Isogamy in large and complex volvocine algae is consistent with the gamete competition theory of the evolution of anisogamy.

Although the gamete competition theory remains the dominant explanation for the evolution of anisogamy, well-known exceptions to its predictions have raised doubts about the completeness of the theory. One of these exceptions is isogamy in large or complex species of green algae. Here, we show that this exception may be explained in a manner consistent with a game-theoretic extension of the original theory: a constraint on the minimum size of a gamete may prevent the evolution of continuously stable anisogamy. We show that in the volvocine algae, both gametes of isogamous species retain an intact chloroplast, whereas the chloroplast of the microgamete in anisogamous species is invariably degenerate. The chloroplast, which functions in photosynthesis and starch storage, may be necessary to provision a gamete for an extended period when gamete encounter rates are low. The single chloroplast accounts for most of the volume of a typical gamete, and thus may constrain the minimum size of a gamete, preventing the evolution of anisogamy. A prediction from this hypothesis, that isogametes should be larger than the microgametes of similar-size species, is confirmed for the volvocine algae. Our results support the gamete competition theory.

A rapid UHPLC-HILIC method for algal guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and the potential separation mechanism.

A fast and facile hydrophilic interaction liquid chromatography (HILIC) method was developed and applied to quantify physiologically important ppGpp and its analogues in a tough sample, the astaxanthin-accumulating alga Hameatococcus pluvialis. The method is able to analyze simultaneously seven nucleotides, including ppGpp at the order of pmol g-1 cells within 12 min. Mechanism on the elution order was investigated. It was found that 1) phosphate salt competed for the amide groups on the HILIC column with the phosphate groups of the nucleotides; 2) intramolecular hydrogen bonds might contribute to the elution order by offsetting and reducing the number of free hydrogen acceptor/donor of the nucleotide molecules interacting with the amide groups. This is the first HILIC method for ppGpp, which is feasible and applicable to a wide range of samples, especially tough samples, e.g., algae and plants.

Biosorption of malachite green onto Haematococcus pluvialis observed through synchrotron Fourier-transform infrared microspectroscopy.

Microalgae have emerged as promising biosorbents for the treatment of malachite green (MG) in wastewater. However, the underlying mechanism for the biosorption of MG onto microalgae is still unclear and needs further intensive study. In this work, synchrotron Fourier-transform infrared (s-FTIR) microspectroscopy in combination with biochemical assay is employed to evaluate MG removal efficiency (95·2%, 75·6% and 66·5%) by three stages of Haematococcus pluvialis. Meanwhile, the various vital changes of algal cells including lipids, proteins, polysaccharides and carotenoids is distinguished and quantified in situ. This study illustrates that s-FTIR microspectroscopy is an effective and powerful tool to scrutinize the mechanism for the interactions between the MG dye and microalgal cells, and it even provides an effective and noninvasive new approach to screen potentially proper biosorbents for the removal of dyes from wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: Microalgae have potential application for their ability to absorb dyes from industrial wastewater. In this study, we initiated the application of synchrotron Fourier-transform infrared (s-FTIR) microspectroscopy to investigate malachite green dye removal efficiency by three stages of Haematococcus pluvialis, demonstrating that s-FTIR is a very powerful tool in exploring the mechanism of the biosorption of dyes onto microalgae.

The Carotenogenic Dunaliella salina CCAP 19/20 Produces Enhanced Levels of Carotenoid under Specific Nutrients Limitation.

