The unicellular green alga Haematococcus pluvialis is the best source of natural astaxanthin (AST) in the world due to its high content under stress conditions. Although high light (HL) can effectively induce AST biosynthesis, the specific mechanisms of light signal perception and transduction are unclear. In the current study, we used transcriptomic data of normal (N), high white light (W), and high blue light (B) to study the mechanisms of light inducing AST accumulation from the point of photoreceptors. The original data of 4.0 G, 3.8 G, and 3.6 G for N, W, and B were obtained, respectively, by the Illumina Hi-seq 2000 sequencing technology. Totally, 51 954 unigenes (at least 200 bp in length) were generated, of which, 20 537 unigenes were annotated into at least one database (NR, NT, KO, SwissProt, Pfam, GO, or KOG). There were 1 255 DEGs in the W vs N, 1 494 DEGs in the B vs N, and 1 008 DEGs in the both W vs N and B vs N. KEGG enrichment analysis revealed that photosynthesis, oxidative phosphorylation, carotenoid biosynthesis, fatty acids biosynthesis, DNA replication, nitrogen metabolism, and carbon metabolism were the significantly enriched pathways. Moreover, a large number of genes encoding photoreceptors and predicted interacting proteins were predicted in Haematococcus transcriptome data. These genes showed significant differences at transcriptional expression levels. In addition, 15 related DEGs were selected and tested by qRT-PCR and the results were significantly correlated with the transcriptome data. The above results indicate that the signal transduction pathway of "light signal - photoreceptors - interaction proteins - (interaction proteins - transcription factor/transcriptional regulator) - gene expression - AST accumulation" might play important roles in the regulation process, and provide reference for further understanding the transcriptional regulation mechanisms of AST accumulation under HL stress.
Chlorophyta/genetics* ; Gene Expression Profiling ; Signal Transduction/genetics* ; Transcriptome/genetics* ; Xanthophylls

Astaxanthin is widely applied as a nutraceutical, pharmaceutical, and aquaculture feed additive because of its high antioxidant activity. Haematococcus pluvialis is a microalgal species that can largely accumulate astaxanthin under adverse environmental conditions. Here we review the research progress of astaxanthin biosynthesis in H. pluvialis, including the induction and regulation of massive astaxanthin, the relationship between astaxanthin synthesis, photosynthesis and lipid metabolism.
Chlorophyceae ; Chlorophyta ; Microalgae ; Xanthophylls

A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2 > 0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency.
Chlorophyta ; chemistry ; Heterotrophic Processes ; High-Throughput Screening Assays ; Lipids ; analysis ; Magnetic Resonance Spectroscopy ; Microalgae ; chemistry ; Staining and Labeling

OBJECTIVETo investigate the inhibitory effect of cocoomyxa gloeobotrydifomis (CGD) on benign prostate hyperplasia (BPH) in aged rats and its underlying mechanism.

METHODSThirty SD male rats aged 21 months were equally randomized to three groups, aged control, low-dose CGD and high-dose CGD, the latter two groups fed on a diet with CGD at 50 and 100 mg per kg per d for 3 months, while the aged controls on normal laboratory chow. Another 10 3-month-old male rats were included in a young control group and fed on the same diet as the aged control rats. At the end of 3 months of CGD treatment, the prostates of all the rats were harvested and weighed. The histomorphological and interstitial changes of the prostatic tissue were observed by HE staining and Masson staining, respectively. The expressions of phosphorylated phosphoinositide-dependent kinase 1 (PDK1), phosphorylated Akt (Ser 473) and phosphorylated PTEN in the rat prostate were determined by Western blotting.

RESULTSThe wet weight and index of the prostate were significantly higher in the aged controls than in the young controls ([1 220 +/- 140] vs [550 +/- 60] mg, P < 0.01; 2.08 +/- 0.17 vs 1.94 +/- 0.10, P < 0.05). High-dose CGD significantly inhibited the increase in the prostatic wet weight and index of the aged rats ([1 080 +/- 97] mg and 1.85 +/- 0.16) as compared with the aged controls (P < 0.01 and P < 0.05). The epithelium and interstitium, particularly the latter, were evidently thicker in the aged control than in the CGD-treated rats. The protein levels of phosphorylated PDK1 and Akt were significantly enhanced, while that of phosphorylated PTEN remarkably down-regulated in the aged rats as compared with the young ones. The expressions of phosphorylated PDK1 and Akt were significantly decreased, whereas that of phosphorylated PTEN markedly increased in both the low-dose and high-dose CGD groups.

