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Nolwenn Gueguen

Characterisation of monogalactosyldiacylglycerol synthases in the model diatom Phaeodactylum tricornutum

Published on 13 May 2022
Thesis presented May 13, 2022

Monogalactosyldiacylglycerol (MGDG) is a membrane lipid class found abundantly in photosynthetic membranes. MGDG is known to play roles in thylakoid biogenesis, and in stabilisation and function of the photosystem. Despite its importance, few information is available on MGDG synthesis in complex plastids found in some eukaryotic algae. In particular, diatoms contain a secondary plastid surrounded by four membranes in which the precise glycerolipid composition is still unknown. In this thesis, we focused on the three MGDG synthase (MGD) isoforms detected by sequence homology in the model diatom Phaeodactylum tricornutum.
Phylogenetic analysis supports that the existence of the three types of MGD identified in P. tricornutum seems a conserved feature in diatoms. GFP-fusion localisation by confocal microscopy showed that each isoform (numbered α, β, and γ) localise to a distinct subcellular compartment. MGDα is located inside the plastid at the vicinity of thylakoids. MGDβ is in the periplastidial vesicular network (known also as the “blob”) and the periplastidial membrane. MGDγ is an intriguing cytosol-localised isoform. We also detected an MGDγ-like sequence in genomic data from basal apicomplexans of the Gregarina phylum, missing the usual vestigial plastid of this clade. Together, these results indicate that in these complex organisms, a MGD has been relocalised to extraplastidial compartments of the cell during evolution. The successful CRISPR-Cas9 edition of targeted MGD genes in P. tricornutum was achieved. Several knock-out mutants were generated for each isoform. Although MGDα is the most expressed isoform and localize at the vicinity of photosynthetic membranes, seeming therefore to act as the main isoform in the cell, MGDα knockout did not lead to any strong alteration in cell growth, photosynthetic activity and membrane integrity. Neither did MGDβ and MGDγ knockout lines. Such phenotype suggest that highly efficient compensation mechanisms exist under optimal growth conditions. In wild type cells cultivated in low nitrogen conditions, MGDα and MGDγ protein levels drop. Cultivation of the MGD mutant strains revealed that MGDβ and MGDγ may play an important role under such abiotic stress. Such specific role should be investigated in the future.
Variation of the fatty acid profiles was observed in different classes of membrane glycerolipids. The impact on MGDG suggests that each isoform has distinct substrate preferences, which might be linked to substrate availability in their respective subcellular compartment. Mutation of the MGDγ isoform led to a strong alteration of the composition of several extraplastidial lipid classes, compared to MGDα and MGDβ isoforms, suggesting a critical role in lipid homeostasis at the whole cell level. Future prospects on MGDG synthesis, localisation and function are proposed.

CRISPR-Cas9, Monogalactosyldiacylglycerol, glycerolipids, diatom, Phaeodactylum tricornutum

On-line thesis.