Dunaliella salina is the popular microalga for ß-carotene production. There is still a growing demand for the best strain identification and growth conditions optimization for maximum carotenoids production. Some strains are noncarotenogenic while other strains may respond differently to applied growth conditions and produce enhanced carotenoid levels. This study tested the carotenogenic ability of Dunaliella salina CCAP 19/20 under sixteen stress conditions and certain biochemical changes in response to specific stress were investigated. This study identified the above strain as carotenogenic, which produces maximum carotenoids under high light (240 µmol photons m-2 sec-1) when combined nitrogen and micronutrients (Cu or CuMn) were limited. Based on the intensity of extracted ions chromatograms, lutein (m/z 568.4357) appears as the major carotenoid followed by ß-carotene (m/z 536.4446) and α-carotene (m/z 536.4435). A polypeptide of 28.3 kDa appeared while another polypeptide of 25.5 kDa disappeared in stress cells as compared to noncarotenogenic cells. Expression levels of antioxidative-enzyme superoxide dismutase-1 (SOD1, H2O2-resistant) remained identical, while the prominent H2O2-sensitive isoforms SOD2 and SOD3 were downregulated during carotenogenic conditions. Overall, increased carotenoids levels might be due to the response of differential expression of specific polypeptides and retention of H2O2-resistant SOD, which eventually might help the organism to thrive in the tested stress conditions.

Taxonomic revision of Chloromonas nivalis (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting Chloromonas species.

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting Chloromonas species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of C. nivalis; however, strains with such vegetative cells in snow-inhabiting species of Chloromonas have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several C. nivalis strains. The morphological data demonstrated that one North American strain could be identified as C. nivalis, whereas three other strains should be re-classified as C. hoshawii sp. nov. and C. remiasii sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of C. nivalis was phylogenetically separated from the Austrian and Japanese specimens previously identified as C. nivalis based on zygote morphology.

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F1FO-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-ß-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V2 and V4) and different respiratory supercomplexes (I/IV6, I/III4, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V4 ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called "respirasome" was able to perform in-vitro oxygen consumption.

Development of Carbon-Based Solid Acid Catalysts Using a Lipid-Extracted Alga, Dunaliella tertiolecta, for Esterification.

Novel carbon-based solid acid catalysts were synthesized through a sustainable route from lipid-extracted microalgal residue of Dunaliella tertiolecta, for biodiesel production. Two carbon-based solid acid catalysts were prepared by surface modification of bio-char with sulfuric acid (H2SO4) and sulfuryl chloride (SO2Cl2), respectively. The treated catalysts were characterized and their catalytic activities were evaluated by esterification of oleic acid. The esterification catalytic activity of the SO2Cl2-treated bio-char was higher (11.5 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹) than that of commercial catalyst silica-supported Nafion SAC-13 (2.3 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹) and H2SO4-treated bio-char (5.7 mmol Prod.∙h⁻¹âˆ™g Cat. ⁻¹). Reusability of the catalysts was examined. The catalytic activity of the SO2Cl2-modified catalyst was sustained from the second run after the initial activity dropped after the first run and kept the same activity until the fifth run. It was higher than that of first-used Nafion. These experimental results demonstrate that catalysts from lipid-extracted algae have great potential for the economic and environment-friendly production of biodiesel.

Chloromonas arctica sp. nov., a psychrotolerant alga from snow in the High Arctic (Chlamydomonadales, Chlorophyta).

With the advent of molecular phylogenetic methods, it has become possible to assess the bioversity of snow algae more accurately. In this study, we focused on a morphological, ultrastructural and taxonomic description of a new Chloromonas-like alga isolated from snow in the High Arctic (Svalbard). Light and transmission electron microscopy revealed broad ellipsoidal or ellipsoidal-cylindrical, occasionally spherical cells with a chloroplast without a pyrenoid, an inconspicuous eyespot and a papilla. The size difference and the aforementioned morphological traits clearly distinguished the alga from its closest counterparts within the genus Chloromonas. Moreover, we were able to cultivate the alga at both 5 and 20 °C, revealing the psychrotolerant nature of the strain. Phylogenetic analyses of the plastid rbcL and nuclear 18S rRNA gene showed that the alga is nested within a clade containing a number of psychrotolerant strains within the Chloromonadinia phylogroup (Chlorophyceae). In the rbcL phylogeny, the alga formed an independent lineage, sister to the freshwater species Chloromonas paraserbinowii. Comparisons of secondary structure models of a highly variable ITS2 rDNA marker showed support for a distinct species identity for the new strain. The ITS2 secondary structure of the new isolate differed from the closest matches 'Chlamydomonas' gerloffii and Choloromonas reticulata by three and five compensatory base changes, respectively. Considering the morphological and molecular differences from its closest relatives, a new psychrotolerant species from the Arctic, Choromonas arctica sp. nov., is proposed.

