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

在项目的前两年，我们可以证明，硅藻海链藻和三角褐指藻调节其脂质组成，以适应不同的环境条件。T.低温胁迫下，类囊体中MGDG含量增加，DGDG含量降低。膜流动性的测定表明，MGDG的积累导致膜流动性增加，即类囊体膜结构的松散。在强光条件下培养强烈增加类囊体膜中SQDG的百分比，并降低MGDG含量。此外，总类囊体脂质浓度，叶绿素（Chl），表现出增加。在一般情况下，每叶绿素的脂质比是显着较高的硅藻类囊体相比，高等植物，这表明无论是减少蛋白质拥挤在硅藻膜或从叶绿素结合蛋白的蛋白质，这是不相关的色素转移。在项目的第三年将更详细地讨论这一重要意见。膜流动性的进一步测量集中在光保护性叶黄素循环色素硅藻黄质（Dtx）的作用上，其在HL条件下通过酶Ddx脱环氧酶从硅藻黄质（Ddx）转化。我们发现，在三角褐指藻的HL类囊体中，与主要含有Ddx的类囊体相比，膜的脂质相中具有大量的游离Dtx，Dtx导致膜刚性增加。在项目的第三年，我们计划对影响硅藻类囊体膜流动性的其他因素进行详细分析，特别关注硅藻膜的脂肪酸组成和阴离子脂质的负电荷。在该项目的前两年，我们已经开发了通过HPLC与CAD检测器耦合分析硅藻脂质和脂肪酸的方法。我们还开发了一种分离天然硅藻脂质和脂肪酸的方法，现在将用于人工膜系统的构建。除了膜流动性的测量，这些脂质体将被用来研究在脂质和脂肪酸组成的差异对Ddx脱环氧化和捕光复合物（FCPs）的结构的影响。在项目的前两年，我们还可以证明FCP的聚集取决于低pH值和Dtx的存在。高浓度的Mg ~（2+）离子导致复合物的进一步聚集。在项目的第三年，高Mg 2+浓度和低pH值也将用于测量膜流动性和Ddx脱环氧化，以补偿脂质SQDG和PG的负电荷。这将使我们能够确定阴离子脂质的负头基是否在这些过程中发挥作用。

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.