High-Light versus Low-Light: Effects on Paired Photosystem II Supercomplex Structural Rearrangement in Pea Plants

Abstract

In plant grana thylakoid membranes Photosystem II (PSII) associates with a variable number of antenna proteins (LHCII) to form different types of supercomplexes (PSII-LHCII), whose organization is dynamically adjusted in response to light cues, with the C₂S₂ more abundant in high-light and the C₂S₂M₂ in low-light. Paired PSII-LHCII supercomplexes interacting at their stromal surface from adjacent thylakoid membranes were previously suggested to mediate grana stacking. Here, we present the cryo-electron microscopy maps of paired C₂S₂ and C₂S₂M₂ supercomplexes isolated from pea plants grown in high-light and low-light, respectively. These maps show a different rotational offset between the two supercomplexes in the pair, responsible for modifying their reciprocal interaction and energetic connectivity. This evidence reveals a different way by which paired PSII-LHCII supercomplexes can mediate grana stacking at diverse irradiances. Electrostatic stromal interactions between LHCII trimers almost completely overlapping in the paired C₂S₂ can be the main determinant by which PSII-LHCII supercomplexes mediate grana stacking in plants grown in high-light, whereas the mutual interaction of stromal N-terminal loops of two facing Lhcb4 subunits in the paired C₂S₂M₂ can fulfil this task in plants grown in low-light. The high-light induced accumulation of the Lhcb4.3 protein in PSII-LHCII supercomplexes has been previously reported. Our cryo-electron microscopy map at 3.8 Å resolution of the C₂S₂ supercomplex isolated from plants grown in high-light suggests the presence of the Lhcb4.3 protein revealing peculiar structural features of this high-light-specific antenna important for photoprotection.

Keywords: cryo-electron microscopy; light acclimation; photosystem II supercomplex; plant thylakoid membranes.

Figure 1

Comparison of the main representative paired PSII-LHCII supercomplexes isolated from plants grown in high-light and low-light. (A) Cryo-EM density map of the (C₂S₂)₂-H at 6.5 Å (light violet) with embedded the cryo-EM density map of the C₂S₂-H at 3.8 Å (yellow). (B) Overlay of the cryo-EM density maps of the (C₂S₂)₂-H at 8.4 Å (orange), (C₂S₂M)₂-H at 11 Å (green mesh) and (C₂S₂M₂)₂-L at 13.1 Å (transparent light blue). In both panels, the side view (on the left) is along the membrane plane; the top view (on the right) shows the PSII from the lumenal side, normal to the membrane plane.

Figure 2

Cryo-electron tomography of isolated stacked thylakoid membranes. (A) Average tomographic slice of a stack of 10 thylakoid membranes in side view. The inset shows a tangential averaged tomographic view of a thylakoid membrane. (B) Higher magnification of the boxed region in A in a slightly different tomographic section. (C) Higher magnification of the boxed region in B showing a paired PSII-LHCII supercomplex in two adjacent thylakoid membranes. (D) Average tomographic slice of a stack of 3 thylakoid membranes in side view. (E) Higher magnification of the boxed region in (D,F,G) are higher magnifications of the boxed regions in E showing paired PSII-LHCII supercomplexes in two adjacent thylakoid membranes. (H) Average tomographic slice of a stack of 7 thylakoid membranes in side view. The inset shows a tangential averaged tomographic view of a thylakoid membrane. (I) Distributions of stromal gap (red) and center-to-center distances (black) between adjacent thylakoid membranes across the stromal gap. In the figure, white and black arrowheads point respectively to the ATP-synthase complexes protruding from the grana end membranes and to the extrinsic subunits of PSII protruding into the lumen; white arrows point to PSII-LHCII supercomplexes.

Figure 3

Peculiar features of proteins and chlorophylls of the C₂S₂ supercomplex isolated from plants grown in high-light. (A) Cryo-EM density map of the C₂S₂-H at 3.8 Å viewed from the lumenal side, normal to the membrane plane, and assigned subunits of the PSII dimer in cyan, LHCII-S trimer in green, Lhcb4 in red and Lhcb5 in orange. (B) Structural superposition of the C-terminal region of Lhcb4 (red) and Lhcb4 from PDB: 5XNL (chain R, purple) [22]. (C) Overview of all chlorophylls a (green) and b (blue) in the supercomplex shown from the lumenal side. (D) Representation of Lhcb4 and Lhcb5 with chlorophyll a (green) and chlorophyll b (blue) molecules. In panel (D), chlorophylls present in the pea C₂S₂ supercomplex from PDB: 5XNL (chain R for Lhcb4 and chain S for Lhcb5) [22] and missing in the C₂S₂-H are highlighted in red, the top view (on the left) and side view (on the right) of proteins are along the membrane plane.

Figure 4

Cryo-EM density maps of paired PSII-LHCII supercomplexes with different antenna size isolated from plants grown in high-light and low-light. Top view towards the lumenal surface of the 3D maps of the supercomplexes (C₂S₂)₂-H at 6.5 Å (A), (C₂S₂M)₂-H at 11 Å (B), (C₂S₂M₂)₂-L at 13.1 Å (C), fitted with high-resolution structures (PDB: 5XNL devoid of one LHCII-M trimer and one Lhcb6 in panel (B); PDB: 5XNL in panel (C)) [22], and corresponding schematic representations (D–F) showing the positions of all fitted supercomplex components and rotational offsets measured between the plane perpendicular to the axis of symmetry of the PSII dimeric core of the lower supercomplex with respect to its counterpart of the upper supercomplex. Subunits coloured as follows: PSII dimer in cyan, LHCII-S trimer in green, Lhcb4 in red, Lhcb5 in orange, LHCII M-trimer and Lhcb6 in pink (dark colours for upper supercomplex, light colours for lower supercomplex). In the scheme, colours match the structures of panels (A–C); solid lines for upper supercomplex, dashed lines for lower supercomplex.

Figure 5

Distance distribution of stromal chlorophylls pairs and PSII excitonic connectivity in paired PSII-LHCII supercomplexes with different antenna size. (A) Distribution of Mg-Mg distances calculated for Chls pairs made by a stromal Chl of the upper supercomplex and a stromal Chl of the lower supercomplex within a cut-off of 90 Å and (B) corresponding box-plot highlighting the contribution of the Chls pairs, belonging exclusively to PSII cores (left) or LHCII antennae (right), in paired PSII-LHCII supercomplexes of different antenna size. (C) Chlorophyll a fluorescence induction curves (i.e., the OJIP transient) plotted on a logarithmic time scale for paired PSII-LHCII supercomplexes isolated from H and L samples. (D) Estimated PSII energetic connectivity as plot of (W_E—W), where W and W_E (shown in Figure S5A) correspond respectively to the normalized O–J phase of the experimental OJIP curve and of the theoretical exponential curve corresponding to the unconnected system (for calculation of W and W_E, see Figure S5D). Graphs in panels C-D display the mean values ± standard deviations of six replicates.