Lipidomic profiling reveals lipid regulation in the snow alga Chlamydomonas nivalis in response to nitrate or phosphate deprivation

The snow alga Chlamydomonas nivalis in the exponential phase was subjected to nitrate or phosphate deprivation for 0–72 h to study its stress responses in lipid profiles analyzed by UPLC/Q-TOF-MS (ultra performance liquid chromatography/quadrupole-time of flight-mass spectrometry). Three clusters were distinguished as the control, nitrate-deprived and phosphate-deprived groups in OPLS-DA (orthogonal projection on latent structure discriminant analysis) score plots based on their lipidome data. Altogether, the lipidomic approach identified twenty-two ions in nitrate-deprived group including nine l, 2-diacylglyceryl-3-O-4′-(N, N, N-trimethyl)-homoserine (DGTSs), one phosphatidylethanolamine (PE), two monogalactosyldiacylglycerols (MGDGs), four digalactosyldiacylglycerols (DGDGs), three phosphatidylglycerols (PGs), two sulfoquinovosyl-diacylglycerols (SQDGs) and one phosphatidylinositiol (PI), and nineteen ions in phosphate-deprived group including four DGTSs, one PE, one MGDG, seven DGDGs, three PGs, two SQDGs and one PG as “differentiating lipid biomarkers”. Moreover, the common and specific biomarkers were found in the two nutrient deprived groups by SUS (shared and unique structure) plot. Biomarkers-based z-score plot and heat map further showed how lipid biomarker expressions deviate from the control. The up- or down-regulation of these lipid biomarkers provided new insights into the lipid metabolism of the snow alga in response to nitrate or phosphate deprivation stress condition.     

Phenolic compounds and antioxidant properties in the snow alga Chlamydomonas nivalis after exposure to UV light

The snow alga Chlamydomonas nivalis was collected from the Sierra Nevada, California, USA, and examined for its ability to produce phenolic compounds, free proline, and provide antioxidant protection factor in response to UV-A and UV-C light. Exposure of C. nivalis cells to UV-A light (365nm) for 5 days resulted in a 5–12% increase in total phenolics, where as exposure to UV-C light (254 nm) resulted in a 12–24% increase in phenolics after 7 days of exposure. Free proline was not affected by UV-A, but increased markedly after UV-C exposure. A three-fold increase in free proline occurred within two days after exposure to UV-C, but then dropped as cells became bleached. Antioxidant protection factor (PF) increased after treatment of cells with UV-A and remained constant throughout UV-C exposure. Spectral analysis of algal extracts revealed a decrease in absorption in the 215–225 nm region, short-term (2day) stimulation of pigment at 280 nm, and an increase in carotenoids (473 nm), after exposure to UV-A. Snow alga exposed to UV-C light had a different spectrum from that of UV-A exposed cells, i.e. an enhancement of three major peaks at 220, 260, and 280 nm, and loss of absorption in the carotenoid region.We report that UV light exposure, especially in the UV-C range, can stimulate phenolic-antioxidant production in aplanospores of C. nivalis effecting biochemical pathways related to proline metabolism. This revised version was published online in August 2006 with corrections to the Cover Date.     

Vertical distribution of the snow alga Chlamydomonas nivalis (Chlorophyta,Volvocales)

Summary Chlamydomonas nivalis commonly forms large blooms, visible as a red coloration, in the snow during summer. Fewer algae are seen in the top layer of snow during days of intense sunlight than on cloudy days. The present experiments were done to investigate this change of vertical distribution of algae. Apparently the algae are able to associate with the watersurface surrounding snow crystals. Due to this association they avoid being washed away by the water from melting snow. Intense sunlight, however, decreases the degree to which the algae associate with the water-surface, and thereby increasing the number of algae being removed from the top layer of snow by the melting water. If the weather becomes cloudy again, the algae do not move upwards, but stay attached to the water-surfaces. Thus when the snow above melts, they will reappear in the top layer.     

