TY - JOUR
AU1 - Kato, Hiroki
AU2 - Tokutsu, Ryutaro
AU3 - Kubota-Kawai, Hisako
AU4 - Burton-Smith, Raymond N.
AU5 - Kim, Eunchul
AU6 - Minagawa, Jun
AB - Abstract Symbiodiniaceae are symbiotic dinoflagellates that provide photosynthetic products to corals. Because corals are distributed across a wide range of depths in the ocean, Symbiodiniaceae species must adapt to various light environments to optimize their photosynthetic performance. However, as few biochemical studies of Symbiodiniaceae photosystems have been reported, the molecular mechanisms of photoadaptation in this algal family remain poorly understood. Here, to investigate the photosynthetic machineries in Symbiodiniaceae, we purified and characterized the PSI supercomplex from the genome-sequenced Breviolum minutum (formerly Symbiodinium minutum). Mass spectrometry analysis revealed 25 light-harvesting complexes (LHCs), including both LHCF and LHCR families, from the purified PSI-LHC supercomplex. Single-particle electron microscopy showed unique giant supercomplex structures of PSI that were associated with the LHCs. Moreover, the PSI-LHC supercomplex contained a significant amount of the xanthophyll cycle pigment diadinoxanthin. Upon high light treatment, B. minutum cells showed increased nonphotochemical quenching, which was correlated with the conversion of diadinoxanthin to diatoxanthin, occurring preferentially in the PSI-LHC supercomplex. The possible role of PSI-LHC in photoprotection in Symbiodiniaceae is discussed. Living corals are associated with symbiotic microalgae. These microalgae are important for oceanic ecosystems because they function as the main primary producer on coral reefs. Members of Symbiodiniaceae, a dinoflagellate family formerly the genus Symbiodinium, provide photosynthetic products to host corals. Thus, optimum photosynthesis in Symbiodiniaceae is critical to sustain ocean ecosystems. However, the symbiotic coral habitat is distributed between the ocean surface to a depth of 150 m, where light intensity largely changes with depth (2,478 μmol photons m−2 s−1 at the ocean surface to 11 μmol photons m−2 s−1 at the depth of 95 m; Lesser et al., 2010). These conditions force symbiotic Symbiodiniaceae under these light environments to efficiently provide photosynthetic products to corals by keeping optimal photosynthetic activity. In the shallow ocean, which is the habitat for many reef-building corals, high-light (HL) stress potentially causes photoinhibition of the symbiotic Symbiodiniaceae. Because photoinhibition results in the collapse of the symbiotic relationship between corals and Symbiodiniaceae, the alga has to protect the photosynthetic apparatus from excess light. To alleviate photoinhibition, Symbiodiniaceae employ a photoprotection mechanism called nonphotochemical quenching (NPQ; Gorbunov et al., 1998), which seems to be similar to the photoprotection mechanism in most other photosynthetic organisms. Symbiodiniaceae possess diadinoxanthin (Ddx) and diatoxanthin (Dtx) as potential NPQ-related xanthophyll pigments (Venn et al., 2006), and it has been suggested that the conversion of Ddx to Dtx under HL conditions contributes to the activation of NPQ in Symbiodiniaceae (Olaizola et al., 1994; Brown et al., 1999). Furthermore, under high-temperature stress conditions, which are believed to trigger coral bleaching (Brown, 1997), Symbiodiniaceae transfer excitation energy directly from PSII to PSI (Slavov et al., 2016). This mechanism is called “spillover,” and the excitation energy transferred to PSI appears to be dissipated as thermal energy at P700+ in Symbiodiniaceae (Slavov et al., 2016). It has been reported that the PSI complexes in both red algae and land plants are associated with the xanthophyll pigment zeaxanthin (Zea), but the physiological role of PSI-associated Zea remains unclear (Suga et al., 2016; Haniewicz et al., 2018). Therefore, photoprotection in Symbiodiniaceae remains poorly understood, mainly owing to the limited number of biochemical studies reported. The structure of PSI complexes is diverse among photosynthetic organisms. In the photosynthetic cyanobacteria Thermosynechococcus elongatus, the crystal structure of the PSI complex