Effects Of Different Nitrogen Levels On Pigment-protein Complexes And Global Protein Profiles Of Oleaginous Scenedesmus Acuminatus

Scenedesmus acuminatusis is a coenobic freshwater alga. Previous research has confirmedthat S.acuminatus can accumulate massive lipids up to54%of dry biomass under lowconcentration of NaNO3(3.6mM), while unit volume total lipid yield in6mM NaNO3showedthat it was the most suitable for S.acuminatus to accumulate oil. To a certain extent, thissuggested that low nitrogen conditions promote the oil accumulation of S.acuminatus cells. Inthis paper, S.acuminatus used as material to research the spectral characteristics of thylakoidmembrane and algal cells, the energy transfer and the change of pigment-prtein of thylakoidmembrane, and differential expression of proteins under different culture conditions with thepurpose to reveal the algal photosynthesis response mechanism during the lipid accumulation.This study could provide theoretical reference for further study in lipid accumulation and itsregulation mechanisms and optimize the culture conditions of S.acuminatus.Main results were as following:(1) Six pigment-protein complex bands were isolated from S.acuminatus cell preparationsby using disc SDS-PAGE electrophoresis. High performance liquid chromatography (HPLC)analysis showed that the pigment compositions of CPⅠ、CPa1、CPa2were mainly containedChla、Chlb and Carotenoid. Light-harvesting pigment protein complexes (LHCP) free pigmentmainly included Chla、Chlb、Lutein、Violaxanthin、Neoxanthin、zeaxanthin and β-car. The resultsof secondary electrophoresis and mass spectrometry showed that CPⅠ、CPⅠa conteined severalpeptides, which were mainly83KD、82KD,42KD、44KD and25-30KD for the next. Therelative molecular weight of peptides in CPa1、CPa2were56KD、50KD and a little of38KD、39KD. The relative molecular weight of peptides in LHCP2were26KD、27KD、20KD、21KD.According to the analysis of77K spectrum characteristics and pigment compositions andpeptides of the complexes bands. We named these complexes as CPⅠa、CPⅠ、CPa1、CPa2、LHCP and FP according to the migration rates from small to large, respectively.(2) The pigment-protein complexes were separated respectively from the S.acuminatus cellpreparations which were cultivated in different time phase (3th、6th、9th、12th、15thday) andconcentrations of NaNO3(18mM、9mM、6mM、3.6mM). The results showed that the color of complexes which were separated from the algae cells cultivated in nitrogen limition becameshallow gradually compared with the replete nitrogen (18mM NaNO3), especially in CPⅠ、CPa1、CPa2change significantly. The CPa1、CPa2disappeared on the9thday when S.acuminatusiswas cultured in6mM NaNO3, whereas they disappeared on the3rd day when cultured in3.6mM NaNO3. The result revealed that the PSⅡof S.acuminatusis was influenced significantly bynitrogen limition.(3) According to77K fluorescence emission spectrum, CPⅠand CPa2cultivated indifferent nitrogen limition were analysised. The result showed that the fluorescence-emission (Fm)of CPⅠa、CPa2became strengthened with the decreasing concentration of nitrogen,especiallythe peak position of Fmin CPa2appeared red shift phenomenon. Nitrogen limitation hadremarkable effected on the energy transfer between the LHCP and the center complexes ofphotosystems.(4)145proteins which included98up-regulation proteins and47down-regulation proteinswere identified by iTRAQ methodology. The up-regulation proteins were mainly involvedCitrate cycle and Glycolysis/Gluconeogenesis which included malate dehydrogenas、aconitatehydratase、fumarate hydratase、Phosphoenolpyruvate/pyruvate domain-containing protein、glycolate dehydrogenase、6-phosphogluconate dehydrogenase、 pyruvate kinase、Glyceraldehyde-3-phosphate dehydrogenase. The result indicated that algal cells provide energysources for basic physiological metabolism by disintegrating carbohydrates. Thedown-regulation proteins were mainly involved Photosynthesis proteins which includephotosystem II47kDa protein (CP47)、light-harvesting chlorophyll-a/b binding protein LhcbM4(LHC)、Photosystem II CP43chlorophyll apoprotein (CP43)、Photosystem Q(B) protein、light-harvesting chlorophyll-a/b binding protein LhcbM3(LHC)、light-harvesting complex Iprotein (LHC I)、Photosystem I P700chlorophyll a apoprotein A2、Photosystem II D2protein.The result indicated that algal cells decreased NADPH by the degradation of photosynthesisrelated proteins to reduce the cell damage and maintain cell basic nitrogen source of growth.