| The South Pole is one of the coldest areas on the earth. Frozen seawater forms a semisolid matrix, permeated by a network of channels and pores, and is filled with brine formed from expelled salts as the ice crystals freeze together. It is within this labyrinth that the sea-ice microalgae live with the only liquid being pockets of concentrated brines. At the same time, it is a low sunlight, poor nutrition and less air exchanges in the ice sheet. In addition, UV-B radiation increases greatly in Antarctic region because of the ozone cavity, which brings great effect on ice microalgae. When sea ice melts in every spring and summer, the environments around the ice microalgae change enormously: temperature rises, salinity reduces, sunlight and UV-B radiation strengthen and air exchange is frequent. Acclimating the frequent freeze-thaw cycles, Antarctic ice microalgae form gradually a series of physiological and biochemical mechanism to adapt these extreme changeable environments.Just because of special physiological characteristic, ice microalgae have been used as fine tested materials to cryobiology and potential source of the new-style active substances. For reasonable development of Antarctic ice microalgae, increase of existing microbial resources, discovery of valuable active substances and products, and the new materials for fundamental researches, further study on the anti-adversity mechanisms and proteomics of Antarctic ice microalgae Chlamydomonas sp. L4 are carried out.(1) Two kinds of temperature microalgae Pheaodaetylum tricornutum and Chlamydomonas monadina as control, the patience to high salinity of 7 Antarctic ice microalgae, Pyramimonas sp. L1, Chlamydomonas sp. L4, Biddulphia sp., Nitzschia sp., Coscinodiscus sp. and Cyanophyceae were detected. The results showed that the patience of ice microalgae was higher than that of temperature microalgae. However, different kinds of ice microalgae had different patience to salinity because of their category, and the resisting order was successively Diatom >Green algae >Blue green alga.(2) Regarding temperature microalga Ch. monadina as control, the membrane lipid superoxidation level of Chlamydomonas sp. L4 stressed by high salinity (99%o)was studied. In normal cases, MDA, SOD and proline contents in Chlamydomonas sp. L4 were higher than that of temperature microalga, in which proline could keep permeating balance inside and outside of the cells, and SOD could remove the active oxygen emerging in cells. But the high content MDA was harmful to the cells. The interaction of intracellular substances maintained the normal metabolism, and maked the cells in a normal level with lower membrane permeability. Under the circumstances that the high salinity was concerned, MDA content in the cell rose, the active oxygen increased, membrane lipid superoxidation took place and membrane permeability increased completely. Additionally, the increased antioxidases, such as SOD, etc. removed the induced active oxygen. Proline, the main osmotic regulator, also increased to regulate the balance outside and inside of cells. Under the regulation of increased substances, the treated cells reached a new balance and kept their normal growth.(3) The relation of membrane lipid superoxidation level and antioxidase systems was analyzed in Chlamydomonas sp. L4 treated with low temperature (—4 ~—6 °C), strong UV-B radiation (70 uE-m"2-s"') and high salinity (99%o). The results were as followed, under the induce of low temperature, the scavengers (such as SOD and POD) could remove O?~ produced by membrane lipid superoxidation, which led to the decrease of MDA and LOX. But the content of CAT was extremely low, and almost had no function in removing the active oxygen. Under the stress of strong UV-B radiation, membrane lipid superoxidation of Chlamydomonas sp. L4 caused by active oxygen instead of LOX increased. In this study, the activity of CAT dropped obviously and SOD increased on a small quantity. Under the stress of high salinity, the superoxidation level of membrane lipid was lower. The content of POD and CAT increased obviously, while the content of SOD reduced, indicating that the main active oxygen were H2O2 and -OH, and 02* ~ was less under high salinity stress. In a word, under the stresses of 3 different adverse circumstances, the antioxidase system displayed high activity in removing free radicals. So, it was suggested that the content of all kinds of antioxidase was the biomarker to adverse acclimation.(4) Cytoplasm membrane is the first location where the environmental stress actions. By the efficient water two-phase system to extract the cytoplasm membrane of Chlamydomonas sp. L4, the changes of fatty acid composition under different adverse circumstance were analyzed. Results indicated that the content of unsaturated fatty acids in Chlamydomonas sp. L4 was higher, which was an adapt response to the Antarctic extreme environments. Under low temperature, the content of saturated fattyacids and monounsaturated fatty acids dropped obviously, but the polyunsaturated fatty acids rose. Additionally, the percentage of fatty acids with chain more than 20 carbons reduced obviously. In addition, one of the most obvious change was that the content of C 18:3 fatty acid obviously increased, and two new fatty acids of C20:l and C20:3 appeared. Therefore, the reducing of saturated fatty acid content and chain length, the increasing of unsaturated fatty acids content and flank chains in the cell membrane, played very important role in anti-freeze of Chlamydomonas sp. L4. Cell response was different under high salinity stress, the contents of saturated fatty acid and monounsaturated fatty acids increased, the polyunsaturated fatty acids dropped, and the fluidity and osmosis of membrane decreased, which reduced the damage of high salinity on membrane.