| Snow algae Chlamydomonas nivalis is a typical species of single-cell green algae which can adapt to stress environments including very low temperature, high light intensity and strong UV radiation for survival. The variation of intracellular lipids has closely relationship with the cell resistance to stress environments. To investigate the lipid changes under stress conditions systematically is critical to find out the typical lipids responding to the stress, and to figure out the physiological mechanism of cells to resist to the stress.In the present work, the log-phase culture of C. nivalis under autotrophic growth with aeration was treated by NaCl with final concentrations of 0.25%, 0.50%, 0.75%, 1.00%, 1.25% or 1.50% for 1, 2, 3, 5, 7, 11, 15, 24 or 48 h, respectively. The lipid components were analyzed and the results were described as follow.1. The growth rate of C. nivalis was increased significantly and 1/3 shorter of growth cycle was obtained when cells grew under aeration condition. The effect of NaCl concentration on inhibition rate of cell growth and mortality rate show an increased inhibition rate was observed with the increase of NaCl concentration. The maximal inhibitory rate (56%) was obtained at 1.5% NaCl, but the mortality rate at this time was only 6.07%, which indicated that mass mortality of cells had not occurred by high inhibition of growth.2. The detection and quantification of fluorescent intensity (FI) in stressed and control samples were carried out by laser scanning confocal microscope (LSCM). The results showed that FI of total lipids at 560~615 nm was increased followed by a decline with stressed time and NaCl concentration after strained by Nile Red and excited at 543nm. The maximal FI reached at 7 h then dropped to to the level of the control at 24 h. At 0.75% NaCl, FI at various time points was higher than that of at other NaCl concentrations with same time points, and the maximal change rate of 211.72% was observed at 7 h.3. The cell size and fluorescent intensity (FI) of neutral and polar lipids in stressed and control samples were quantified after stained by Nile Red and excited at 488 nm by flow cytometry (FCM). The results showed that FI of neutral (FL1, 530nm) and polar lipids (FL2,575 nm) was increased followed by a decline with stressed time and NaCl concentration. The maximal variation of neutral lipid up to 68.16 times was observed at 1.00% NaCl for 7 h, and 10.27 times for polar lipid was at 1.25% NaCl for 5 h treatment, respectively. At the same NaCl concentration, the cells size (SSC signal) enlarged quickly during 3 to 5 h, and then stopped enlarging. At the same stress time, no significant change of cells size occurred at different NaCl concentration.4. Fatty acid components in free fatty acids (FFAs) and neutral lipids (NL) before and after NaCl treatment were analyzed by GC-MS. The results showed C16:0, C18:3 and C18:1 were the main and typical fatty acids in FFAs, which consist of 75% for total FFAs, 70% for total unsaturated fatty acids and 65% for total lipid unsaturated degree in FFAs, respectively. In neutral lipids, C16:0, C18:3 and phytol are the typical components, which consist of 75% for total FAs, 78% for total unsaturated fatty acids and 85% for total lipid unsaturated degree in NL. These components were the main regulated fatty acids under high-salt condition and play an important role in the resistance to the high-salt stress.5. The above results revealed the physiological mechanism of resistance to NaCl stress in C. nivalis. By regulating the biosysthesis of trpical fatty acid in cells, the ratio of saturated and unsaturated fatty acids was changed fast in several hours, resulting in the variation of fluidity in biomembrane and osmotic pressure in cells. It led to a serious of rapid responses of cells to high salt stress and resistance. The rapid increase of chlorophyll also facilitates to get more energe to resist high salt stress.
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