Effects Of Acetic Acid, Nacl And Na₂CO₃Stresses On Chlamydomonas Reinhardtii

At present, water body acidification is one of the most dramaticallyenvironmental problems in the world. Besides inorganic acids, organic acids are otherreasons for the acidification, and acetic acid is the dominant component of organicacids. It widely spreads in nature, and its content can accumulate up to millimolarconcentrations, so it is a tremendous potential danger for the growth of algae. Besidesthat, water body in arid and semi-arid regions is becoming salinization mainly causedby high evaporation, so the algae often face this stress. As the acidification andsalinization are seriously affecting the ecosystem balance and environment safety inwater body, we investigated the effects of acetic acid, NaCl and Na₂CO₃stresses onthe growth of Chlamydomonas reinhardtii, a model organism of single cell, and therelationship between those stresses and photorespiration of the alga. We furtherstudied the induction of PCD by acetic acid in C. reinhardtii cells and release ofVOCs during the cell death. The possible role of VOCs as a signal was alsoinvestigated. Meanwhile, we investigated the VOCs from the cells stressed by NaCland Na₂CO₃, too. We finally tried to find whether ROS could be produced by theoperation of photorespiration under NaCl stress. The results are summarized asfollows:1. The degradation of photosynthetic pigments in C. reinhardtii cells was foundunder the stress of acetic acid at pH5.0, of NaCl at300mM, and of Na₂CO₃at25mM,50mM or150mM, and the degradation was independent of light condition. Thephotosynthetic characters were also changed by acetic acid, NaCl and Na₂CO₃stresses, and the Fv/Fm, Y（II） and ETR were declined in some degree. The qP wasincreased under lower strength stresses, but was decreased under stronger stresses.The NPQ was increased under all stresses in a certain time.2. The stresses of acetic acid, NaCl and Na₂CO₃induced the burst of O₂ˉ.andH₂O₂in C. reinhardtii cells, and the burst happened at10min under acetic acid stressand at30min under NaCl and Na₂CO₃stresses. The activities of SOD, POD and CATwere also changed with the increase of ROS content, and all raised in some degree under lower strength stresses.3. In both the cell line of control （CW-Arg） and the SGAT gene knocked downcell lines of C. reinhardtii （line T and A）, the SGAT activity, as well as the content ofSer and Gly were increased under salt stress, and the increase was accompanied withthe stress strengthening. The growth rate of T and A cell lines was lower than that ofCW-Arg （control） under normal and stress conditions, however, the stress effects ofacetic acid, NaCl and Na₂CO₃on the growth of T and A was stronger than that ofCW-Arg. Those indicated that alga photorespiration closely related with its capabilityof anti-stress. The stress promoted the photorespiration in algae, and their capabilityof stress resistance was markedly declined after the photorespiration was affected.4. When the glycolate-quinone oxidoreductase system was inhibited by SHAMor glycine decarboxylase was inhibit by MOA, the content of Ser in C. reinhardtiicells was significantly reduced, but Gly was significantly increased, indicating thatthe photorespiration was markedly affected. Under NaCl stress, ROS content in thecells treated with the two inhibitors was dramatically lower than that in the untreatedcells, with the SHAM treated cells being the lowest. This suggested that the reactioninhibited by SHAM might be the ROS production site. Thus, the decrease in ROScontent after inhibition of glycine decarboxylase by MOA might also result from thereduction of the conversion of glycolate to glyoxylate, which was caused by theinhibition of its downstreem reaction, the conversion of glycine to serine. Because thephotorespiration pathway in A cell line was markedly inhibited in the conversion ofserine to glycine, its ROS content was also significantly lower than that in CW-Arg（cell line of control） under NaCl stress. Those results demonstrated that ROS was alsoproduced during the operation of photorespiration in this alga, and that the productionsite was the conversion of glycolate to glyoxylate catalyzed by glycolate-quinoneoxidoreductase system.5. The usage of extracellular L-Ser and L-Lys by C. reinhardtii cells was not viadirect absorption, but via extracellular deamination. The deamination was dependenton nitrogen starvation, and acetic acid can promote this process. C. reinhardtii cellscan use L-Cys as the sole nitrogen source, and its intracellular content was increasedwith the increase of extracellular content and prolonging the feeding time, indicating that L-Cys was taken up via passive diffusion. The uptake of L-Cys was not affectedby Leu, indicating that their absorptions are independent.6. Acetic acid at pH5.0induced DNA degradation after30min treatment, and thedegradation was strengthened gradually during the treatment and finally resulted infragments of about150-350bp after6h treatment. The degradation of DNA can bedelayed by ZnSO₄treatment, and the pretreatment with1mM had greater inhibitioneffect on DNA degradation than with0.5mM. The cells treated with pH5.0aceticacid had TUNEL positive nuclei, and the positive nuclei appeared in10%,95%and100%cells after1h,2h and4h treatment, respectively. Those results suggested thatpH5.0acetic acid induced PCD in C. reinhardtii cells.7. Acetic acid （34.3mM） at pH5.0induced PCD in C. reinhardtii cells, whileH₂SO₄, HCl, H₃PO₄, phosphate buffer and propionic acid did not, but killed the cellsat pH₃.0, indicating that the induction of PCD did not mainly relate with the mediumacidity, but related with its chemical property of acetic acid. When the cells were keptin the culture medium containing50mM acetic acid and at pH6.0, they were alsokilled. However, the cells could survive in the medium containing150mM acetic acidand at pH7.0, at which most of the acetic acid molecules are in ionic form. Thisdemonstrated that there was no relationship between the cell death and the CH₃COOˉform in the medium. The results showed that PCD induced by acetic acid was mostprobably related with both pH and CH₃COOH form.8. Lots of VOCs were released from the C. reinhardtii cells in PCD induced byacetic acid, and their components and content reached the highest level after2htreatment. At least34different compounds were determined and are among thealdehydes, alcohols, esters, ketones, alkanes, alkenes and terpenoids. Besides aceticacid,300mM NaCl or150mM Na₂CO₃stress also induced VOCs release from C.reinhardtii cells. Hexanal （GLVs） was induced by both acetic acid and NaCl stresses,with the peak areas of23.49×10⁷and3.14×10⁷, respectively. However, Na₂CO₃stressdid not induce hexanal, but induced special components of3,4-dimethyl-hexane and5-methyl-2-heptene. Longifolene was the main terpenoid under the3kinds of stresses,with peak areas of5.82×10⁷,3.11×10⁷and4.00×10⁷, respectively, for acetic acid,NaCl and Na₂CO₃. 9. When normal C. reinhardtii cells were exposed to those VOCs from the cellsstressed by acetic acid, NaCl or Na₂CO₃, the cell growth was significantly inhibited,but the chlorophyll content, Fv/Fm, H₂O₂content and antioxidant enzyme activitieswere significantly increased, indicating that VOCs from algae under abiotic stress cantransfer information between algae.