Elucidating the interplay of PMF determinants in mitochondria and chloroplasts in folio

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Mitochondria and chloroplasts have been shaping eukaryotic life as we know it. At the center of the function of both organelles is the proton motive force (PMF). The PMF connects redox chemistry of electron transport with ATP synthesis, but also acts as a regulation hub that determines the function and the makeup of the organelle. In plants and algae, the PMF of both mitochondria and chloroplasts are interconnected via the cellular metabolic network. While efficient photosynthesis requires chloroplast and the mitochondrial bioenergetics to work in concert, how exactly this interplay is achieved and underpinned in vivo remains insufficiently understood. In P07, we will elucidate how the subcellular dynamics of protons (H+), potassium ions (K+) and calcium ions (Ca2+) determine PMF characteristics in mitochondria and chloroplasts in vivo. Using a mature rosette leaf of Arabidopsis thaliana as a model, we will exploit a set of fluorescent protein-based biosensors and high-resolution imaging to generate an atlas of H+, K+ and Ca2+ in the subcompartments of the chloroplasts and the mitochondria in folio. The atlas will not only comprise three-dimensional molecular information in living leaf tissues, but also responses to different illumination regimes. It will provide a hitherto unique reference and will be used to further corroborate the significance of candidate proteins for H+, K+ and Ca2+ transport across the organelle membranes in determining PMF characteristics in folio. A selection of genetic mutants of candidate proteins of the mitochondria and the chloroplasts to set organelle H+, K+ and Ca2+ dynamics will be tested for their impact on organellar PMF characteristics in folio. The role of several of those players, such as KEA3 and NHD1, will be assessed in close collaboration within this consortium, while that of others such as the MCUs, MICU, UCP1 and AOX1a will be assessed based on the recent advances by our lab. The atlas approach and the mutants combined will allow to address the central question to what extent changes in the PMF characteristics in one organelle affect the other. The significance of altered PMF dynamics for photosynthetic metabolism, organelle ultrastructure, and whole plant performance will become accessible through collaboration with leading experts within the consortium. An advanced understanding of PMF regulation in vivo and the relative significance of specific candidate protein players in setting PMF characteristics will be established as a result.