment, chloroplast arrangement and ultrastructure, and is relatively large. Increased size is a feature of the wider clade of "Group D" snow algae. A synthesis of field and laboratory investigations may be needed to unravel the life cycle and correct the systematics of this group.

DsLCYB Directionally Modulated ß-Carotene of the Green Alga Dunaliella salina under Red Light Stress.

Carotenoids, which are natural pigments found abundantly in wide-ranging species, have diverse functions and high industrial potential. The carotenoid biosynthesis pathway is very complex and has multiple branches, while the accumulation of certain metabolites often affects other metabolites in this pathway. The DsLCYB gene that encodes lycopene cyclase was selected in this study to evaluate ß-carotene production and the accumulation of ß-carotene in the alga Dunaliella salina. Compared with the wild type, the transgenic algal species overexpressed the DsLCYB gene, resulting in a significant enhancement of the total carotenoid content, with the total amount reaching 8.46 mg/g for an increase of up to 1.26-fold. Interestingly, the production of α-carotene in the transformant was not significantly reduced. This result indicated that the regulation of DsLCYB on the metabolic flux distribution of carotenoid biosynthesis is directional. Moreover, the effects of different light-quality conditions on ß-carotene production in D. salina strains were investigated. The results showed that the carotenoid components of ß-carotene and ß-cryptoxanthin were 1.8-fold and 1.23-fold higher than that in the wild type under red light stress, respectively. This suggests that the accumulation of ß-carotene under red light conditions is potentially more profitable.

Phylotranscriptomic and Evolutionary Analyses of the Green Algal Order Chaetophorales (Chlorophyceae, Chlorophyta).

Considering the phylogenetic differences in the taxonomic framework of the Chaetophorales as determined by the use of nuclear molecular markers or chloroplast genes, the current study was the first to use phylotranscriptomic analyses comparing the transcriptomes of 12 Chaetophorales algal species. The results showed that a total of 240,133 gene families and 143 single-copy orthogroups were identified. Based on the single-copy orthogroups, supergene analysis and the coalescent-based approach were adopted to perform phylotranscriptomic analysis of the Chaetophorales. The phylogenetic relationships of most species were consistent with those of phylogenetic analyses based on the chloroplast genome data rather than nuclear molecular markers. The Schizomeriaceae and the Aphanochaetaceae clustered into a well-resolved basal clade in the Chaetophorales by either strategy. Evolutionary analyses of divergence time and substitution rate also revealed that the closest relationships existed between the Schizomeriaceae and Aphanochaetaceae. All species in the Chaetophorales exhibited a large number of expanded and contracted gene families, in particular the common ancestor of the Schizomeriaceae and Aphanochaetaceae. The only terrestrial alga, Fritschiella tuberosa, had the greatest number of expanded gene families, which were associated with increased fatty acid biosynthesis. Phylotranscriptomic and evolutionary analyses all robustly identified the unique taxonomic relationship of Chaetophorales consistent with chloroplast genome data, proving the advantages of high-throughput data in phylogeny.

Research of Fluridone's Effects on Growth and Pigment Accumulation of Haematococcus pluvialis Based on Transcriptome Sequencing.

