Research On Glycerolipid Metabolism In Nitrogen-deprived Chlamydomonas Reinhardtii

As the major component of cellular biological membranes,lipids are highly sensitive to variable environment and play crucial roles in adaption to stressed environmental conditions.Nitrogen deprivation induces microalgal cells to elicit global metabolic shifts,during which the accumulation of storage carbon molecule,e.g.triacylglycerol,occurred.Importantly,TAG biosynthesis involves chloroplast and endoplasmic reticulum pathways and is presumed to be closely related with polar glycerolipids.Polar glycerolipids in microalgae specifically reside in various organelles.In this dissertation,the model microalga,Chlamydomonas reinhardtii,was used to comprehensively investigate TAG accumulation and the metabolic response of the individual polar lipid under distinct periods of nitrogen stress on a basis of the analysis platform of glycerolipidomics and the technique of intact chloroplast isolation.In addition,the effects of autotrophic and mixotrophic culture on TAG biosynthesis and metabolism of polar lipids were studied.The distinct contributions of the individual polar lipid to TAG accumulation were further explored for different stages of nitrogen stress.The platform regarding qualitative and quantitative analyses of glycerolipids was developed based on thin layer chromatography and gas spectrometry(TLC and GC)and liquid chromatography-mass spectrometry(LC-MS)techniques in C.reinhardtii.The constituents of polar glycerolipids and their fatty acids were first analyzed using TLC and GC technique.MS scanning by UPLC-ESI-Q-Trap/MS and MS/MS scanning by UPLC-ESI-Orbitrap/MS2,were separately applied to identify a total of 109 molecular species.Further,the targeted quantitative analysis based on external standards was performed for the 45 molecular species by UPLC-ESI-Q-Trap/MS in multiple reaction monitoring(MRM)mode.An extended approach to rapidly quantify microalgal TAG was developed using characteristic fatty acid based on that the amounts of neutral lipid,TAG,were verified to be linearly correlated with the relative abundance or the absolute content of the characteristic fatty acid.Given that the chloroplast was difficult to be isolated from stress-induced microalgae hampering study of the subcellular TAG accumulation mechanism,stressed chloroplasts were successfully isolated from C.reinhardtii subjected to 4 h of nitrogen stress for the first time.A novel quality assessment was proposed in terms of the integrity and purity of chloroplast based on the fatty acid biomarker analysis.The subcellular lipidomic analysis showed that TAG accumulated exclusively outside the chloroplast during 4 h of nitrogen stress.In addition,the betaine lipid,DGTS,and the glycolipid,DGDG,were found to reside inside and outside the chloroplast,respectively,as well.The newly synthesized DGDG and DGTS prominently increased at the extra-chloroplastidic compartments and served as the major precursors for TAG biosynthesis.In particular,DGDG contributed to the extra-chloroplastidic TAG assembly mainly in form of DAG and DGTS in form of acyl groups.The chloroplastidic membrane lipid,MGDG,was presumed to primarily offer DAG for TAG formation outside the chloroplast.The glycerolipidome responses to nitrogen starvation were compared in autotrophically and mixotrophically grown C.reinhardtii.The results revealed that there were minor differences in the amounts of glycerolipids and their fatty acid compositions between the two culture modes.The level of the individual polar lipid first augmented and subsequently declined.The main component of polar lipids converted from MGDG to DGDG together with DGTS.In addition,the distribution of carbon flux to polar lipid varied gradually.The carbon flux mainly flowed to DGDG during 0-4 h of nitrogen stress and DGTS during 4-24 h of nitrogen stress,respectively.The proportion of carbon flux to polar lipid decreased during 24-48 h of nitrogen stress.These findings indicated the vital roles of newly formed DGDG and DGTS on regulation of lipid metabolism in C.reinhardtii under nitrogen stress.The source for TAG accumulation was analyzed for mixotrophically grown C.reinhardtii during the entire phase of nitrogen starvation.The results demonstrated that multiple PUFAs derived from polar lipids were successively biosynthesized and converted to TAG via turnover of MGDG,DGDG and DGTS,resulting in the contribution of polar lipid turnover based on PUFA to TAG accumulation gradually increased.Among them,DGDG and DGTS were found to be the primary polar lipids contributing to accumulation of polyunsaturated TAG.