| The snow alga Chlamydomonas nivalis is a typical microalgal species that can adapt andresist to natural habitats in the polar region and similar extreme environments. Among varioushostile living conditions, high salinity and nutrient limitation are the common stress which C.nivalis should adapt to for survival. The regulation of lipid biosynthesis in microalgae is themost crucial strategy in resistant physiology. Especially, the alteration of fatty acids in cellularlipids, which is related to the functions of cell membrane and metabolic processes, is knownto play a vital role for cell survival and reproduction. The other acclimation and regulatoryresponses including the changes of metabolic flux (increased catabolism and decreased anab-olism, etc.) also enable the algae to survive in unfavorable conditions. In this work, NaCl waschosen as the representative salt in scientific work to provide salinity stress. Nitrogen andphosphorus were deprived to simulate nutrients deficiency in living C. nivalis cells. Themain results were as follows:1. In cytomic study, confocal laser scanning microscopy (CLSM), flow cytometry (FCM)and spectral scanning multimode microplate reader (SSMMR) were used to rapidly screenand quantify the total lipids, polar lipids and neutral lipids. The algae were stressed for1,2,3,5,7,11,15,24,48h at NaCl concentration of0.25%,0.50%,0.75%,1.00%,1.25%,1.50%.The biomass first increased and then began to decrease after24h, indicating a lagtime forNaCl stress to inhibit the cell growth. Total lipids by CLSM increased to the maximum at7hand then decreased at all NaCl stress groups except1.5%NaCl which increased to the peak at5h. The fluorescent intensity of neutral and polar lipids also showed an increase and then adecrease trend. For different NaCl concentrations, the lipids signal peaked at different time. Inall, the lipids change is closely related to cell volume changes and glycerol production, whichis help the algae to adapt to NaCl stress. The decrease of biomass could lead to the decrease oflipids content.2. The algae cells under nitrogen-deprivation, phosphate-deprivation and nitro-gen/phosphate co-deprivation were stressed for6,12,24,48,72h. The biomass was sharplydecreased after24h. The contents of total lipids, neutral and polar lipids reached the maxi-mum at24h. In the earlier nutrients deprivation condition, the cell could use intracellular nu-trients storage for cell growth, and then the biomass decreased after nutrients consumed up.Initially, the lipids increased after nutrients deprivation induction and then decreased due tocell growth inhabitation. 3. An integrated approach of GC/MS coupled with multivariate statistical analysis wasdeveloped to discriminate the fatty acids profiles between different stress conditions and dis-cover the fatty acid biomarkers in response to various stresses. Pattern recognition by orthog-onal projection on latent structure discriminant analysis (OPLS-DA) showed that algae sam-ples between different groups could be separated by their different fatty acids profiles. Sixfatty acids, C16:0, C16:3, C18:0, C18:1, C18:2and C18:3, in NaCl stressed group; one fattyacids, C18:1, in nitrogen deprivation group; four fatty acids. C16:1, C18:0, C18:1and C16:0,in phosphate deprivation group; three fatty acids, C16:1, C18:1and C16:0, in nitrogen andphosphate co-deprivation group were selected as the biomarkers respectively. The alterationof these fatty acid biomarkers resulted in the decreased ratio of unsaturated fatty ac-ids/saturated fatty acids (UFA/SFA), leading to a decreased degree of lipid unsaturation (DLU)in the corresponding group. Therefore, the cells decreased cell membrane fluidity and perme-ability to enhance cell tolerance to NaCl and nutrient deprivation stress.4. An integrated approach of UPLC/Q-TOF coupled with multivariate statistical analysiswas developed to discriminate the lipid profiles between different stress groups and discoverthe lipid biomarkers in response to various stresses. Pattern recognition by OPLS-DA andPLS-DA (partial least squares discriminant analysis) showed that algae samples between dif-ferent groups could be separated by their different lipid profiles. Thirty-five, twenty-two andnineteen lipid biomarkers were selected in NaCl stress, nitrogen, and nitrogen/phosphateco-deprivation group respectively. All these biomarkers were structured and identified thatthey belongs to seven types of polar lipid molecules, MGDG, DGDG, DGTS, PG, SQDG, PEand PI. The functions of these biomarkers in relation to the regulation of cell membrane sta-bilities, signal transduction and photosynthesis efficiency under various stress conditions arediscussed.5. By using GC/TOF-MS based approach, major metabolite profiles in samples from ni-trogen or phosphate deprivation stress groups were obtained, then171metabolites which be-long to8classes of carbohydrates, non-amino acids organic acids, polyols, nitrogen(N)-containing compounds, phosphates, amino acids, fatty acids and other compounds wereidentified. During a72-h time course experiment, five groups (control, nitrate-deprivation for6and12h, nitrate-deprivation for24,48and72h, phosphate-deprivation for6and12h,phosphate-deprivation for24,48and72h) were clearly clustered in PCA and OPLS-DA pat-tern recognition model.30metabolites were selected as the metabolic biomarkers in responseto nitrogen-deprivation and39metabolites to phosphate-deprivation stress condition. Thechanges of these biomarkers contents in cells showed the increase of carbohydrate and fatty acids compounds and the decrease of N-compounds and organic acids in both stress condi-tions. The up-or down-regulation of these metabolic biomarkers biosynthesis were closelyrelated to the regulation of amino acid metabolism, fatty acid biosynthesis, glycolysis and cit-ric acid cycle pathway. Based on these regulations of metabolic pathway in cells, the snowalga can response and adapt to unfavorable stress conditions very quickly.This is the first report for the comprehensive understanding of C. nivalis in response andadaptation to NaCl and nutrient deprivation stress by analyzing the whole lipidome andmetabolome based on “omics” technology, indicating the cytomic, lipidomic, and metabo-lomic technologies has paved the way for a systematically biological understanding of algalstress phenomena. |
PDF Full Text Request