has been reported to be a trimer of PSI core complexes (Jordan et al., 2001), whereas the structures of the PSI core complexes associated with light-harvesting complexes (LHCs), PSI-LHC supercomplexes, have been reported in several eukaryotic photosynthetic organisms. Four light-harvesting complex Is (LHCIs; encoded by Lhca1– Lhca4 genes) and eight to 10 LHCIs (encoded by LHCA1– LHCA9 genes) have been reported to be associated with a monomeric PSI core complex in land plants (Mazor et al., 2017) and green algae, respectively (Drop et al., 2011; Kubota-Kawai et al., 2019; Su et al., 2019; Suga et al., 2019). In the moss Physcomitrella patens, large PSI supercomplex structures associated with 12 LHCs including a LHCII trimer were reported (Iwai et al., 2018; Pinnola et al., 2018). The red alga Cyanidioschyzon merolae possesses three Lhcr genes (Lhcr1–Lhcr3; Matsuzaki et al., 2004; Busch et al., 2010) and forms PSI-LHC supercomplexes with three to five LHCRs in response to the incident light (Pi et al., 2018; Antoshvili et al., 2019). Nannochloropsis gaditana, a secondary symbiotic alga in the same red photosynthetic lineage as Symbiodiniaceae, was reported to associate five to 11 LHCs with the PSI core complex (Alboresi et al., 2017; Bína et al., 2017). As described above, PSI-LHCI supercomplexes have been extensively investigated in many photosynthetic organisms, and the structures show great variation. Light-harvesting antennae associated with PSI are also diversified among photosynthetic organisms. The antennae are composed of membrane-intrinsic proteins called LHCIs in the green plants and LHCRs in red algae (Hoffman et al., 2011). Symbiodiniaceae has two types of light-harvesting antennae called peridinin (Per)-chlorophyll (Chl) a-binding proteins (Norris and Miller, 1994) and the Chl a-Chl c 2-Per protein complexes (Hiller et al., 1993), with the latter acting as the membrane-intrinsic antenna similar to LHCs in many eukaryotic photosynthetic organisms. LHCs (Chl a-Chl c 2-Per protein complexes) in Symbiodiniaceae are encoded by 145 genes, and those genes are phylogenetically classified into 10 groups (Maruyama et al., 2015). Five groups are recognized as a Lhcf-type family, which is exclusively conserved in algal species that harbor secondary plastids of red algal origin, and another five groups are classified as a Lhcr-type family, of which members are known to be associated with PSI in red algal plastids and secondary plastids of red algal origin (Shoguchi et al., 2013; Maruyama et al., 2015). Although there are relatively large numbers of LHC-coding genes in Symbiodiniaceae compared to other photosynthetic organisms, it has not been fully investigated how these genes contribute to light harvesting in symbiotic algae. Given that Symbiodiniaceae species have to acclimate to various light conditions in coral habitats, the qualitative and quantitative regulation of those LHCs may be indispensable for both algal survival and the maintenance of symbiotic relationships. In this study, to explore the molecular mechanisms of photoacclimation in symbiotic microalgae, we purified and characterized the PSI-LHC supercomplex from a genome-sequenced Symbiodiniaceae, Breviolum minutum (formerly, Symbiodinium minutum). A combination of Suc density gradient (SDG) ultracentrifugation and ferredoxin (Fd) affinity purification enabled us to successfully purify the PSI-LHC supercomplexes from B. minutum. Further analysis using a combination of mass spectroscopy and electron microscopy (EM) revealed that the PSI-LHC in B. minutum forms large supercomplexes composed of numerous LHCs encoded by both Lhcf and Lhcr. Interestingly, significant amounts of Ddx/Dtx pigments were detected in the PSI-LHC supercomplex, suggesting that the supercomplex may be involved in photoprotection in B. minutum. RESULTS AND DISCUSSION Purification of the PSI-LHCI Supercomplex The goal of this study was to characterize the PSI-LHC supercomplex in B. minutum. To achieve this goal, we first isolated the PSI-LHC supercomplexes from this alga. Initially, the thylakoid membranes were prepared from large-scale culture of B. minutum and solubilized with n-dodecyl-α-d-maltoside (α-DDM) as described