Investigating the structure and function of the plant photosynthetic membrane using atomic force microscopy and Monte Carlo simulations

The relative spatial organisation of protein complexes involved in the light reactions of photosynthesis in plants, (photosystem II (PSII), cytochrome b6f (cytb6f), photosystem I (PSI), and ATP synthase) remains unknown, but is crucial as it determines the diffusive path of mobile electron carriers plastoquinone (PQ) and plastocyanin (PC). Protocols were developed for purifying grana and stromal lamellae membranes for atomic force microscopy (AFM) in model plants spinach (Spinacia oleracea) and Arabidopsis thaliana. The development of methods to purify stromal lamellae from spinach for AFM allowed for the nanoscale imaging of PSI and ATP synthase for the first time. The 3.2 nm, stroma-protruding subunits of PSI were clearly visible in the AFM images, facilitating its identification. Contrary to the consensus that PSI in plants is monomeric, 25% of PSI were found to exist as dimers, which may facilitate diffusion of protein complexes within the stromal lamellae. ATP synthase was present and intact within the purified stromal lamellae membranes and showed the same height and width as purified Bos taurus ATP synthase incorporated into a DOPC lipid bilayer. The implications of the observed organisation of PSII, cytb6f, and PSI for the rate of electron transport were investigated using Monte Carlo simulations. AFM data revealed that long-range (>100 nm) diffusion distances are required of the thylakoid luminal protein plastocyanin (PC). The simulations revealed that the diffusion of PC is significantly restricted due the crowded nature of the thylakoid lumen and was affected by the size of the grana/stromal lamellae interface. It was proposed that the size of the interface is, therefore, the result of a compromise between a large interface, which maximises the rate of PC transport, and a small interface which minimises the excitation energy spillover from PSII to PSI.