CONCLUSIONCGD can significantly inhibit BPH in aged rats through down-regulating the PI3K/Akt pathway.

Animals ; Chlorophyta ; chemistry ; Down-Regulation ; Male ; Phosphatidylinositol 3-Kinases ; metabolism ; Polysaccharides ; pharmacology ; Prostate ; metabolism ; Prostatic Hyperplasia ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley

Botryococcus braunii is a unique colonial green microalga and a great potential renewable resource of liquid fuel because of its ability to produce lipids. Due to the dense cell colonies and rigidly thick cell wall of B. braunii, the traditional Nile red method is usually of low sensitivity and bad repeatability and hard for the determination of lipid content in the cells. By dispersing the colony with ultrasonic, assisting permeation of Nile red across the cell wall with dimethyl sulfoxide and optimizing the staining conditions, we established an improved detection method. The details were as follows: after the colonial algal sample was treated by ultrasonic at 20 kHz for 20 s, 100 W transmitting power and with 1 s on/1 s off intermittent cycle, the equivoluminal 15% (V/V) dimethyl sulfoxide and 3 microg/mL Nile red were successively added and mixed evenly, then the staining system was incubated in dark at 40 degrees C for 10 min, and subsequently was measured by fluorescence spectroscopy detection with an excitation wavelength of 490 nm. Compared with the traditional method, the improved one not only had higher detection sensitivity which was increased by 196.6%, but also had obviously better detection repeatability whose characteristic parameter - relative standard deviation (RSD) was decreased from 10.91% to 1.84%. Therefore, the improved method could provide a rapid and sensitive detection of lipid content for B. braunii breeding and cultivation.
Chlorophyta ; chemistry ; Lipids ; analysis ; Microalgae ; chemistry ; Spectrometry, Fluorescence ; methods ; Ultrasonics

In order to lower the cost of lipid production of microalgae and reduce greenhouse gas emissions, microalgae Chlorococcum alkaliphilus MC-1 with the characteristics of rapid pH drift and high pH adaptability, was cultivated with bubbling of flue gas. The experiment was first performed in the photobioreactor (15 L) in three groups (control group, CO2 group and flue gas group), then, in the open raceway pond (24 m2). The adaptability of microalgae MC-1 to the cultivation with flue gas was studied. The results showed that the maximum biomass concentration, growth rate, total lipid content and CO2 fixation rate were (1.02+/-0.07) g/L, (0.12+/-0.02) g/(L.d), (37.84+/-0.58)% and (0.20+/-0.02) g/(L.d) in the photobioreactor treated with flue gas, 36%, 33.33%, 15.34% and 33.33% higher than those of the CO2 group, respectively. In the open raceway pond with aeration of flue gas, the maximum biomass concentration, growth rate, total lipid content and CO2 fixation rate were 147.40 g/m2, 14.73 g/(m2.d), 35.72% and 24.01 g/(m2.d), respectively, which were similar to the cultivation with pure CO2. The toxic heavy metal contents (Pb, As, Cd and Cr) in the biomass of MC-1 treated with flue gas were all below the legal limits. Additionally, the absorptive effect of CO2, NO and SO2 were determined. In the photobioreactor and open raceway pond, the average absorption ratios of these gases were all higher than previous studies. Therefore, our study showed that MC-1 can adapt to the cultivation with flue gas, and it is feasible to enlarge the outdoor cultivation of MC-1 for lipid production coupling with emissions reduction of flue gas.
Adaptation, Physiological ; physiology ; Carbon Dioxide ; chemistry ; Chlorophyta ; classification ; growth & development ; physiology ; Culture Media ; metabolism ; Culture Techniques ; methods ; Gases ; chemistry ; Microalgae ; classification ; growth & development ; physiology ; Nitric Oxide ; chemistry ; Sulfur Dioxide ; chemistry