Haematococcus pluvialis has high economic value because of its high astaxanthin-producing ability. The mutation breeding of Haematococcus pluvialis is an important method to improve the yield of astaxanthin. Fluoridone, an inhibitor of phytoene dehydrogenase, can be used as a screening reagent for mutation breeding of Haematococcus pluvialis. This study describes the effect of fluridone on the biomass, chlorophyll, and astaxanthin content of Haematococcus pluvialis at different growth stages. Five fluridone concentrations (0.00 mg/L, 0.25 mg/L, 0.50 mg/L, 1.00 mg/L, and 2.00 mg/L) were set to treat Haematococcus pluvialis. It was found that fluridone significantly inhibited the growth and accumulation of astaxanthin in the red dormant stage. In addition, transcriptome sequencing was used to analyze the expression of genes related to four metabolic pathways in photosynthesis, carotenoid synthesis, fatty acid metabolism, and cellular antioxidant in algae after fluridone treatment. The results showed that six genes related to photosynthesis were downregulated. FPPS, lcyB genes related to carotenoid synthesis are downregulated, but carotenoid ß-cyclic hydroxylase gene (LUT5), which plays a role in the conversion of carotenoid to abscisic acid (ABA), was upregulated, while the expression of phytoene dehydrogenase gene did not change. Two genes related to cell antioxidant capacity were upregulated. In the fatty acid metabolism pathway, the acetyl-CoA carboxylase gene (ACACA) was downregulated in the green stage, but upregulated in the red stage, and the stearoyl-CoA desaturase gene (SAD) was upregulated. According to the transcriptome results, fluridone can affect the astaxanthin accumulation and growth of Haematococcus pluvialis by regulating the synthesis of carotenoids, chlorophyll, fatty acids, and so on. It is expected to be used as a screening agent for the breeding of Haematococcus pluvialis. This research also provides an experimental basis for research on the mechanism of astaxanthin metabolism in Haematococcus pluvialis.

When you Like Other Algae: Adglutina synurophila gen. et sp. nov. (Moewusinia, Chlorophyceae), a Clingy Green Microalga Associated with Synura Colonies.

Unicellular green biflagellates of the order Volvocales (Chlorophyceae, Chlorophyta) are common inhabitants of various types of habitats, and can also form peculiar interspecific relationships. Most of their morphological diversity has historically been assigned to the two prominent genera Chlamydomonas and Chloromonas. Ongoing reclassification of these algae, aided by molecular phylogenetics, has resulted in numerous newly proposed genera, but there are certainly brand-new taxa awaiting recognition. In this study, based on morphological and ultrastructural observations together with sequence data of the nuclear 18S and ITS2 rDNA and the plastid rbcL gene, we describe Adglutina synurophila gen. et sp. nov., a volvocalean biflagellate isolated from colonies of the golden-brown alga Synura petersenii (Chrysophyceae). Phylogenetic analyses placed Adglutina in the phylogroup Moewusinia as a sister lineage to the acidophilic "Chlamydomonas" species. It is characterised by having oval to broadly ellipsoidal cells with a low keel-shaped papilla and a cup-shaped chloroplast lacking a pyrenoid, but possessing a lateral eyespot of a variable position. The unique set of features, together with its Synura-loving nature, anchor Adglutina as a well distinguishable phylogenetic lineage within the Moewusinia. The novel alga has a widespread distribution; it has been found in three European countries to date.

Inhibitory properties of crude microalgal extracts on the in vitro replication of cyprinid herpesvirus 3.

Microalgae are possible sources of antiviral substances, e.g. against cyprinid herpesvirus 3 (CyHV-3). Although this virus leads to high mortalities in aquacultures, there is no treatment available yet. Hence, ethanolic extracts produced with accelerated solvent extraction from six microalgal species (Arthrospira platensis, Chlamydomonas reinhardtii, Chlorella kessleri, Haematococcus pluvialis, Nostoc punctiforme and Scenedesmus obliquus) were examined in this study. An inhibition of the in vitro replication of CyHV-3 could be confirmed for all six species, with the greatest effect for the C. reinhardtii and H. pluvialis crude extracts. At still non-cytotoxic concentrations, viral DNA replication was reduced by over 3 orders of magnitude each compared to the untreated replication controls, while the virus titers were even below the limit of detection (reduction of 4 orders of magnitude). When pre-incubating both cells and virus with C. reinhardtii and H. pluvialis extracts before inoculation, the reduction of viral DNA was even stronger (> 4 orders of magnitude) and no infectious viral particles were detected. Thus, the results of this study indicate that microalgae and cyanobacteria are a promising source of natural bioactive substances against CyHV-3. However, further studies regarding the isolation and identification of the active components of the extracts are needed.

Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

The Papain-like Cysteine Protease HpXBCP3 from Haematococcus pluvialis Involved in the Regulation of Growth, Salt Stress Tolerance and Chlorophyll Synthesis in Microalgae.

