Study On Photoprotection Mechanisms In An Antarctic Microalga, Chlamydomonas Sp. ICE-L

Light is necessary for photosynthesis, but excess light can causephotoinactivation and even result in cell death. For this reason photosyntheticorganisms have evolved multiple protection mechanisms with protection from thedamaging effects of light. Non-photochemical quenching (NPQ) has been found tohave the fastest response to excess light and consists in the thermal dissipation ofenergy absorbed in excess. Interestingly, NPQ is present in both plants and greenalgae and yet its activation relies on different gene products, whereas the PSBSprotein is crucial in plants, the green alga Chlamydomonas reinhardtii requires adistinct Lhc-like polypeptide called LHCSR. In the timescale of several minutes,strong illumination also activates the synthesis of the xanthophylls zeaxanthin frompre-existing violaxanthin by activation of violaxanthin de-epoxidase (VDE), whichcalled the xanthophyll cycle. It has been demonstrated that the amount of zeaxanthinis strongly correlated with NPQ in various plants under different conditions.In the Antarctic region, seasonal changes in light intensity, UV-B radiation andhigh salinity of ice bubbles restrict photosynthesis in polar oceans. Accordingly,elucidating the molecular mechanism of acclimation and regulation of photosynthesisin Antarctic algae is very important. Chlamydomonas sp. ICE-L is a primary greenalga found in the Antarctic region, and its responsive mechanisms of adaptation tostress provide an important basis for study of the Antarctic environment and species.In this study, two different LhcSR genes, LhcSR1and LhcSR2from Chlamydomonassp. ICE-L, were cloned from the total cDNA and characterized in response to highlight (HL), low light (LL), UV-B radiation and high salinity (HS). In parallel, theeffects of those conditions on photophysiological parameters and de-epoxidation state of the violaxanthin cycle were examined.Maximum PSII photochemical efficiency (Fv/Fm) and NPQ of cells subjected todifferent conditions (HL, LL, UV-B radiation and HS) were examined by Dual-PAM.The lower Fv/Fm as well as the associated induction of NPQ was observed under highlight, UV-B radiation and high salinity. The data showed that Chlamydomonas sp.ICE-L was under stress and NPQ plays an important role in the thermal dissipation ofexcess absorbed energy. Under HS conditions, Fv/Fm reached its lowest point from1to3h, while the NPQ increased, after which they returned to their normal levelsslowly. These results demonstrated that short-term HS conditions were stresscondition for Chlamydomonas sp. ICE-L, but the algal cells can accommodate thelonger periods of HS stress. Fv/Fm and NPQ changed only slightly under LLcondition, which indicated that Chlamydomonas sp. ICE-L was not obviouslystressed.Primers for partial cDNA of LhcSR1and LhcSR2were designed from thenucleotide conserved regions of LhcSR1and LhcSR2from C. reinhardtii, respectivily.Based on the partial sequences of the two genes, the3’-and5’-ends of the cDNAwere obtained by the RACE approach. The full-length of LhcSR1and LhcSR2were1118bp and1122bp, respectively. The amino acid sequence of LHCSR1was62%homologous with LHCSR1of C. reinhardtii, while LHCSR2shared64%homologywith LHCSR2/3of C. reinhardtii. There were three possible transmembrane helices inthe two proteins, among which the first and third helices of the LhcSR proteinsincluded the well-conserved generic LHC motif.The expression of LhcSR genes under different stress conditions (HL, LL, UV-Bradiation and HS) was intensively studied by quantitative real-time PCR (qRT-PCR).The expressions of LhcSR1and LhcSR2did not obviously increase during the first0.5h of HL. However, they increased rapidly from0.5h and peaked at3h. This longerresponse to HL might relate to the unique physiology of polar environments. UnderLL, the expression of LhcSR1decreased gradually, while LhcSR2was up-regulatedduring the first0.5h and then decreased. LhcSR2might be necessary to collectphotons as efficiently as possible under LL conditions. The up-regulation of LhcSR1 and LhcSR2, which was strongly induced by UV-B and HS, was accompanied by ahigh level of NPQ. These findings indicated that LhcSR1and LhcSR2might play aprimary role in reducing the excited state of excess photons to avoid photodamage.The expression of LhcSR1and LhcSR2were both up-regulated under HL, UV-Bradiation and HS stress conditions, which enable a better understanding of thephysiological function of Chlamydomonas sp. ICE-L under stress conditions. Theprotein of LHCSR1and LHCSR2were up-regulated under stress conditions by thewestern blot analysis, suggesting that the two proteins participate the photoprotect ofChlamydomonas sp. ICE-L and LHCSR1plays an important role in the thismechanism.Recombinant of prokaryotic expression vectors pET-28a-LHCSR wereconstructed and transformed to E.coli BL21to express LHCSR1and LHCSR2protein.The expression condition was0.5mmol/L concentration of IPTG,15℃and5hours.SDS-PAGE results indicated that the molecular weight of the target proteins weresimilar to theoretic molecular weight. ICE-L-LHCSR1and ICE-L-LHCSR2proteinwas expressed as inclusion body. It is very helpful for further research of thereconstitution of protein-pigment complexes in vitro.The concentration of xanthophyll cycle pigments (violaxanthin (V),antheraxanthin (A) and zeaxanthln (Z)) was determined using reversed phase HPLC.The de-epoxidation state of the violaxanthin cycle was calculated as (Z+0.5A)/(V+A+Z). The value of (Z+0.5A)/(V+A+Z) increased under stress conditions (HL, HS).So, xanthophyll cycle plays an important role in photoprotect of Chlamydomonas sp.ICE-L.