Exploring the Role of LHC Protein Structure and Function in the Evolution of NPQ Mechanisms in Eukaryotic Photosynthetic Organisms

The initial step of photosynthesis occurs when light energy is absorbed by an array of pigments surrounding the photosynthetic reaction center. Chlorophyll and carotenoid molecules are coordinated by members of a family of intrinsic thylakoid membrane proteins known as Light-Harvesting Complex (LHC) proteins. LHCs are essential for stabilizing and tuning the spectroscopic characteristics of each individual pigment, as well as the pigment array, to allow for efficient energy transfer. Despite a high degree of sequence conservation within the protein family, each of the LHCs performs a different role in modulating the energy landscape of the thylakoid membrane. Two stress response LHC proteins, LHCSR and PSBS, are known to be essential for non-photochemical quenching in some photosynthetic eukaryotes. It has been shown that LHCSR is necessary for quenching in the green alga Chlamydomonas reinhardtii. Homologs of this stress-response protein are present in many photosynthetic eukaryotes, even those with chloroplasts that are of a red algal origin, but LHCSR is absent from terrestrial vascular plants. Likewise, PSBS is necessary for quenching in the plant Arabidopsis thaliana, but PSBS is only found in the green lineage of photosynthetic organisms. The molecular mechanism of both of these proteins is still unknown./nThis work describes the relationship between protein structure and quenching function in the stress response protein LHCSR. I show the distinct contributions to non-photochemical quenching from each of the two LHCSR protein isoforms, LHCSR1 and LHCSR3, in C. reinhardtii. I also contributed to the identification of three lumen-exposed residues in LHCSR3 that are required for quenching function. By using directed mutagenesis and expression in heterologous and native organisms, this shows that while LHCSR is necessary for nearly all non-photochemical quenching in its native cellular environment within C. reinhardtii, it is not sufficient to activate quenching in plants when expressed heterlogously. By altering some of the pigment-binding sites within LHCSR3, however, the heterologously expressed mutant LHCSR3 can activate additional quenching in a plant system./nThis also explores possible roles of the PSBS protein from C. reinhardtii, which has not previously been shown to have a function. Here, I present data showing that the algal PSBS protein can enhance non-photochemical quenching when expressed heterologously in plants. Since we do not see the same behavior in the alga, if PSBS is expressed in C. reinhardtii it most likely plays a different role or is not sufficient to induce quenching alone. Heterologous co-expression of both LHCSR and PSBS from C. reinhardtii results in enhanced quenching in the tobacco, Nicotiana benthamiana. The enhanced quenching phenotype is dependent upon PSBS being pH-sensitive. While no evidence has yet been obtained showing an interaction between PSBS and LHCSR in C. reinhardtii, this motivates further exploration./nIn order to better understand the evolution of photoprotective mechanisms employed by photosynthetic eukaryotes, I have also contributed to a genome annotation project aimed at establishing the marine heterokont alga, Nannochloropsis oceanica CCMP 1779, as a new model alga for studying photosynthesis. I identified three potential stress-response LHCs in the organism’s genome based on sequence homology, whose functionality and expression characteristics are being investigated by other researchers.

Advisor: Niyogi, Krishna K