Speaker
Description
In photosynthesis, light drives electron transfer reactions that conserve reducing power and energy. Electron transfer is coupled to proton translocation into the chloroplast thylakoid lumen, building up a proton motive force (PMF) that drives ATP synthesis. Regulating PMF is essential for rapidly adjusting photosynthesis to fluctuations in light intensity, enabling a dynamic balance between protective mechanisms, such as non-photochemical quenching, and efficient light utilization. Even though the general principles that underpin the PMF in photosynthesis are well-understood, how exactly the makeup of the PMF is regulated in vivo and at the level of the individual chloroplast remains unclear.
The PMF consists of two components: the membrane potential (∆Ψ) and the proton gradient (∆pH). Both components can be measured – however the available approaches are indirect and are limited in spatial resolution, which is why a detailed picture of PMF heterogeneity between individual cells, chloroplasts or even thylakoid membranes is lacking. To address this knowledge gap, we are developing in vivo biosensing methodologies for real-time PMF monitoring and optimize high-resolution light microscopy techniques to visualize single chloroplasts and photosynthetic membranes. To this end we have expressed different genetically encoded biosensors in Arabidopsis thaliana to directly measure pH dynamics at either side of the thylakoid membranes in response to varying light conditions. This approach will be expanded to Chlamydomonas reinhardtii and is bolstered by the in-depth characterization of the biochemical and biophysical properties of each of the sensors in vitro and in vivo, ensuring specificity and minimizing measurement artifacts. Furthermore, we will advance super-resolution imaging techniques to investigate the thylakoid membrane in order to explore the structural basis of the PMF, its partitioning and potential heterogeneity. To this end we have started to develop advanced protocols for protein labeling and single-particle localization microscopy.
By integrating real-time biosensing with cutting-edge imaging technologies, we will contribute to a new level of understanding to PMF dynamics and photosynthetic efficiency. Additionally, once established these tools and techniques will be made available to the community to foster further discoveries.