The papain-like cysteine proteases (PLCPs), the most important group of cysteine proteases, have been reported to participate in the regulation of growth, senescence, and abiotic stresses in plants. However, the functions of PLCPs and their roles in stress response in microalgae was rarely reported. The responses to different abiotic stresses in Haematococcus pluvialis were often observed, including growth regulation and astaxanthin accumulation. In this study, the cDNA of HpXBCP3 containing 1515 bp open reading frame (ORF) was firstly cloned from H. pluvialis by RT-PCR. The analysis of protein domains and molecular evolution showed that HpXBCP3 was closely related to AtXBCP3 from Arabidopsis. The expression pattern analysis revealed that it significantly responds to NaCl stress in H. pluvialis. Subsequently, transformants expressing HpXBCP3 in Chlamydomonas reinhardtii were obtained and subjected to transcriptomic analysis. Results showed that HpXBCP3 might affect the cell cycle regulation and DNA replication in transgenic Chlamydomonas, resulting in abnormal growth of transformants. Moreover, the expression of HpXBCP3 might increase the sensitivity to NaCl stress by regulating ubiquitin and the expression of WD40 proteins in microalgae. Furthermore, the expression of HpXBCP3 might improve chlorophyll content by up-regulating the expression of NADH-dependent glutamate synthases in C. reinhardtii. This study indicated for the first time that HpXBCP3 was involved in the regulation of cell growth, salt stress response, and chlorophyll synthesis in microalgae. Results in this study might enrich the understanding of PLCPs in microalgae and provide a novel perspective for studying the mechanism of environmental stress responses in H. pluvialis.

Lipid accumulation by Coelastrella multistriata (Scenedesmaceae, Sphaeropleales) during nitrogen and phosphorus starvation.

A novel freshwater strain of Coelastrella multistriata MZ-Ch23 was discovered in Tula region, Russia. The identification is based on morphological features, phylogenetic analysis of SSU rDNA gene and ITS1-5.8S rDNA-ITS2 region and predicted secondary structure of the ITS2. Phylogenetic analysis places the novel strain in the "core" Coelastrella clade within the Chlorophyceae. This is the first record of Coelastrella multistriata in the algal flora of Russia. Cultivation experiments were carried out to evaluate growth dynamics of the newly identified strain and the impact of nitrogen and/or phosphorus depletion on the fatty acid profiles and lipid productivity. On the fully supplemented Bold's basal medium and under phosphorus-depleted conditions as well, the fatty acid profiles were dominated by α-linolenic acid (29.4-38.1% of total fatty acids). Depletion of either nitrogen or both nitrogen and phosphorus was associated with increased content of oleic acid (32.9-33.7%) and linoleic acid (11.9%). Prolongation of the growth to two months (instead of 25 days) resulted in increased content and diversity of very long-chain fatty acids including saturated species. The total very long-chain fatty acid content of 9.99% achieved in these experiments was 1.9-12.3-fold higher than in stress experiments. The highest variation was observed for oleic acid (3.4-33.7%). The novel strain showed the ability to accumulate lipids in amounts up to 639.8 mg L-1 under nitrogen and phosphorus starvation, which exceeds the previously obtained values for most Coelastrella strains. Thus, the newly identified MZ-Ch23 strain can be considered as a potential producer of omega-3 fatty acids on fully supplemented Bold's basal medium or as a source of biomass with high content of saturated and monounsaturated fatty acids after nitrogen and phosphorus starvation.

Ancient origin of fucosylated xyloglucan in charophycean green algae.

The charophycean green algae (CGA or basal streptophytes) are of particular evolutionary significance because their ancestors gave rise to land plants. One outstanding feature of these algae is that their cell walls exhibit remarkable similarities to those of land plants. Xyloglucan (XyG) is a major structural component of the cell walls of most land plants and was originally thought to be absent in CGA. This study presents evidence that XyG evolved in the CGA. This is based on a) the identification of orthologs of the genetic machinery to produce XyG, b) the identification of XyG in a range of CGA and, c) the structural elucidation of XyG, including uronic acid-containing XyG, in selected CGA. Most notably, XyG fucosylation, a feature considered as a late evolutionary elaboration of the basic XyG structure and orthologs to the corresponding biosynthetic enzymes are shown to be present in Mesotaenium caldariorum.

