The effects of proton motive force on thylakoid architecture and function

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Thylakoid membranes scaffold photosynthetic complexes, which couple electron transfer to proton movement across the membrane, thereby generating the proton motive force (PMF, composed of Delta-pH and Delta-Psi) that drives the production of ATP. Thylakoid architecture is intimately linked to efficient photosynthesis. Thylakoids are flat instead of spherical, increasing the concentration of photosynthetic complexes per luminal area to drive the PMF, while providing directionality for the diffusion of the soluble electron carrier plastocyanin through the thylakoid lumen. However, thylakoid architecture is not static and responds to changes in light quality and quantity. Preliminary electron microscopy observations in cyanobacteria, green algae, and plants have shown that the thylakoid lumen swells under increased light intensity. A logical hypothesis would be that lumen acidification (Delta-pH) and ion flux-driven changes in membrane potential (Delta-Psi) may be coupled to osmotic forces that expand the lumen. However, the precise dynamics and mechanism of this architectural change remain uncharacterized. The goal of this project is to understand the mechanistic basis and physiological consequences of light-induced thylakoid swelling, and to specifically test how this swelling is modulated by different components of the PMF. This study will be enabled by our advanced cryo-electron tomography (cryo-ET) workflow, which we have developed to measure native thylakoid architecture (membrane and lumen width) with sub-nanometer precision and analyze the organization of each photosynthetic complex along these membranes. First, we aim (i) to characterize the kinetics of thylakoid swelling in green algae (Chlamydomonas reinhardtii) and cyanobacteria (Synechocystis sp. 6803) by performing a detailed cryo-ET time-series of wild-type cells after transition from darkness to different light intensities. Next, we aim (ii) to examine the contributions of Delta-pH and Delta-Psi to thylakoid lumen expansion by combining cryo-ET with small molecule ionophores and genetic perturbations of ATP synthase and KEA3 ion antiporters. In parallel, we aim (iii) to test the specific PMF contributions of cytochrome b6f to thylakoid lumen dynamics. Finally, we aim (iv) to create an integrated model of light-dependent lumen expansion by combining our cryo-ET data with functional measurements of PMF and photosynthetic function from other members of the consortium. The focus of our project is to deliver on the overall aim 3 of GoPMF (Structural dynamics of thylakoid membranes in remodeling of energy conversion systems). Our work will also contribute to the overall aims 1, 2, and 4.