Identification of astaxanthin diglucoside diesters from snow alga Chlamydomonas nivalis by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

A method is described for the identification of astaxanthin glucoside esters from snow alga Chlamydomonas nivalis by means of liquid chromatography-mass spectrometry with atmospheric pressure chemical ionization (LC-MS/APCI). The method is based on the use of preparative HPLC and subsequent identification of astaxanthin diglucoside diesters by microbore LC-MS/APCI. The combination of these two techniques was used to identify more than 100 molecular species. The astaxanthin diglucoside diester, i.e. (all-E)-[di-(6-O-oleoyl-beta-D-glucopyranosyloxy)]-astaxanthin, was also synthesized to unambiguously confirm its structure.     

Seasonal and diel changes in photosynthetic activity of the snow alga Chlamydomonas nivalis (Chlorophyceae) from Svalbard determined by pulse amplitude modulation fluorometry

The seasonal and diel dynamics of the physiological state and photosynthetic activity of the snow alga Chlamydomonas nivalis were investigated in a snowfield in Svalbard. The snow surface represents an environment with very high irradiation intensities along with stable low temperatures close to freezing point. Photosynthetic activity was measured using pulse amplitude modulation fluorometry. Three types of cell (green biflagellate vegetative cells, orange spores clustered by means of mucilaginous sheaths, and purple spores with thick cell walls) were found, all of them photosynthetically active. The pH of snow ranged between 5.0 and 7.5, and the conductivity ranged between 5 and 75 microS cm(-1). The temperature of snow was stable (-0.1 to +0.1 degrees C), and the incident radiation values ranged from 11 to 1500 micromol photons m(-2) s(-1). The photosynthetic activity had seasonal and diel dynamics. The Fv/Fm values ranged between 0.4 and 0.7, and generally declined over the course of the season. A dynamic response of Fv/Fm to the irradiance was recorded. According to the saturating photon fluence values Ek, the algae may have obtained saturating light as deep as 3 cm in the snow when there were higher-light conditions, whereas they were undersaturated at prevalent low light even if on the surface.     

Regulation of glycerol and starch metabolism in Chlamydomonas pulsatilla in response to changes in salinity

Abstract. Chlamydomonas pulsatilla rapidly adjusts its level of osmoregulatory solute, glycerol, when exposed to increased (upshock) or decreased (downshock) salinities. Rates of degradation, or loss, of glycerol are similar in light and dark, while rates of net synthesis proceed about 20% faster in the light than in the dark. Synthesis of glycerol in the dark is correlated with degradation of starch, and starch appears also to be utilized for glycerol synthesis in response to extensive salinity upshocks in the light, where photosynthesis is strongly inhibited. Under conditions of moderate upshocks in the light, photosynthesis is not affected, and net increases in both glycerol and starch were observed. Glycerol turnover takes place both under iso-osmotic conditions, and during period of adjustments to osmotic downshocks, where there is a rapid net decrease in the glycerol pool. Half times for glycerol turnover were estimated from rates of incorporation of photoassimilated ¹⁴C into glycerol and glycerol pool sizes to be 20 and 74 min for cells incubated at 50 and 100% artificial seawater, respectively.     

Lipid metabolism and identification of biomarkers in asthma by lipidomic analysis

