The Significance of Organellar Membrane Contact Sites in Photosynthetic Metabolism of Plant Cells

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Plant cell metabolism can switch between autotrophic and heterotrophic growth modes, depending on environmental conditions. In the light, metabolic cooperativity between several organelles is necessary for plant survival due to the process of photorespiration. Here, noxious glycolate produced by the oxygenase activity of Ribulose-1,5-bisphosphat carboxylase/oxygenase (RuBisCO) is recycled via eight enzymes partitioned between peroxisomes, mitochondria and chloroplasts.The established knowledge on the biochemical level of photorespiration faces a lack of knowledge regarding the function of spatial organelle organisation and the formation of specific membrane contacts sites (MCS). Whereas close associations between chloroplasts, mitochondria and peroxisomes have been monitored microscopically since decades, their molecular structure and their relevance for plant metabolism and performance remains unknown.Here we investigate the hypothesis that MCSs in plants underpin metabolic remodelling and provide the basis for reorganising the cell to respond effectively to changing environmental conditions. To address the question of specific and dynamic MCS formations between the photorespiratory organelles, we establish a set of genetically-encoded in vivo proximity biosensors, based on Bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer/ fluorescence lifetime imaging (FRET/FLIM), respectively. In addition, to address the molecular identity and function of MCS-resident proteins, we created a set of candidate proteins that will be expanded by labelling MCS-resident proteins using a promiscuous biotin ligase construct (BioID technique). By designing synthetic tethering constructs for organelle pairs, as well as by investigating mutant plant lines of candidate MCS-resident proteins, we will perturb MCS formation. In order to quantify the biological relevance of specific MCSs, we use plant growth phenotypes, photorespiratory capacity and metabolites, as well as photosynthetic parameters as a readout.Our results will solve the long-standing question of formation and physiological relevance of MCSs regarding photorespiration and plant performance during light/dark transitions. We will define were organelle proximity is based on specific association rather than spatial constraints. We anticipate our work to create links between cell biology, pathway partitioning between organelles, and plant metabolic plasticity. Furthermore, as photorespiration is a major target to understand and optimize photosynthetic plant performance, we expect our work to be relevant for future biotechnological approaches.