Growth and metabolic characteristics of oleaginous microalgal isolates from Nilgiri biosphere Reserve of India.

BACKGROUND: Renewable energy for sustainable development is a subject of a worldwide debate since continuous utilization of non-renewable energy sources has a drastic impact on the environment and economy; a search for alternative energy resources is indispensable. Microalgae are promising and potential alternate energy resources for biodiesel production. Thus, our efforts were focused on surveying the natural diversity of microalgae for the production of biodiesel. The present study aimed at identification, isolation, and characterization of oleaginous microalgae from shola forests of Nilgiri Biosphere Reserve (NBR), the biodiversity hot spot of India, where the microalgal diversity has not yet been systematically investigated. RESULTS: Overall the higher biomass yield, higher lipid accumulation and thermotolerance observed in the isolated microalgal strains have been found to be the desirable traits for the efficient biodiesel production. Species composition and diversity analysis yielded ten potential microalgal isolates belonging to Chlorophyceae and Cyanophyceae classes. The chlorophytes exhibited higher growth rate, maximum biomass yield, and higher lipid accumulation than Cyanophyceae. Among the chlorophytes, the best performing strains were identified and represented by Acutodesmus dissociatus (TGA1), Chlorella sp. (TGA2), Chlamydomonadales sp. (TGA3) and Hindakia tetrachotoma (PGA1). The Chlamydomonadales sp. recorded with the highest growth rate, lipid accumulation and biomass yield of 0.28 ± 0.03 day-1 (µexp), 29.7 ± 0.69% and 134.17 ± 16.87 mg L-1 day-1, respectively. It was also found to grow well at various temperatures, viz., 25 °C, 35 °C, and 45 °C, indicating its suitability for open pond cultivation. The fatty acid methyl ester (FAME) analysis of stationary phase cultures of selected four algal strains by tandem mass spectrograph showed C16:0, C18:1 and C18:3 as dominant fatty acids suitable for biodiesel production. All the three strains except for Hindakia tetrachotoma (PGA1) recorded higher carbohydrate content and were considered as potential feed stocks for biodiesel production through hydrothermal liquefaction technology (HTL). CONCLUSIONS: In conclusion, the present investigation is a first systematic study on the microalgal diversity of soil and water samples from selected sites of NBR. The study resulted in isolation and characterization of ten potent oleaginous microalgae and found four cultures as promising feed stocks for biodiesel production. Of the four microalgae, Chlamydomonadales sp. (TGA3) was found to be significantly thermo-tolerant and can be considered as promising feedstock for biodiesel production.

Transcriptome Analysis in Haematococcus pluvialis: Astaxanthin Induction by High Light with Acetate and Fe2.

Haematococcus pluvialis is a commercial microalga, that produces abundant levels of astaxanthin under stress conditions. Acetate and Fe2+ are reported to be important for astaxanthin accumulation in H. pluvialis. In order to study the synergistic effects of high light stress and these two factors, we obtained transcriptomes for four groups: high light irradiation (HL), addition of 25 mM acetate under high light (HA), addition of 20 µM Fe2+ under high light (HF) and normal green growing cells (HG). Among the total clean reads of the four groups, 156,992 unigenes were found, of which 48.88% were annotated in at least one database (Nr, Nt, Pfam, KOG/COG, SwissProt, KEGG, GO). The statistics for DEGs (differentially expressed genes) showed that there were more than 10 thousand DEGs caused by high light and 1800-1900 DEGs caused by acetate or Fe2+. The results of DEG analysis by GO and KEGG enrichments showed that, under the high light condition, the expression of genes related to many pathways had changed, such as the pathway for carotenoid biosynthesis, fatty acid elongation, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms and so on. Addition of acetate under high light significantly promoted the expression of key genes related to the pathways for carotenoid biosynthesis and fatty acid elongation. Furthermore, acetate could obviously inhibit the expression of genes related to the pathway for photosynthesis-antenna proteins. For addition of Fe2+, the genes related to photosynthesis-antenna proteins were promoted significantly and there was no obvious change in the gene expressions related to carotenoid and fatty acid synthesis.

Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species.

Structural basis of proton translocation and force generation in mitochondrial ATP synthase.

ATP synthases produce ATP by rotary catalysis, powered by the electrochemical proton gradient across the membrane. Understanding this fundamental process requires an atomic model of the proton pathway. We determined the structure of an intact mitochondrial ATP synthase dimer by electron cryo-microscopy at near-atomic resolution. Charged and polar residues of the a-subunit stator define two aqueous channels, each spanning one half of the membrane. Passing through a conserved membrane-intrinsic helix hairpin, the lumenal channel protonates an acidic glutamate in the c-ring rotor. Upon ring rotation, the protonated glutamate encounters the matrix channel and deprotonates. An arginine between the two channels prevents proton leakage. The steep potential gradient over the sub-nm inter-channel distance exerts a force on the deprotonated glutamate, resulting in net directional rotation.

Isolation and characterization of peroxiredoxin 1 gene of Dunaliella salina.

Peroxiredoxin 1 (Prdx1) is a ubiquitously expressed protein in eukaryotic cells, and plays an important role in cell proliferation, differentiation, apoptosis, and redox signaling. Although Prdx1 has been better studied in yeasts and humans, only few Prdx1 genes have been cloned in green algae. The microalga Dunaliella salina (D. salina) is a model for the study of a variety of human cilia-related diseases. In this study, a suppression subtractive hybridization cDNA library of D. salina was constructed, and 6 flagellum-associated genes including D. salina Prdx1 (DsPrdx1) were isolated and identified. A 956bp full-length cDNA of DsPrdx1 was cloned using rapid amplification of cDNA end (RACE). The open reading frame (ORF) of this DNA sequence encodes a polypeptide of 201 amino acids with a predicted molecular weight of 22kDa and a theoretical isoelectric point (pI) of 5.27. Sequence comparison showed that Prdx1 is highly evolutionarily conserved from the unicellular green alga D. salina to human. To our knowledge, this is the first reported full-length sequence of Prdx1 in D. salina. Interestingly, the protein expression of DsPrdx1 was obviously increased during flagellar disassembly in D. salina. Additionally, a yeast two-hybrid assay showed interaction between Prdx1 and RNA, and suggested that DsPrdx1 can protect RNA from degradation by RNase. Taken together, DsPrdx1 not only participates in flagellar disassembly, but also protects RNA from degradation.

Assessing intragenomic variation of the internal transcribed spacer two: Adapting the Illumina metagenomics protocol.

Primary and secondary structural data from the internal transcribed spacer two (ITS2) have been used extensively for diversity studies of many different eukaryotic organisms, including the green algae. Ease of amplification is due, at least in part, to the fact that ITS2 is part of the tandemly-repeated rRNA array. The potential confounding influence of intragenomic variability has yet to be addressed except in a few organisms. Moreover, few of the assessments of intragenomic variation have taken advantage of the deep sequencing capacity of sequence-by-synthesis protocols. We present results from our adaptation of the 16S Metagenomics Sequencing Library Preparation/Illumina protocol for deep sequencing of the ITS2 genes in selected isolates of the green algal genus, Haematococcus. Deep sequencing yielded from just under 20,000 to more than 500,000 merged reads, outpacing results from recent pyrosequencing efforts. Furthermore, a conservative evaluation of these data revealed a range of three to six ITS2 sequence haplotypes (defined as unique sets of nucleotide polymorphisms) across the taxon sampling. The frequency of the dominant haplotype ranged from 0.35 to 0.98. In all but two cases, the haplotype with the greatest frequency corresponded to a sequence obtained by the Sanger method using PCR templates. Our data also show that results from the sequencing-by-synthesis approach are reproducible. In addition to advancing our understanding of ribosomal RNA variation, the results of this investigation will allow us to begin testing hypotheses regarding the maintenance of homogeneity across multi-copy genes.