To study the growth effects of differing concentrations of artemisinin on green algae and to evaluate the ecological risk. The effects of artemisinin on the growth and the content change of chlorophyll, protein, oxygen, conductivity, SOD, CAT, MDA in Chlorella pyrenoidosa and Scenedesmus oblique were studied through 96 h toxicity tests. Artemisinin accelerated the growth of algae at a lower concentration ( <40 microg . L-1) with content increase of chlorophyll or protein and so on, and it inhibited the growth of algae at higher concentration ( >80 microg . L-1). The content of chlorophyll or protein in algae cells reduced with the increasing concentration of artemisinin, exhibiting the good concentration-effect relationship. SOD and CAT activity was stimulated at low concentrations ( <40 microg . L-1 ) and inhibited at high concentrations ( >80 microg . L- 1). However, MDA content increased significantly with the increase of concentration. According to the seven kinds of indicators changes, the time-response and dose-response suggested that the surfactant first hurt in Ch. pyrenoidosa was damaging membrane by changing membrane lipid molecules soluble. And primary mechanism on Chlorophyta cells might be related to the oxidation damage of lipid and other biological large molecules caused by artemisinin. The large-scale intensive planting of Artemisia annua may reduce the surrounding water productivity.
Artemisinins ; pharmacology ; Chlorophyll ; metabolism ; Chlorophyta ; drug effects ; metabolism

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and long-term response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fixes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.
Amino Acid Sequence ; Amino Acid Substitution ; Arabidopsis ; metabolism ; Arabidopsis Proteins ; chemistry ; genetics ; metabolism ; Binding Sites ; Catalytic Domain ; Chlorophyta ; enzymology ; Crystallography, X-Ray ; Cyclin-Dependent Kinase 2 ; chemistry ; metabolism ; Molecular Sequence Data ; Phosphorylation ; Protein Structure, Secondary ; Protein-Serine-Threonine Kinases ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity

In the forensic practice, the reliability of diatom test as a supportive measure to diagnose drowning is still disputed, from trustworthy to worthless. Some of the reason for the controversy is low sensitivity of the test, possibility of postmortem contamination and the detection of diatom in the tissues of non-drowned body. However, there is a variation of the diatom flora by season and by locale and it is strongly correlated with the frequency of positive diatom test outcomes. Therefore, if there is a profile of the diatom flora at a site, it can be compared with the diatom genera found in tissues of the immersed bodies, and also the test result can be predicted or verified. On each season, at three aquatic locations where drowning victims are often found, the author collected water samples and examined the plankton species of the samples, including dominant species and total number of plankton by site and by season. The examination result showed 16 species of diatoms, 20 species of green algae, 6 species of cyanobacteria, and 6 species of other algae. There is an enormous difference of population of algae by site(39 cells ~ 37,180 cells), but conspicuous periodicity of types and numbers of algae is not noted by season and by depth.
Chlorophyta ; Cyanobacteria ; Diatoms ; Drowning ; Periodicity ; Plankton ; Seasons ; Water

Developing a high-throughput screening method is of great importance for directed evolution of atrazine chlorohydrolase. A mutagenesis library of atzA from Pseudomonas sp. ADP and Arthrobacter sp. AD1 was constructed using error-prone PCR and DNA shuffling. Candidate mutants were screened through Haematococcus pluvialis expression system, using atrazine as selection pressure. Sequence analysis showed that mutations in the obtained 12 mutants with enhanced activity were all point-substitutions and scattered throughout the gene. Enzymatic activity analysis showed that the mutants all had higher activities than that of the wild type. The activities were 1.8-3.6 fold of the wild-type enzyme when cultured in BBM medium with 1 mg/L atrazine, whereas 1.8-2.6 fold with 2 mg/L atrazine. These results indicated that Haematococcus pluvialis expression system is an ideal high throughput screening system for directed evolution of atrazine chlorohydrolase.
Amidohydrolases ; genetics ; Atrazine ; metabolism ; Bacterial Proteins ; genetics ; Biodegradation, Environmental ; Chlorophyta ; genetics ; metabolism ; Herbicides ; metabolism ; High-Throughput Screening Assays ; Hydrolases ; biosynthesis ; genetics ; Mutagenesis, Insertional ; Pseudomonas ; enzymology ; genetics