BackgroundLipids participate in many important biological functions through energy storage, material transport, signal transduction, and molecular recognition processes. Studies have reported that asthmatic patients have abnormal lipid metabolism. However, there are limited studies on the characterization of lipid metabolism in asthmatic patients by lipidomics.MethodsWe characterized the plasma lipid profile of 28 healthy controls and 33 outpatients with asthma (18 mild, 15 moderate) by liquid chromatography mass spectrometry/mass spectrometry-based lipidomics.ResultsWe determined 1338 individual lipid species in the plasma. Significant changes were identified in ten lipid species in asthmatic patients than in healthy controls (all P < 0.05). Phosphatidylethanolamine (PE) (18:1p/22:6), PE (20:0/18:1), PE (38:1), sphingomyelin (SM) (d18:1/18:1), and triglyceride (TG) (16:0/16:0/18:1) positively correlated with the severity of asthma (all P < 0.05). Phosphatidylinositol (PI) (16:0/20:4), TG (17:0/18:1/18:1), phosphatidylglycerol (PG) (44:0), ceramide (Cer) (d16:0/27:2), and lysophosphatidylcholine (LPC) (22:4) negatively correlated with the severity of asthma (all P < 0.05). Correlation analysis showed a significant correlation between all ten lipid species (all P < 0.05). From the area under the curve of the receiver operating characteristic curve analysis, PE (38:1) was the major lipid metabolite that distinguished asthmatic patients from healthy controls, and may be considered a potential lipid biomarker. PE (20:0/18:1) and TG (16:0/16:0/18:1) might be related to IgE levels in asthmatic patients.ConclusionsOur results indicated the presence of abnormal lipid metabolism, which correlated with the severity and IgE levels in asthmatic patients.     

Physiological and morphological processes in the Alpine snow alga Chloromonas nivalis (Chlorophyceae) during cyst formation

Amongst a specialised group of psychrophilic microalgae that have adapted to thrive exclusively in summer snow fields, Chloromonas nivalis has been reported as a species causing green, orange or pink blooms in many alpine and polar regions worldwide. Nevertheless, the cytology, ecophysiology and taxonomy of this species are still unresolved. Intracellular processes during cyst formation, which is the dominant stage on snow fields, were examined with samples from the European Alps to better understand the cellular strategies of a green alga living in this harsh habitat. We show with two different methods, i.e. oxygen optode fluorometry and by chlorophyll fluorescence, that the cysts are photosynthetically highly active, although they do not divide, and that Chloromonas nivalis can cope with low as well as high light conditions. During cyst formation, the chloroplast is fragmented into several smaller parts, enlarging the surface to volume ratio. The pool of xanthophyll-cycle pigments is significantly enlarged, which is different from other snow algae. The cytoplasm is filled with lipid bodies containing astaxanthin, a secondary carotenoid that causes the typical orange colour. The cyst wall surface possesses characteristic elongate flanges, which are assembled extracellulary by accumulation of material in the periplasmatic interspace. Comparison of Chloromonas nivalis samples from different locations (Austrian Alps, Spitsbergen) by molecular methods indicates genetic variations due to spatial isolation, while a North American strain has no close relationship to the taxon.     

Regulation of plants metabolism in response to salt stress: an omics approach

In recent era, some man-made and natural activities are responsible for causing salt stress that affects each component of environment. Among several components, plant is one of the essential components, and with the consequence of excess salts accumulation in the environment their metabolic activities may get affected. Plants exhibit hyper-osmotic stress and ion disturbance in the presence of excess salt accumulation. To combat this stress situation, some metabolites/or stress-responsive gene(s) and proteins are synthesized by the plants to mitigate the salt toxicity. Therefore, to reduce the impact of salt stress on yield and other physiological activities of plants it is essential to know the whole pathway through which plant’s metabolism get affected in the presence of salt. The present review is also dealing with the same objective and special focus is given to the omics tools, such as genomics, proteomics and metabolomics to discuss the different ways of tolerance mechanism in the plant system against salt toxicity.     

Regulation of molybdenum cofactor species in the green alga Chlamydomonas reinhardtii

