Photosynthetic Dynamics: in situ AFM Analyses

In nature, photosynthetic efficiency is often limited because of the inability of plants to maintain high photosynthetic rates at high intensity irradiation. In addition, light incident on photosynthetic organisms fluctuates rapidly between high and low intensity due to dynamic shading and cloud cover. Furthermore, much of the light energy absorbed during mid-day can be in excess of what can be used for CO2 fixation, and ultimately cause cellular damage, especially to the photosynthetic apparatus. To protect the photosynthetic apparatus from photodamage, plants have evolved a set of dynamic processes, including nonphotochemical quenching, which dissipates much of the excess absorbed light energy as heat. While there is clearly biochemical/biophysical reorganization of the photosynthetic machinery within thylakoid membranes, our view of the the movement/re-organization of complexes within the membranes and their dynamic interactions would provide us with a new view of the networks that govern photosynthetic yields

It is difficult to study these dynamic changes in the organization of protein complexes within membranes using fluorescence-tagged live-cell imaging because the optical resolution is spatially limited and there is often an extensive fluorescence background generated by the chromophores that are required for photosynthetic function (e.g. chlorophyll). To overcome both of these issues, we are using Atomic Force Microscopy in liquid medium. At this point, we have resolved and characterized dimeric PS II complexes, each monomer having two lumenal protruding masses (of different heights); these protruding masses represent the PS II Oxygen-Evolving Complex (PSII-OEC) monomer (Figure 1). We have also been able to localize the cytochrome b6f complex within the granal membranes. Some of this work was recently published [1]. We are now in the process of using AFM to observe the dynamics of these complexes, under different light conditions and redox states, in real time.


Figure 1. High resolution CM-AFM images of PSII-OEC dimers in liquid medium. Top, left. High resolution topography image. Brightness of the white areas of the image corresponds to the height, allowing for qualitatively distinction of the small and large protruding sub-particles within PS II-OEC. .Top, right. Digital enlargement of a PSII-OEC particle. Bottom, left. Height profile of PSII-OEC particle across two monomers. Bottom, right. Height profile of PSII-OEC particle along a single monomer. Note that the transects in the Top, right image are chosen such that they pass the maximum height of the visible protrusions and that the zero level in the bottom panels is defined as the minimum height associated with the individual profiles. 

[1]  Phuthong, W., Huang, Z., Wittkopp, T.M., Sznee, K., Heinnickel, W.L., Dekker, J.P., Frese, R.N., Prinz, F.B., Grossman, A.R. (2015) The use of contact mode atomic force microscopy in an aqueous medium for structural analyses of photosynthetic complexes in spinach grana membranes. Plant Physiol. 169: 1318-32.