ROS and calcium signaling mediated pathways involved in stress responses of the marine microalgae Dunaliella salina to enhanced UV-B radiation.

UV-B ray has been addressed to trigger common metabolic responses on marine microalgae, however, the upstream events responsible for these changes in marine microalgae are poorly understood. In the present study, a species of marine green microalgae Dunaliella salina was exposed to a series of enhanced UV-B radiation ranging from 0.25 to 1.00 KJ·m-2 per day. The role of ROS and calcium signaling in the D. salina responses to UV-B was discussed. Results showed that enhanced UV-B radiation markedly decreased the cell density in a dose-dependent manner, but the contents of protein and glycerol that were essential for cell growth increased. It suggested that it was cell division instead of cell growth that UV-B exerted negative effects on. The subcellular damages on nuclei and plasmalemma further evidenced the hypothesis. The nutrient absorption was affected with UV-B exposure, and the inhibition on PO43- uptake was more serious compared to NO3- uptake. UV-B radiation promoted reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) contents, decreased the redox status and altered the antioxidant enzyme activities. The addition of the ROS scavenger and the glutathione biosynthesis precursor N-acetyl-l-cysteine (NAC) alleviated the stress degree, implying ROS-mediated pathway was involved in the stress response to UV-B radiation. Transient increase in Ca2+-ATPase was triggered simultaneously with UV-B exposure. Meanwhile, the addition of an intracellular free calcium chelator aggravated the damage of cell division, but exogenous calcium and ion channel blocker applications did not, inferring that endogenously initiated calcium signaling played roles in response to UV-B. Cross-talk analysis showed a relatively clear relationship between ROS inhibition and Ca2+-ATPase suppression, and a relation between Ca2+ inhibition and GPx activity change was also observed. It was thus presumed that ROS-coupled calcium signaling via the glutathione cycle was involved in the response of marine microalgae to UV-B stimuli.

Transcriptome analysis illuminates the nature of the intracellular interaction in a vertebrate-algal symbiosis.

During embryonic development, cells of the green alga Oophila amblystomatis enter cells of the salamander Ambystoma maculatum forming an endosymbiosis. Here, using de novo dual-RNA seq, we compared the host salamander cells that harbored intracellular algae to those without algae and the algae inside the animal cells to those in the egg capsule. This two-by-two-way analysis revealed that intracellular algae exhibit hallmarks of cellular stress and undergo a striking metabolic shift from oxidative metabolism to fermentation. Culturing experiments with the alga showed that host glutamine may be utilized by the algal endosymbiont as a primary nitrogen source. Transcriptional changes in salamander cells suggest an innate immune response to the alga, with potential attenuation of NF-κB, and metabolic alterations indicative of modulation of insulin sensitivity. In stark contrast to its algal endosymbiont, the salamander cells did not exhibit major stress responses, suggesting that the host cell experience is neutral or beneficial.

Utilization of Starch-Enriched Brewery (Rice Wine) Waste for Mixotrophic Cultivation of Ettlia Sp. YC001 Used in Biodiesel Production.

Starch-enriched brewery waste (SBW), an unexplored feedstock, was investigated as a nutritious low-cost source for the mixotrophic cultivation of Ettlia sp. YC001 for biodiesel production. Stirring, autoclaving, and sonication were assessed for the SBW, in conjunction with pH. Stirring at 55 °C was found to be the best, in terms of the effectiveness of starch hydrolysis and yeast disintegration as well as cost. The treated solutions were found to support the mixotrophic growth of microalgae: 20 g/L of glucose medium resulted in the highest biomass production of 9.26 g/L and one with 10 g/L of glucose showed the best lipid productivity of 244.2 mg/L/day. The unsaturated fatty acids increased in the resulting lipid and thus quality well suited for the transportation fuel. All these suggested that SBW, when treated properly, could indeed serve as a cheap feedstock for microalgae-based biodiesel production.