Molybdenum cofactor (MoCo) of molybdoenzymes is constitutively produced in cells of the green alga Chlamydomonas reinhardtii grown in ammonium media, under which conditions certain molybdoenzymes are not synthesized. In soluble form, MoCo was found to be present in several forms: (i) as a low Mr free species; (ii) bound to a MoCo-carrier protein of about 50 kDa that could release MoCo to directly reconstitute in vitro nitrate reductase activity in the nit-1 mutant of Neurospora crassa, but not to Thiol-Sepharose which, in contrast, bonded free MoCo; and (iii) bound to other proteins, putatively constitutive molybdoenzymes, which only released MoCo after a denaturing treatment. The amount of total MoCo (free, carrier-bound and heat releasable forms) was dependent on the growth phase of cell cultures. Constitutive levels of total MoCo in ammonium-grown cells markedly increased when cells were transferred to media lacking ammonium (nitrate, urea or nitrogen-free media). This increase did not require de novo protein synthesis and was stimulated by light. Levels of both total MoCo and free plus carrier-bound MoCo seemed to be unrelated to either nitrate reductase synthesis or functioning of nit-1 and nit-2 genes responsible for nitrate reductase structure and regulation, respectively. Results suggest that MoCo is continuously synthesized in C. reinhardtii and that its levels are regulated by ammonium in a way independent of nitrate reductase synthesis.     

Retained duplicate genes in green alga Chlamydomonas reinhardtii tend to be stress responsive and experience frequent response gains

Background

Green algae belong to a group of photosynthetic organisms that occupy diverse habitats, are closely related to land plants, and have been studied as sources of food and biofuel. Although multiple green algal genomes are available, a global comparative study of algal gene families has not been carried out. To investigate how gene families and gene expression have evolved, particularly in the context of stress response that have been shown to correlate with gene family expansion in multiple eukaryotes, we characterized the expansion patterns of gene families in nine green algal species, and examined evolution of stress response among gene duplicates in Chlamydomonas reinhardtii.

Results

Substantial variation in domain family sizes exists among green algal species. Lineage-specific expansion of families occurred throughout the green algal lineage but inferred gene losses occurred more often than gene gains, suggesting a continuous reduction of algal gene repertoire. Retained duplicates tend to be involved in stress response, similar to land plant species. However, stress responsive genes tend to be pseudogenized as well. When comparing ancestral and extant gene stress response state, we found that response gains occur in 13% of duplicate gene branches, much higher than 6% in Arabidopsis thaliana.

Conclusion

The frequent gains of stress response among green algal duplicates potentially reflect a high rate of innovation, resulting in a species-specific gene repertoire that contributed to adaptive response to stress. This could be further explored towards deciphering the mechanism of stress response, and identifying suitable green algal species for oil production.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1335-5) contains supplementary material, which is available to authorized users.     

Regulation of glycerol metabolism in Zygosaccharomyces rouxii in response to osmotic stress

Summary Enzyme analyses indicated that the metabolism of glycerol by Zygosaccharomyces rouxii occurred via either glycerol-3-phosphate (G3P) or dihydroxyacetone (DHA). The route via DHA is significant in osmoregulation. The specific activities of glycerol dehydrogenase (GDHG) and DHA kinase, which metabolize glycerol via DHA, increased nine- and fourfold respectively during osmotic stress [0.960 water activity (a_w) adjusted with NaCl] when compared to non-stressed conditions (0.998 a_w). Both pathways are under metabolic regulation. Glycerol kinase, mitochondrial G3P dehydrogenase and DHA kinase are induced by glycerol while the latter is also repressed by glucose. Cells treated with cycloheximide prior to osmotic upshock showed significantly lower DHA kinase and GDHG levels and lower intracellular glycerol concentrations when compared to untreated control cells. Thus protein synthesis is essential for osmotic adaptation. Offprint requests to: B. A. Prior     

Induction of tolerance to oxidative stress in the green alga, Chlamydomonas reinhardtii, by abscisic acid

The effect of abscisic acid (ABA) on the tolerance to oxidative stress in a freshwater green alga, Chlamydomonas reinhardtii, was investigated. Exogenously added ABA enhanced the growth of this alga, which was observed under continuous illumination but not in the dark. The cells treated with ABA for 24 h showed tolerance to oxidative stress caused by exposure to paraquat or hydrogen peroxide. In the ABA‐treated cells, the activities of two antioxidant enzymes, catalase (CAT) and ascorbate peroxidase (APX), were significantly higher than those in the untreated control. The result suggests that ABA plays a role in the enhancement of tolerance to oxidative stress by increasing the activity of antioxidant enzymes.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii

