Quantitative Proteomics Of Thylakoid Membranes In Carotenoids Accumulating Chlorophytes

Green algae evolved at the position between higher plants and lower plants. Andthey are widely distributed both on land and in the sea. The versatile and toughhabitats green algae live in enable them to survive and acclimate. Many green algaespecies synthesize and accumulate carotenoids in the process of stress response.Carotenoids include a wide array of secondary metabolites. For example, the freshwater unicellular microalgae Haematococcus pluvialis are characterized with theability of accumutaing astaxanthin under stress. And Dunaliella salina is able toaccumulate β-carotene intracellularly under stress. However, some macroalgae, Ulvaprolifera for example, show high stress tolerance ability without any carotenoidsaccumulated. Consequently, carotenoids accumulstion and stress acclimation areinterelated, with some synergy effects existed.Thylakoid membrane is where the main process of photosynthesis occurs, makingit important for green algae. In this research, the three green algae, including H.pluvialis, D. salina and U. prolifera were comparatively studied from the thylakoidproteomic perspective. H. pluvialis, which firstly synthesizes β-carotene and convertsit to astaxanthin under stress, was extensively investigated in this research. However,D. salina synthesizes and accumulates β-carotene under stresses. Hence, D. salinacould be used as a suitable comparative object in H. pluvialis photoprotection research.Besides, the macroalga U. prolifera, which shows neither large amount of β-carotenenor astaxanthin accumulation under high light stress, could also cope with high light.The elucidation of high light responsive pattern in U. prolifera might provide someclue revealing the carotenoids role in high light response. Conseqently, the thylakoidmembranes of three green algae were comparatively studied under high light stress.The main research work and conclusions are as follows:(1) The relation of carbon fixation and astaxanthin synthesis in H. pluvialisi wasinvestigated. Among the four types of cells, GV (green vegetative) cells showed the highest CA (carbonic anhydrase) activity and the lowest astaxanthin content level.Radioactive labeling and tracing results also confirmed GV showed highest carbonfixation capability. Comparatively, RC (red cyst) cells were highest in astaxanthincontent while lowest in CA activity. Notably, OR (orange resting) cells showedhighest capability of converting fixed carbon into astaxanthin. Those findings were inaccordance with the fact that OR cells were in the process of rapid astaxanthinsynthesis in H. pluvialis.(2) Comparative analysis using2D DIGE identified some differential expressedproteins among the four types of H. pluvialis cells. The rapid proliferation andbiomass accumulation characteristics GV cells were supported by some up-regulatedproteins. And the other three types of cells, which were under various stress, showedhigher level of proteins involving in low cellular metabolism. Thylakoid membraneproteomics research using BN-PAGE and sucrose gradient ultracentrifugationidentified some regulatory proteins and results also showed that H. pluvialis indifferent stages conserved the photosynthetic proteins. However, RC cells showed thepossibility of LHC-PS super-complex forming as revealed by continuous sucrosegradient ultracentrifugation.(3) By quantitatively monitoring the abundance variations of thylakoid membraneproteins, H. pluvialis showed great high light acclimation capability. Based on thefindings in this study and inspired by results from others, a hyperthetical carbon flowreorientation strategy was proposed, by which the H. pluvialis employed during highlight acclimation. It was theoretically possible that phosphate pentose pathway wasinvolved in stress induced metabolisms including starch and astaxanthin synthesis.Comparatively, U. prolifera showed inbalanced energy distribution pattern betweenthe two photosystems as indicated by the abundance alteration of LHCI and LHCII.Moreover, PsbS protein was monitored to be significantly up-regulated under highlight. Thus it is possible that PsbS activated and enhanced NPQ to funnel excessenergy for photoprotection.