Bioenergetic mechanisms in Physcomitrium patens: analysis of alternative respiratory pathways and in vivo ATP dynamics

Respiration is a metabolic process that utilizes a mitochondrial electron transport chain (mETC) and consumes O2, where complexes I to IV establish a proton gradient across the inner mitochondrial membrane to drive ATP synthesis. In addition to this phosphorylation pathway, plants possess alternative respiratory mechanisms, whose function is to prevent the over-reduction of mETC and the consequent formation of the harmful reactive oxygen species (ROS). One pathway involves the alternative oxidase (AOX), which transfers electrons from ubiquinone to oxygen, bypassing the proton-pumping sites complex III and IV, decreasing ATP production. Another mechanism is mediated by uncoupling proteins (UCPs) that translocate protons from the intermembrane space to the matrix, bypassing the ATP synthase, uncoupling respiration from ATP production, releasing energy as heat. In plant cells, also chloroplasts are important bioenergetic organelles, serving as the primary site of photosynthesis. Chloroplast electron transport chain (cETR) occurs at the level of the thylakoid membranes and generates a light-dependent proton gradient that drives stromal ATP synthesis. As cellular energy currency, ATP fuels all life processes, and its production by both mitochondria and chloroplasts is dynamically modulated in response to variable environmental conditions. This study used the moss Physcomitrium patens, a model organism ideal for functional analysis due to its strategic evolutionary position and efficient homologous recombination, to study ATP dynamics in vivo. Moreover, its single-layer photosynthetic tissue also allows the clear study of ATP kinetics within the chloroplast. The first part of the thesis was aimed to characterize the UCP protein family, whose role in bryophytes remains largely unknown. Through a bioinformatic search based on sequence homology with known UCPs, in particular Arabidopsis thaliana, and the analysis of conserved protein domains, six putative UCPs were identified in P. patens genome. Phylogenetic analysis revealed that these proteins are distributed across three major groups and suggested an ancient origin and early functional diversification of UCPs, predating the emergence of vascular plants. This investigation provides a fundamental framework for future functional studies on mitochondrial physiology and energy regulation. The second goal was to investigate in vivo ATP dynamics using lines containing the fluorescent protein biosensor ATeam 1.03-nD/nA. First, a time-based MgATP2- specific Fluorescence Resonance Energy Transfer (FRET) protocol was optimized using confocal microscopy to examine the light-dark cycle response. In wild-type (WT) lines, the ATeam 1.03-nD/nA was either expressed in plastids or in the cytosol to compare ATP dynamics under dark-to-light and light-to-dark transitions in different cell compartments. The ATP concentration in plastid stroma is lower than in the cytosol and responds faster to light-to-dark transitions. To investigate the contribution of mitochondrial electron transport to ATP dynamics, inhibitors of respiratory complex III (Antimycin A) and AOX (SHAM) were used. Furthermore, to understand how AOX impacts the ATP dynamics in different cell compartments, aox knockout lines expressing the ATeam 1.03-nD/nA in the cytosol and the plastid stroma were generated.