(5) By optical microscope, scan and transmission microscopes, the morphology and ultrastructure of Chlamydomonas sp. L4 treated with low temperature (—4 ~ —6 °C), strong UV-B radiation (70 uE-m'^s"1) and high salinity (99%o) were studied. Comparing with normal temperature green microalgae, the main characteristics of Chlamydomonas sp. L4 were as followed: relatively thick cell wall, obvious plasmolysis phenomena at the end; much black particles between cell wall and membrane. In addition, much lipid and starch particles, as the important storage components, played an important role in the survival of Chlamydomonas sp. L4 in Antarctic area. The chloroplast of Chlamydomonas sp. L4 was irregular and spread all over the cell, and all of these were the adaptation to extreme environment. Through 3 kinds of adverse stresses of low temperature, UV-B radiation and high sanity, the morphology of Chlamydomonas sp. L4 changed scarcely under low temperature, only with a small amount of starch particles consumption; Secondly, it was UV-B radiation that caused the secretion of extracellular substances and increasing thickness of cell wall to shield UV-B rightly, so as to protect the intracellular organs; while in high salinity, the algal morphology changed greatly, with the membrane producing a large amount of folds and consuming a large number of starch particles. We also found that the thylakoid, mitochondria and cell nucleus were always steady under different adversity, which maintained the fundamental metabolism of organisms.(6) Through reduplicative practices, an efficient method of 2-DE analyses was founded to study the anti-adversity proteins of Chlamydomonas sp. L4. Liquid nitrogen grinding, together with quartz sand grinding buffer, could break the microalgal cells completely. Improved Trichloroacetic acid (TCA)-acetone fractionalprecipitation method was the effective extraction method for soluble protein, which could get rid of pigment, fat and salt in ice microalgae. The sample quality of 150 ug was more suitable for IPG gel of 13 cm, in which proteins could be separated effectively without overlapped protein spots. The equilibration time of 15 min was reasonable in the equilibration, which not only made protein transfer to SDS gel completely, but less lost protein into buffer. Silver-stained for mass spectrometric analysis method was feasible, which could detect low-abundant protein, and silver-stained proteins could be analyzed directly by mass spectrometry.(7) Anti-adversity proteins of Chlamydomonas sp. L4 treated with low temperature (—4 ~—6 °C), UV-B radiation (70 uE-m^-s'1) and high salinity (99%o) were analyzed by means of 2-DE. 356 and 325 spots were obtained in ice microalga before and after the treatment of strong UV-B radiation. Under the stress of UV-B radiation, the synthesis of 12 spots increased and 16 decreased. In addition, 7 spots disappear and 2 spots (20 kDa, 21 kDa) were newly synthesized. By searching in protein database, one of the newly-synthesized proteins is primarily confirmed as Mn-SOD, an important active oxygen and free radicals scavenger. The other is the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which acts as the transporter of electrons in the energy supply. 626 and 652 spots were obtained in Chlamydomonas sp. L4 before and after the treatment of high salinity. Under the stress of high salinity, the synthesis of 18 spots increased and 28 decreased. In addition, 8 spots disappeared and 1 spot (MW 51 kDa, pi 6.90) was newly synthesized. By searching in protein database, the newly-synthesized protein is primarily confirmed as the processor of light reaction center protein CP43 in photosystem II, which increase photosynthesis ability of Chlamydomonas sp. L4 treated with high salinity. 467 and 429 spots were obtained in ice microalga before and after the treatment with low temperature. Under the stress of low temperature, the synthesis of 11 spots increased and 6 decreased. In addition, 4 spots disappeared and 2 spots were newly-synthesized.(8) By PMF and database researching methods, 2 new protein spots induced by low temperature (—4 ~— 6 °C) in Chlamydomonas sp. L4 were identified, and the results showed they were respectively isopropylmalate/homocitrate/citramalate syntheses and glutathione S-transferase. Isopropylmalate/homocitrate/citramalate syntheses play an important role in the synthesis of ammonia acids and proteins, which compensate the decreasing activity of protease caused by low temperature. Glutathione S-transferase can scavenge the active oxygen and radicals caused by low temperature. These changes brought about good conditions for the existence ofChlamydomonas sp. L4 in low temperature.(9) Ice-active substances (IASs) are a kind of glycoproteins with the function of ice binding, recrystallization and croprotective activity. IASs of 5 Antarctic ice microalgae were determined, and the results indicated that different microalgae had different activity of recrystallization inhibition, and their activity were successively for Chlamydomonas sp. L4 > Biddulphia sp. > Nitzschia sp. > Coscinodiscus sp. > Auricula castrracane B7. The determination of recrystallization inhibition activity showed that solution containing IASs formed relatively little particles, which could lighten the frozen injury to organisms.In a word, the author discusseed the adaptability of Chlamydomonas sp. L4 to low temperature (—4 ~— 6 °C), high salinity (99%o) and strong UV-B radiation (70 uE-m"2-s "]) from the angle of physiological and biochemical changes, morphology and ultrastructure, membrane fatty acids, proteins and IASs changes, which provided that Antarctic ice microalgae could adapt the extreme environments bitterly. In fact, Chlamydomonas sp. L4 lives in channels and pores of high salt, low illumination and strong radiation, containing a large amount of active oxygen at the same time. So, all adversities must be considered synthetically to understand accurately the acclimation of Antarctic ice microalgae to extreme environments.
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