Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalgaostreococcus tauri

Abstract

Short title: plastidic Δ6-desaturase in the green lineage 15One sentence summary: Osteococcus tauri plastidic lipid C18-PUFA remodelling 16 involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for 17 both head-group and acyl-chain. 18 Footnotes: 19Author Contributions 20 CDG performed the work and analyses on O. tauri (cloning, transgenic screening, TCL, GC-FID, MS/MS); RD performed the work and analyses on N. benthamiana OE 22 (cloning, FAMES analysis); CL performed most of the lipid analyses of 23 O. tauri and N. benthamiana (agro-transformation, HP-TLC, GC-FID); NP performed the 24 work and analysis on Synechocystis (transformation, screening, TCL, GC-FID); SM designed, 25 2 performed and analyzed the photosynthesis experiments; KT performed cloning and qPCR 26 experiments; JeJ performed the work on DES localization and qPCR analyses; JuJ performed 27 MS/MS analyses; JG performed the work on DES localization; IS supervised the work on 28Synechocystis; FD performed and supervised the work on N. benthamiana, helped to organize 29 the MS; FC designed, supervised and performed the research, analyzed the data (O. tauri, N. 30 benthamiana, Synechocystis), wrote the paper. 31The authors responsible for distribution of materials integral to the findings presented in 32 this article in accordance with the policy described in the Instructions for Authors are: 33 Florence Corellou, florence.corellou@u-bordeaux.fr and Iwane Suzuki, 34 iwanes6803@biol.tsukuba.ac.jp concerning Synechocystis PCC 6803 lines. 35 3 ABSTRACT 36 Eukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 37 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several 38 microalgae, including O. tauri, plastidic C18-PUFA are specifically regulated by 39 environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. 40 Sequence retrieval from O. tauri desaturases, highlighted two putative Δ6/Δ8-desaturases 41 sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 42 desaturases. Their overexpression in heterologous hosts, including N. benthamiana and 43 Synechocystis, unveiled their Δ6-desaturation activity and plastid localization. O. tauri lines 44 overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount 45 under phosphate starvation but didn't show any obvious physiological alterations. Detailed 46 lipid analyses from the various overexpressing hosts, unravelled that the substrate features 47 involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely 48 the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the 49 substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity 50for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 51 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native h...