DOI: 10.5281/zenodo.15188812
title: Dataset for publication: "Photosystem II supercomplexes lacking light-harvesting antenna protein LHCB5 and their organization in the thylakoid membrane"

description: Data repository for "Photosystem II supercomplexes lacking light-harvesting antenna protein LHCB5 and their organization in the thylakoid membrane".

FIGURE 1 Phenotype and photosynthetic characteristics of the lhcb5 mutant. (A) Phenotype of Arabidopsis thaliana wild type (WT) and lhcb5 mutant plants grown at controlled conditions for 6 weeks (8 h light/16 h dark cycle; 22/20°C; 110 µmol photons m-2 s-1; 60% humidity). (B) Immunoblot analysis of thylakoid membranes of WT and lhcb5 mutant plants with antibody directed against LHCB5. (C) Content of light-harvesting proteins LHCB1-6 evaluated relatively to the content of CP43 protein in the WT and the lhcb5 mutant. The protein content was determined in isolated thylakoid membranes by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The columns represent means ± SD, individual points show technical replicates. All data passed the normality and equal variance tests and according to Student t-test the datasets of WT and lhcb5 were not significantly different (α ≤ 0,05), except for the relative content of LHCB5/CP43. (D) Protein ratios of photosynthesis-related thylakoid membrane proteins of the WT and the lhcb5 mutant. The protein content was determined by LC-MS/MS in isolated thylakoid membranes. PSII represents the sum of relative PG intensities of D1, D2, CP43, and CP47 proteins, LHCII - LHCB1–3 proteins, PSI - PSAA and PSAB proteins, LHCI - LHCA1–4 proteins, ATPS - α and β subunits of ATP synthase, and cyt f represents cytochrome f component of cytochrome b6f complex. The columns represent means ± SD, individual points show technical replicates. All data passed the normality and equal variance tests and according to Student t-test the datasets of WT and lhcb5 are not significantly different (α ≤ 0,05).

FIGURE 2 Separation and structural characterization of PSII supercomplexes from lhcb5 mutant plants. (A) Separation of pigment–protein complexes from thylakoid membranes from Arabidopsis thaliana WT and lhcb5 mutant plants by clear native polyacrylamide gel electrophoresis. Thylakoid membranes were solubilized by n-dodecyl α-D-maltopyranoside (detergent/chlorophyll mass ratio of 10). (B) Electron density maps of characteristic PSII supercomplexes from the separated green gel bands of the lhcb5 mutant designated as C2S2M2, C2S2M and C2SM. Projection maps are fitted by corresponding structural high-resolution models of PSII supercomplexes (Van Bezouwen et al., 2017) without LHCB5. Individual PSII subunits are color-coded according to (E). (C), (D) Comparison of structural models of the PSII C2S2M2 supercomplexes from Arabidopsis thaliana WT and the lhcb5 mutant. (C) Projection map of the PSII C2S2M2 supercomplex from Arabidopsis thaliana wild type (Ilíková et al., 2021) fitted by the high-resolution structure from Van Bezouwen et al. (2017). (D) Overlay of structural models of the PSII C2S2M2 supercomplex from Arabidopsis thaliana wild type (surface representation, partially transparent) and the lhcb5 mutant shows a specific shift of the S and M LHCII trimers as well as the monomeric antenna LHCB6 (see arrows in the corresponding colors) due to the absence of LHCB5. Individual PSII subunits are color-coded according to (E). (E) Legend of individual PSII subunits, which are color-coded as follows: PSII core complex in green, S and M LHCII trimers in red and blue, respectively, and the monomeric antenna proteins, LHCB4, LHCB5, LHCB6, in yellow, cyan, and dark orange, respectively.

FIGURE 3 Organization of photosystem II in thylakoid membranes of the lhcb5 mutant. (A, B) Examples of electron micrographs of negatively stained thylakoid membrane isolated from the lhcb5 mutant with densities corresponding to the PSII core complex. Representative picture of PSII supercomplexes “randomly” organized (A) and organized into 2D semi-crystalline array (B). (C, D, E) Projection maps of PSII megacomplexes obtained using image analysis of PSII particles in thylakoid membranes. Three specific associations of PSII supercomplexes are shown and fitted by the model of PSII supercomplex C2S2M2 without LHCB5 (see Figure 2B). Megacomplexes are averaged projections of 1 925 (C), 2 241 (D), and 2 305 (E) particles. (F) Isolated PSII particle from thylakoid membranes with “randomly” organized PSII as an average projection of 3 741 particles fitted by the model of PSII supercomplex C2S2M2 without LHCB5 (see Figure 2B). (G) PSII supercomplexes organized into 2D semi-crystalline array as an average projection of 418 sub-areas together with the fitted model of PSII C2S2M2 supercomplexes (see Figure 2B). Projection maps of PSII supercomplexes show core complexes in green, S trimers in red, M trimers in blue, LHCB4 in yellow, and LHCB6 in dark orange color.