High light (HL) stress adversely affects growth, productivity and viability of photosynthetic organisms. The green alga Chlamydomonas reinhardtii is a model system to study photosynthesis and light stress. Comparative proteomics of wild-type and two very high light (VHL)-resistant mutants, VHL(R)-S4 and VHL(R)-S9, revealed complex alterations in response to excess light. A two-dimensional reference map of the soluble subproteome was constructed representing about 1500 proteins. A total of 83 proteins from various metabolic pathways were identified by peptide mass fingerprinting. Quantitative comparisons of 444 proteins showed 105 significantly changed proteins between wild type and mutants under different light conditions. Commonly, more proteins were decreased than increased, but different proteins were affected in each genotype. Proteins uniquely altered in either VHL(R) mutant may be involved in VHL resistance. Such candidate proteins similarly altered without light stress, thus possibly contributing to "pre-adaptation" of mutants to VHL, included decreased levels of a DEAD box RNA helicase (VHL(R)-S4) and NAB1 and RB38 proteins (VHL(R)-S9), and increased levels of an oxygen evolving enhancer 1 (OEE1) isoform and an unknown protein (VHL(R)-S4). Changes from increased levels in HL to decreased levels in excess light, included OEE1 (VHL(R)-S9) or the reverse change for NAB1, RB38, beta-carbonic anhydrase and an ABC transporter-like protein (VHL(R)-S4).     

Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth

Isocitrate lyase is a key enzyme of the glyoxylate cycle. This cycle plays an essential role in cell growth on acetate, and is important for gluconeogenesis as it bypasses the two oxidative steps of the tricarboxylic acid (TCA) cycle in which CO₂ is evolved. In this paper, a null icl mutant of the green microalga Chlamydomonas reinhardtii is described. Our data show that isocitrate lyase is required for growth in darkness on acetate (heterotrophic conditions), as well as for efficient growth in the light when acetate is supplied (mixotrophic conditions). Under these latter conditions, reduced acetate assimilation and concomitant reduced respiration occur, and biomass composition analysis reveals an increase in total fatty acid content, including neutral lipids and free fatty acids. Quantitative proteomic analysis by ¹⁴N/¹⁵N labelling was performed, and more than 1600 proteins were identified. These analyses reveal a strong decrease in the amounts of enzymes of the glyoxylate cycle and gluconeogenesis in parallel with a shift of the TCA cycle towards amino acid synthesis, accompanied by an increase in free amino acids. The decrease of the glyoxylate cycle and gluconeogenesis, as well as the decrease in enzymes involved in β–oxidation of fatty acids in the icl mutant are probably major factors that contribute to remodelling of lipids in the icl mutant. These modifications are probably responsible for the elevation of the response to oxidative stress, with significantly augmented levels and activities of superoxide dismutase and ascorbate peroxidase, and increased resistance to paraquat.     

Molybdenum metabolism in the alga Chlamydomonas stands at the crossroad of those in Arabidopsis and humans

Molybdenum (Mo) is a very scarce element whose function is fundamental in living beings within the active site of Mo-oxidoreductases, playing key roles in the metabolism of N, S, purines, hormone biosynthesis, transformation of drugs and xenobiotics, etc. In eukaryotes, each step from Mo acquisition until its incorporation into a biologically active molybdenum cofactor (Moco) together with the assembly of this Moco in Mo-enzymes is almost understood. The deficiency in function of a particular molybdoenzyme can be critical for the survival of the organism dependent on the pathway involved. However, incapacity in forming a functional Moco has a pleiotropic effect in the different processes involving this cofactor. A detailed overview of Mo metabolism: (a) specific transporters for molybdate, (b) the universal biosynthesis pathway for Moco from GTP, (c) Moco-carrier and Moco-binding proteins for Moco transfer and (d) Mo-enzymes, is analyzed in light of recent findings and three systems are compared, the unicellular microalga Chlamydomonas, the plant Arabidopsis and humans.