Alleviation of metabolic bottleneck by combinatorial engineering enhanced astaxanthin synthesis in Saccharomyces cerevisiae.

Highly efficient biosynthesis of the commercially valuable carotenoid astaxanthin by microbial cells is an attractive alternative to chemical synthesis and microalgae extraction. With the goal of enhancing heterologous astaxanthin production in Saccharomyces cerevisiae, metabolic engineering and protein engineering were integrated to improve both the expression and activity of rate-limiting enzymes. Firstly, to increase the supply of ß-carotene as a key precursor for astaxanthin, a positive mutant of GGPP synthase (CrtE03M) was overexpressed together with three other rate-limiting enzymes tHMG1, CrtI and CrtYB. Subsequently, to accelerate the conversion of ß-carotene to astaxanthin, a color screening system was developed and adopted for directed evolution of ß-carotene ketolase (OBKT), generating a triple mutant OBKTM (H165R/V264D/F298Y) with 2.4-fold improved activity. After adjusting copy numbers of the above-mentioned rate-limiting enzymes to further balance the metabolic flux, a diploid strain YastD-01 was generated by mating two astaxanthin-producing haploid strains carrying the same carotenogenic pathway. Finally, further overexpression of OCrtZ and OBKTM in YastD-01 resulted in accumulation of 8.10mg/g DCW (47.18mg/l) of (3S, 3'S)-astaxanthin in shake-flask cultures. This combinatorial strategy might be also applicable for alleviation of metabolic bottleneck in biosynthesis of other value-added products, especially colored metabolites.

An on-chip imaging droplet-sorting system: a real-time shape recognition method to screen target cells in droplets with single cell resolution.

A microfluidic on-chip imaging cell sorter has several advantages over conventional cell sorting methods, especially to identify cells with complex morphologies such as clusters. One of the remaining problems is how to efficiently discriminate targets at the species level without labelling. Hence, we developed a label-free microfluidic droplet-sorting system based on image recognition of cells in droplets. To test the applicability of this method, a mixture of two plankton species with different morphologies (Dunaliella tertiolecta and Phaeodactylum tricornutum) were successfully identified and discriminated at a rate of 10 Hz. We also examined the ability to detect the number of objects encapsulated in a droplet. Single cell droplets sorted into collection channels showed 91 ± 4.5% and 90 ± 3.8% accuracy for D. tertiolecta and P. tricornutum, respectively. Because we used image recognition to confirm single cell droplets, we achieved highly accurate single cell sorting. The results indicate that the integrated method of droplet imaging cell sorting can provide a complementary sorting approach capable of isolating single target cells from a mixture of cells with high accuracy without any staining.

Analysis of carotenogenic genes promoters and WRKY transcription factors in response to salt stress in Dunaliella bardawil.

The unicellular alga Dunaliella bardawil is a highly salt-tolerant organism, capable of accumulating glycerol, glycine betaine and ß-carotene under salt stress, and has been considered as an excellent model organism to investigate the molecular mechanisms of salt stress responses. In this study, several carotenogenic genes (DbCRTISO, DbZISO, DbLycE and DbChyB), DbBADH genes involved in glycine betaine synthesis and genes encoding probable WRKY transcription factors from D. bardawil were isolated, and promoters of DbCRTISO and DbChyB were cloned. The promoters of DbPSY, DbLycB, DbGGPS, DbCRTISO and DbChyB contained the salt-regulated element (SRE), GT1GMSCAM4, while the DbGGPS promoter has another SRE, DRECRTCOREAT. All promoters of the carotenogenic genes had light-regulated elements and W-box cis-acting elements. Most WRKY transcription factors can bind to the W-box, and play roles in abiotic stress. qRT-PCR analysis showed that salt stress up-regulated both carotenogenic genes and WRKY transcription factors. In contrast, the transcription levels of DbBADH showed minor changes. In D. bardawil, it appears that carotenoid over-accumulation allows for the long-term adaptation to salt stress, while the rapid modulation of glycine betaine biosynthesis provides an initial response.