The molecular basics of rapid photoprotection and its influence on competitive fitness in Phaeodactylum tricornutum

• Applicant: Professor Dr. Bernard Lepetit
• Subject Area: Plant Physiology ; Plant Biochemistry and Biophysics
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 290346585

Diatoms are successful microalgae in most aquatic environments. The pronounced photoprotection capacity based on the energy dependent fluorescence quenching (qE) is assumed to be a major reason for their competitive advantage. While the importance of the xanthophyll diatoxanthin, synthesized during high light exposure in the xanthophyll cycle, for qE is known since decades, the relevance of Lhcx proteins for qE of diatoms was revealed only some years ago. In our previous DFG project we demonstrated that Lhcx1, x2, and x3, but not Lhcx4, all provide qE in Phaeodactylum tricornutum. This enables P. tricornutum to adjust qE capacity depending on the environmental constraints. We also showed that Lhcx proteins provide qE by reducing the functional absorption cross section of photosystem II via uncoupling of the outer antennae. Out of this DFG project, new questions emerged and some need to be addressed with different experimental approaches. The follow-up DFG proposal has three objectives. We want to:i) Reveal which amino acids and peptide motifs are essential to functionally distinguish an Lhcx from a classical light harvesting protein. We will use the P. tricornutum null qE x1KO line, transform several modified Lhcx proteins into this line and check for qE recovery. Besides mutated version of the P. tricornutum Lhcx proteins, we will also transform Lhcx/Lhcsr proteins from other organisms as well as chimeras of Lhcx and Lhcf/Lhcr proteins.ii) Identify protein interaction partners of Lhcx proteins and thus obtain insights into their localization within the thylakoids. For this, classical biochemical approaches based on pigment protein complex separation, followed by protein identifications with western blot and mass spectrometry (MS) , were only partly successful in P. tricornutum. Therefore, we will apply membrane permeable crosslinkers, followed by mass spectrometry analyses (XL-MS), as well as pull-down assays, followed by western blot and MS.iii) Address the impact of qE for competitive advantage of cells under prolonged light stress conditions more thoroughly. As the previous experiments showed that also x1KO cells develop qE during prolonged light stress due to up-regulation of Lhcx2 and x3, we will produce triple Lhcx1/x2/x3 KO lines with a CRISPR-Cas ribonucleoprotein or episome based CRISPR-Cas approach. Additionally, we want to create KO mutants of the xanthophyll cycle, as the absence of diatoxanthin results in a complete loss of qE capacity in P. tricornutum. The mutant lines will then be co-cultivated under various stress conditions with a P. tricornutum strain which contains an eGFP but otherwise possesses normal qE capacity, and the respective growth will be assessed using flow cytometry. These different experiments will provide major insights into the molecular characteristics of Lhcx mediated qE as well as its impact on competitive advantage of diatoms.

• DFG Programme: Research Grants