Conspicuous chloroplast with light harvesting-photosystem I/II megacomplex in marine Prorocentrum cordatum

Abstract

Marine photosynthetic (micro)organisms drive multiple biogeochemical cycles and display a large diversity. Among them, the bloom-forming, free-living dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) stands out with its distinct cell biological features. Here, we obtained insights into the structural properties of the chloroplast and the photosynthetic machinery of P. cordatum using microscopic and proteogenomic approaches. High-resolution FIB/SEM analysis revealed a single large chloroplast (∼40% of total cell volume) with a continuous barrel-like structure, completely lining the inner face of the cell envelope and enclosing a single reticular mitochondrium, the Golgi apparatus, as well as diverse storage inclusions. Enriched thylakoid membrane fractions of P. cordatum were comparatively analyzed with those of the well-studied model-species Arabidopsis (Arabidopsis thaliana) using 2D BN DIGE. Strikingly, P. cordatum possessed a large photosystem-light harvesting megacomplex (>1.5 MDa), which is dominated by photosystems I and II (PSI, PSII), chloroplast complex I, and chlorophyll a-b binding light harvesting complex proteins. This finding parallels the absence of grana in its chloroplast and distinguishes from the predominant separation of PSI and PSII complexes in A. thaliana, indicating a different mode of flux balancing. Except for the core elements of the ATP synthase and the cytb6f-complex, the composition of the other complexes (PSI, PSII, and pigment-binding proteins, PBPs) of P. cordatum differed markedly from those of A. thaliana. Furthermore, a high number of PBPs was detected, accounting for a large share of the total proteomic data (∼65%) and potentially providing P. cordatum with flexible adaptation to changing light regimes.

Figure 1.

3D reconstruction of subcellular structures of P. cordatum based on 605 FIB/SEM micrographs. The single, expansive chloroplast has a large, continuous, barrel-like structure and is pictured from (A) front, (B) back, and (C) bottom. The structure of thylakoid-membranes of the chloroplast are highlighted for (D) loose vs. (E) tightly packed areas. F) Presumptive starch storage vesicles (white) reside exclusively within the chloroplast. G) Reconstruction of the single, reticular mitochondrium (red), the nucleus (blue), the Golgi apparatus (yellow), phosphate storage bodies (black), and lipid droplets (brown). Subcellular structures enclosed by the chloroplast as viewed from (H) front and (I) top. *, for spatial matching between parts (A), (C), and (H).

Figure 2.

Analysis of thylakoid membrane-protein complexes from P. cordatum by 2-dimensional blue native difference gel electrophoresis (2D BN DIGE) and 2D BN/SDS-PAGE. A) Co-separation and comparison of fluorescently labeled (CyDyes) of P. cordatum (green, Cy2) and A. thaliana (red, Cy3) applying 2D BN DIGE. The molecular masses (kDa) were estimated according to the sizes of standard protein complexes from A. thaliana given in Schröder et al. (2022). B) Separation of unlabeled samples of P. cordatum by 2D BN/SDS-PAGE poststained with Coomassie, displaying first and second dimension gels. C) Distribution profiles of thylakoid membrane protein complexes and PBPs of P. cordatum. The profiles are displayed as relative shares (%) across the entire gel based on the analysis of 24 vertical segments (indicated by the blue lines). Detailed protein data are provided at the GelMap portal online (https://www.gelmap.de/2657). *100%. Abbreviations: PBP, pigment binding protein; PSII, photosystem II; Cytb₆f, cytochromeb₆f-complex; PSI, photosystem I; CCI, chloroplast complex I (formerly NDH-1 complex); FNR, ferredoxin:NADP⁺ oxidoreductase; ATP syn, cF₁/F₀ ATP synthase; FCP, fucoxanthin-chla–c binding protein, FCP; CCBP, caroteno-chla–c binding protein; LhcP, chla–b binding light harvesting complex protein; GPR, green-light absorbing proteorhodopsin. Further details on assigned proteins are provided in Supplementary Fig. S5.

Figure 3.

Illustration of the photosynthetic apparatus of A. thaliana superimposed by the current findings with P. cordatum. A) Sequence similarities between photosynthetic proteins of A. thaliana and P. cordatum; blue coloring reflects ranking results from sequence analyses. B) Identified proteins of P. cordatum matching the photosynthetic proteins of A. thaliana; brown coloring reflects protein abundance (based on the sum of detected compounds), light green coloring indicates the ambiguous assignment of Lhcs. Abbreviations are as defined in legend to Fig. 2.

Figure 4.

Abundance profiles of PBPs of P. cordatum based on blue native separation of protein complexes. A) 1D BN PAGE gel annotated with identified (mega/super) complexes. B) Protein abundance profiles as determined via two different proteomic approaches. First, separation via 2D BN/SDS PAGE coupled to 3D iontrap MS (left faces of box double rows); here abundances are indicated as sum of detected compounds from excised protein spots (Supplementary Figs. S7 and S8). Second, separation via 1D BN PAGE coupled to orbitrap MS (right faces of box double rows); here abundances are indicated as IBAQ values. Note that the top-down order of PBPs reflects their clustering (left part) as also used in subsequent Fig. 5 on PBPs classification. Abbreviations are as defined in legend to Fig. 2.

Figure 5.

Classification of PBPs of P. cordatum identified based on 2D BN/SDS-PAGE. The four categories of PBPs are displayed in orange (CCBP), light green (FCP), purple (LhcP), and dark green (GPR). A) Number of PBPs identified (in total 83) vs. predicted only (in total 57) according to the four categories. B) Relative share of identified PBPs across the four categories. C) Clustering of all identified PBPs based on multiple sequence alignments and factoring in the individual category affiliation.

Figure 6.

Genetic variability and domain structure of PBPs of P. cordatum. A) Gene models of four different PBPs encoded on scaffold 158 of the genome of P. cordatum. B) Structure and arrangement of chlorophyll-binding domains in the four selected PBPs. C) Clustering of the 12 chlorophyll-binding domains possessed by PBP82. Domains are sorted in phylogenetic order based on their sequence identity. D) Number of chlorophyll-binding domains of the predicted 140 PBPs. Footnotes: ^a, g5095 encodes PBP88; ^b, g5096 encodes PBP56; ^c, g5092 encodes PBP83; and ^d, g50194 encodes PBP82. Abbreviations are as defined in legend to Fig. 2.

Figure 7.

Schematic representation of the photosynthetic apparatuses of P. cordatum and A. thaliana. A) Homogenous thylakoid membranes in P. cordatum. B) Grana stacks vs. stroma-like thylakoid membranes in A. thaliana. C) PSI/II–LHC–CCI megacomplex in P. cordatum. D) Spatial separation of PSI and PSII complexes in A. thaliana. E) Hypothesized mode of electron transfer between PSI and PSII in P. cordatum. F) Transfer of mobile LHCs between PSI and PSII mediated by (de)phosphorylation in A. thaliana.