De novo transcriptome assembly of the green alga Ankistrodesmus falcatus.

Ankistrodesmus falcatus is a globally distributed freshwater chlorophyte that is a candidate for biofuel production, is used to study the effects of toxins on aquatic communities, and is used as food in zooplankton research. Each of these research fields is transitioning to genomic tools. We created a reference transcriptome for of A. falcatus using NextGen sequencing and de novo assembly methods including Trinity, Velvet-Oases, and EvidentialGene. The assembled transcriptome has a total of 17,997 contigs, an N50 value of 2,462, and a GC content of 64.8%. BUSCO analysis recovered 83.3% of total chlorophyte BUSCOs and 82.5% of the eukaryotic BUSCOs. A portion (7.9%) of these supposedly single-copy genes were found to have transcriptionally active, distinct duplicates. We annotated the assembly using the dammit annotation pipeline, resulting in putative functional annotation for 68.89% of the assembly. Using available rbcL sequences from 16 strains (10 species) of Ankistrodesmus, we constructed a neighbor-joining phylogeny to illustrate genetic distances of our A. falcatus strain to other members of the genus. This assembly will be valuable for researchers seeking to identify Ankistrodesmus sequences in metatranscriptomic and metagenomic field studies and in experiments where separating expression responses of zooplankton and their algal food sources through bioinformatics is important.

Reassessment of suitable markers for taxonomy of Chaetophorales (Chlorophyceae, Chlorophyta) based on chloroplast genomes.

Filamentous green algae Chaetophorales present numerous taxonomic problems as many other green algae. Phylogenetic analyses based on nuclear genes have limited solutions. Studies with appropriate chloroplast molecular markers may solve this problems; however, suitable molecular markers for the order Chaetophorales are still unknown. In this study, 50 chloroplast genomes of Chlorophyceae, including 15 of Chaetophorales, were subjected to single protein-coding gene phylogenetic analyses, and substitution rate and evolutionary rate assays, and PCR amplification verification was conducted to screen the suitable molecular markers. Phylogenetic analyses of three chloroplast representative genes (psaB, tufA, and rbcL) amplified from 124 strains of Chaetophorales showed that phylogenetic relationships were not improved by increasing the number of samples, implying that the genes themselves, rather than limited samples, were the reason for the unsupported Topology I. Seven genes (atpF, atpI, ccsA, cemA, chlB, psbB, and rpl2) with robust support were selected to be the most suitable molecular markers for phylogenetic analyses of Chaetophorales, and the concatenated seven genes could replace the time-consuming and labor-intensive phylogenetic analyses based on chloroplast genome to some extent. To further solve the taxonomic problems of Chaetophorales, suitable chloroplast markers combined with more taxon-rich approach could be helpful and efficient.

Molecular Mechanism of Lipid Accumulation and Metabolism of Oleaginous Chlorococcum sphacosum GD from Soil under Salt Stress.

The oleaginous microalgae species Chlorococcum sphacosum GD is a promising feedstock for biodiesel production from soil. However, its metabolic mechanism of lipid production remains unclear. In this study, the lipid accumulation and metabolism mechanisms of Chlorococcum sphacosum GD were analyzed under salt stress based on transcriptome sequencing. The biomass and lipid content of the alga strain were determined under different NaCl concentrations, and total RNA from fresh cells were isolated and sequenced by HiSeq 2000 high throughput sequencing technology. As the salt concentration increased in culture medium, the algal lipid content increased but the biomass decreased. Following transcriptome sequencing by assembly and splicing, 24,128 unigenes were annotated, with read lengths mostly distributed in the 200-300 bp interval. Statistically significant differentially expressed unigenes were observed in different experimental groups, with 2051 up-regulated genes and 1835 down-regulated genes. The lipid metabolism pathway analysis showed that, under salt stress, gene-related fatty acid biosynthesis (ACCase, KASII, KAR, HAD, FATA) was significantly up-regulated, but some gene-related fatty acid degradation was significantly down-regulated. The comprehensive results showed that salt concentration can affect the lipid accumulation and metabolism of C. sphacosum GD, and the lipid accumulation is closely related to the fatty acid synthesis pathway.

