Artificial membranes as model systems to investigate the temperature adaptation of diatoms

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In the first two years of the project we could show that the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum modulate their lipid composition in adaption to varying environmental conditions. Cultivation of T. pseudonana under lower temperatures induced an increase of the MGDG amount in the thylakoids accompanied by a decreased concentration of DGDG. Measurements of the membrane fluidity indicated that the accumulation of MGDG led to increased membrane fluidity, i.e. loosening of the thylakoid membrane structure. Cultivation under high light conditions strongly increased the percentage of SQDG in the thylakoid membranes and reduced the MGDG content. Furthermore, the total thylakoid lipid concentration, in relation to chlorophyll (Chl), showed an increase. In general, the lipid per Chl ratio is significantly higher in diatom thylakoids compared to higher plants, which indicates either a decreased protein crowding in the diatom membrane or a shift from Chl binding proteins to proteins which are not associated with pigments. This important observation will be addressed in more detail in the third year of the project. Further measurements of the membrane fluidity concentrated on the role of the photoprotective xanthophyll cycle pigment diatoxanthin (Dtx), which is converted from diadinoxanthin (Ddx) by the enzyme Ddx de-epoxidase under HL conditions. We found that in HL thylakoids of P. tricornutum, with a high amount of free Dtx in the lipid phase of the membrane, Dtx leads to increased membrane rigidity in comparison to thylakoids which mainly contain Ddx. In the third year of the project we plan a detailed analysis of the other factors which influence the fluidity of the diatom thylakoid membrane with a special focus on the fatty acid composition and the negative charge of the anionic lipids which dominate the diatom membrane. In the first two years of the project we have developed methods for the analysis of diatom lipids and fatty acids by means of HPLC coupled with a CAD detector. We have also developed a method for the isolation of native diatom lipids and fatty acids which will now be used for the construction of artificial membrane systems. Besides measurements of the membrane fluidity, these liposomes will be used to study the effect of differences in the lipid and fatty acid composition on Ddx de-epoxidation and the structure of light-harvesting complexes (FCPs). During the first two years of the project we could also show that aggregation of the FCPs depends on low pH-values and the presence of Dtx. High concentrations of Mg2+ ions lead to further aggregation of the complexes. In the third year of the project high Mg2+ concentrations and low pH-values will also be used in measurements of the membrane fluidity and Ddx de-epoxidation to compensate the negative charges of the lipids SQDG and PG. This will allow us to determine if the negative head-groups of the anionic lipids play a role in these processes.