FIGURE 4 Distribution of mutual distances between neighboring photosystem II particles in thylakoid membranes of Arabidopsis thaliana WT and the lhcb5 mutant. The distances between two closest neighboring PSII supercomplexes were analyzed using EM. Histograms are normalized to the maximum.

SUPPORTING FIGURE 1 Analysis of chosen photosystem I and II photosynthesis related parameters. (A) Quantum yield of photochemistry of PSI - Y(I). (B) Quantum yield of photochemistry of PSII - Y(II). (C) Non-photochemical quenching – NPQ. Parameters were measured during actinic light exposure (800 µmol photons m-2 s-1) and dark relaxation using saturating light pulses (300 ms, 10 000 µmol photons m-2 s-1) in WT and lhcb5 mutant plants. Results represent mean values ± SD from 4 measurements. Plants were dark-adapted for 30 min before the measurement.

SUPPORTING FIGURE 2 Single-particle image analysis and classification of protein complexes from CN−PAGE C2S2M2 band from the Arabidopsis lhcb5 mutant (Figure 2A). Number of averaged projections in given classes are indicated.

SUPPORTING FIGURE 3 Single-particle image analysis and classification of protein complexes from CN−PAGE C2S2M band from the Arabidopsis lhcb5 mutant (Figure 2A). Number of averaged projections in given classes are indicated.

SUPPORTING FIGURE 4 Single-particle image analysis and classification of protein complexes from CN−PAGE C2SM band from the Arabidopsis lhcb5 mutant (Figure 2A). Number of averaged projections in given classes are indicated.

SUPPORTING FIGURE 5 A histogram of the relative abundance of PSII semi-crystalline arrays in thylakoid membranes of Arabidopsis lhcb5 mutant. The bars represent the number of electron micrographs where the 2D arrays cover the indicated percentage of the membrane. The histogram was obtained by evaluation of 50 randomly selected images.

SUPPORTING TABLE 1 Physiological parameters of Arabidopsis WT and lhcb5 mutant plants.

SUPPORTING TABLE 2 Density of bands corresponding to LHCB5-less PSII supercomplexes evaluated relatively to WT.

Figure 1 C-D - source data for Figure 1. (panels C-D) Documentation of similar physiology of Arabidopsis thaliana wild type (WT), and its mutant with loss of LHCB5 protein subunit (lhcb5): (C) relative content of photosysthesis related proteins in thylakoid membranes of Arabidopsis thaliana lhcb5 genotype normalised to WT determined by LC-MS/MS; (D) relative protein ratios normalised to WT of photosynthesis related thylakoid membrane proteins of Arabidopsis thaliana lhcb5 genotype determined in isolated thylakoid membranes by LC-MS/MS.

Figure 4 - source data for Figure 4. Relative distribution of photosystem II (PSII) distances in thylakoid grana membranes of Arabidopsis thaliana wild type (WT) and mutant with missing LHCB5 protein (lhcb5).

Supporting figure 1 Source data for supporting figure 1 Photosynthesis related parametres describing PSI and PSII function. (A) quantum yield of photochemistry of PSI (Y(I)) in Arabidopsis thaliana WT and lhcb5 genotype leaves during red acitinic light exposure and dark relaxation; (B) quantum yield of photochemistry of PSII (Y(II)) in Arabidopsis thaliana WT and lhcb5 genotype leaves during red acitinic light exposure and dark relaxation; (C) non-photochemical quenching of Arabidopsis thaliana genotypes: The level of NPQ estimated during red acitinic light exposure and dark relaxation of WT and lhcb5 leaves.

Supporting figure 5 Source data for Supplement figure 4. Relative abundance of 2D PSII arrays in the thylakoid membranes of Arabidopsis thaliana lhcb5 mutant from 50 randomly selected images.

Supporting table 1 - source data Source data for supporting table 1. Physiological parameters of witl type (WT) Arabidopsis thaliana and its mutant lacking LHCB5 protein (lhcb5): Repetitions of data measured for each genotypes.

Supporting table 2 - source data Source data for supporting table 2. Density of bands corresponding to LHCB5-less PSII supercomplexes evaluated relatively to WT: Repetitions of data measured for each genotypes.

Figure 1B source WB Source WB picture for FIGURE 1B.

Figure 1B source WB, marker Source WB picture with molecular marker for FIGURE 1B.