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Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea.

ABSTRACT: Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37 -C40) ketones (alkenones) with 2-4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as "alkenone body (AB)." Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft-associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.

SUBMITTER: Shi Q

PROVIDER: S-EPMC5034830 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea.

Shi Qing Q Araie Hiroya H Bakku Ranjith Kumar RK Fukao Yoichiro Y Rakwal Randeep R Suzuki Iwane I Shiraiwa Yoshihiro Y

Proteomics 20150702 23-24

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37 -C40) ketones (alkenones) with 2-4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant ...[more]

PMID: 25914246

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Project description:Dynamic xanthophyll cycle (XC) related non-photochemical quenching (NPQd, also called qE) is present in most phototrophs. It allows dissipating excess light energy under adverse growing conditions. Generally, NPQd rapidly reverses for photosynthesis to resume when light intensity decreases back toward optimal intensity. Under certain environmental conditions and/or in some species, NPQ can be strongly sustained (NPQs showing hours-to-days relaxation kinetics). Tisochrysis lutea is a South Pacific haptophyte phytoplankton with a strong potential for aquaculture and biotechnology applications. It was previously reported to show a surprisingly low NPQd capacity while synthesizing large amounts of diatoxanthin (Dt), a pigment involved in the XC. In order to better understand this paradox, we investigated the characteristics of NPQ in T. lutea under various growth conditions of light and nutrient availability (different photoperiods, low and high light, nutrient starvations). We found a strong NPQs, unmeasurable with usual fluorometry protocols. Along with confirming the involvement of Dt in both NPQd and NPQs (by using the dithiothreitol inhibitor), we highlighted a strong relationship between Dt and the maximum quantum yield of photochemistry (Fv/Fm) across growing conditions and during relaxation experiments in darkness. It suggests that changes in Fv/Fm, usually attributed to the 'photoinhibitory' quenching (qI), are simultaneously largely impacted by photoprotective NPQ. The overlap of xanthophyll pigments-related photoprotective NPQ with several other mechanisms involved in the cell response (Photosystem II photoinactivation, changes in pigments composition, and detoxification by antioxidants) to energy unbalance is further discussed. Our findings question both how widespread NPQs is in the global ocean, particularly in nutrient starved environments (oligotrophic waters) and situations (post-bloom), and the use of adapted active fluorescence protocols (i.e. with extended NPQ relaxation period prior to measurement).

Project description:The production of polyunsaturated fatty acids (PUFA) in Tisochrysis lutea was studied using the gradual incorporation of a 13C-enriched isotopic marker, 13CO2, for 24 h during the exponential growth of the algae. The 13C enrichment of eleven fatty acids was followed to understand the synthetic pathways the most likely to form the essential polyunsaturated fatty acids 20:5n-3 (EPA) and 22:6n-3 (DHA) in T. lutea. The fatty acids 16:0, 18:1n-9 + 18:3n-3, 18:2n-6, and 22:5n-6 were the most enriched in 13C. On the contrary, 18:4n-3 and 18:5n-3 were the least enriched in 13C after long chain polyunsaturated fatty acids such as 20:5n-3 or 22:5n-3. The algae appeared to use different routes in parallel to form its polyunsaturated fatty acids. The use of the PKS pathway was hypothesized for polyunsaturated fatty acids with n-6 configuration (such as 22:5n-6) but might also exist for n-3 PUFA (especially 20:5n-3). With regard to the conventional n-3 PUFA pathway, Δ6 desaturation of 18:3n-3 appeared to be the most limiting step for T. lutea, "stopping" at the synthesis of 18:4n-3 and 18:5n-3. These two fatty acids were hypothesized to not undergo any further reaction of elongation and desaturation after being formed and were therefore considered "end-products". To circumvent this limiting synthetic route, Tisochrysis lutea seemed to have developed an alternative route via Δ8 desaturation to produce longer chain fatty acids such as 20:5n-3 and 22:5n-3. 22:6n-3 presented a lower enrichment and appeared to be produced by a combination of different pathways: the conventional n-3 PUFA pathway by desaturation of 22:5n-3, the alternative route of ω-3 desaturase using 22:5n-6 as precursor, and possibly the PKS pathway. In this study, PKS synthesis looked particularly effective for producing long chain polyunsaturated fatty acids. The rate of enrichment of these compounds hypothetically synthesized by PKS is remarkably fast, making undetectable the 13C incorporation into their precursors. Finally, we identified a protein cluster gathering PKS sequences of proteins that are hypothesized allowing n-3 PUFA synthesis.

Dataset Information

Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea.

Publications

Proteomic analysis of lipid body from the alkenone-producing marine haptophyte alga Tisochrysis lutea.

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