Characterization of type-2 diacylglycerol acyltransferases in Haematococcus lacustris reveals their functions and engineering potential in triacylglycerol biosynthesis.

BACKGROUND: Haematococcus lacustris is an ideal source of astaxanthin (AST), which is stored in oil bodies containing esterified AST (EAST) and triacylglycerol (TAG). Diacylglycerol acyltransferases (DGATs) catalyze the last step of acyl-CoA-dependent TAG biosynthesis and are also considered as crucial enzymes involved in EAST biosynthesis in H. lacustris. Previous studies have identified four putative DGAT2-encoding genes in H. lacustris, and only HpDGAT2D allowed the recovery of TAG biosynthesis, but the engineering potential of HpDGAT2s in TAG biosynthesis remains ambiguous. RESULTS: Five putative DGAT2 genes (HpDGAT2A, HpDGAT2B, HpDGAT2C, HpDGAT2D, and HpDGAT2E) were identified in H. lacustris. Transcription analysis showed that the expression levels of the HpDGAT2A, HpDGAT2D, and HpDGAT2E genes markedly increased under high light and nitrogen deficient conditions with distinct patterns, which led to significant TAG and EAST accumulation. Functional complementation demonstrated that HpDGAT2A, HpDGAT2B, HpDGAT2D, and HpDGAT2E had the capacity to restore TAG synthesis in a TAG-deficient yeast strain (H1246) showing a large difference in enzymatic activity. Fatty acid (FA) profile assays revealed that HpDGAT2A, HpDGAT2D, and HpDGAT2E, but not HpDGAT2B, preferred monounsaturated fatty acyl-CoAs (MUFAs) for TAG synthesis in yeast cells, and showed a preference for polyunsaturated fatty acyl-CoAs (PUFAs) based on their feeding strategy. The heterologous expression of HpDGAT2D in Arabidopsis thaliana and Chlamydomonas reinhardtii significantly increased the TAG content and obviously promoted the MUFAs and PUFAs contents. CONCLUSIONS: Our study represents systematic work on the characterization of HpDGAT2s by integrating expression patterns, AST/TAG accumulation, functional complementation, and heterologous expression in yeast, plants, and algae. These results (1) update the gene models of HpDGAT2s, (2) prove the TAG biosynthesis capacity of HpDGAT2s, (3) show the strong preference for MUFAs and PUFAs, and (4) offer target genes to modulate TAG biosynthesis by using genetic engineering methods.

Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide.

Raphidocelis subcapitata is one of the most frequently used species for algal growth inhibition tests. Accordingly, many microalgal culture collections worldwide maintain R. subcapitata for distribution to users. All R. subcapitata strains maintained in these collections are derived from the same cultured strain, NIVA-CHL1. However, considering that 61 years have passed since this strain was isolated, we suspected that NIVA-CHL1 in culture collections might have acquired various mutations. In this study, we compared the genome sequences among NIVA-CHL1 from 8 microalgal culture collections and one laboratory in Japan to evaluate the presence of mutations. We found single-nucleotide polymorphisms or indels at 19,576 to 28,212 sites per strain in comparison with the genome sequence of R. subcapitata NIES-35, maintained at the National Institute for Environmental Studies, Tsukuba, Japan. These mutations were detected not only in non-coding but also in coding regions; some of the latter mutations may affect protein function. In growth inhibition test with 3,5-dichlorophenol, EC50 values varied 2.6-fold among the 9 strains. In the ATCC 22662-2 and CCAP 278/4 strains, we also detected a mutation in the gene encoding small-conductance mechanosensitive ion channel, which may lead to protein truncation and loss of function. Growth inhibition test with sodium chloride suggested that osmotic regulation has changed in ATCC 22662-2 and CCAP 278/4 in comparison with NIES-35.