in “Materials and Methods.” In addition to the free LHC fraction (A1), which was possibly dissociated from both PSI and PSII photosystems, the PSI-LHC supercomplex fraction (A2) was obtained as a result of SDG ultracentrifugation (Fig. 1A; Supplemental Fig. S1). Interestingly, the PSI-LHC supercomplex fraction obtained from B. minutum was banded at a higher-density position than that of the unicellular green alga Chlamydomonas reinhardtii, indicating that the M r of B. minutum PSI-LHC supercomplex was larger than that of C. reinhardtii, which harbors 10 LHCIs (Kubota-Kawai et al., 2019). Figure 1. Open in new tabDownload slide Purification of the B. minutum PSI-LHC supercomplex. A, SDG ultracentrifugation of thylakoid membranes from C. reinhardtii and B. minutum following solubilization with 1.0% α-DDM. B, SDS-PAGE analysis of the thylakoid membranes, the PSI-LHC fraction from SDG ultracentrifugation, and Fd affinity-purified PSI-LHC. One microgram of Chl from each PSI sample was loaded and the gel was stained with Coomassie Brilliant Blue R-250. The samples were subjected to immunoblotting with antibodies specific to D1 protein. C, Protein compositions of PSI-LHC from C. reinhardtii and B. minutum. One microgram of Chl from each samples was loaded and the gel was stained with Coomassie Brilliant Blue R-250. Each protein band was identified by LC-MS/MS analysis. Figure 1. Open in new tabDownload slide Purification of the B. minutum PSI-LHC supercomplex. A, SDG ultracentrifugation of thylakoid membranes from C. reinhardtii and B. minutum following solubilization with 1.0% α-DDM. B, SDS-PAGE analysis of the thylakoid membranes, the PSI-LHC fraction from SDG ultracentrifugation, and Fd affinity-purified PSI-LHC. One microgram of Chl from each PSI sample was loaded and the gel was stained with Coomassie Brilliant Blue R-250. The samples were subjected to immunoblotting with antibodies specific to D1 protein. C, Protein compositions of PSI-LHC from C. reinhardtii and B. minutum. One microgram of Chl from each samples was loaded and the gel was stained with Coomassie Brilliant Blue R-250. Each protein band was identified by LC-MS/MS analysis. To further purify the PSI-LHC supercomplex, we performed affinity purification using a Fd column as reported previously (Kubota-Kawai et al., 2018). Fd is known to be reduced by PSI, and this reaction is mediated via a physical interaction of Fd with PSI subunits, namely PsaA, PsaC, and PsaE (Fischer et al., 1998; Kubota-Kawai et al., 2018). Accordingly, we expected that Fd affinity purification would enable us to purify PSI complexes with their associating proteins. The Fd affinity column maintained a greenish color after the initial washing step, suggesting that Fd-1 from C. reinhardtii used in this study also interacted with the PSI subunit(s) in B. minutum. SDS-PAGE analysis of the final eluate showed that polypeptides from PSII and nonphotosynthetic proteins were decreased (Fig. 1B). We thus conclude that PSI was further concentrated by Fd affinity column purification. We next evaluated the intactness of the Fd-purified PSI-LHC supercomplex by measuring the photochemical activity of the supercomplex. Since O2 uptake activity in the presence of diaminodurene and methyl viologen reflects PSI activity (Vernon and Cardon, 1982), we measured the O2 consuming capability of the purified B. minutum PSI-LHC. The maximal O2 consumption rate we observed was remarkably higher (2.02 ± 0.34 mmol O2 mg Chl−1 h−1; Table 1) than that of the monomeric and trimeric PSI isolated from Synechocystis sp. PCC 6803 (Kubota et al., 2010), trimeric PSI in Thermosynechococcus elongatus and PSI-LHCI supercomplex in C. merolae (Haniewicz et al., 2018). These data suggest that the Symbiodiniaceae PSI-LHC supercomplex obtained in this study retained functional integrity. Photosynthetic activity of PSI-LHC in B. minutum compared with PSI monomer and trimer in Synechocystis sp. PCC 6803 Table 1. Photosynthetic activity of PSI-LHC in B. minutum compared with PSI monomer and trimer in Synechocystis sp. PCC 6803 Values are represented as means ± sd (n = 3). Species . Preparation . O2 Absorption Rate . mmol O 2 mg Chl −1 h −1 B. minutum (this study) PSI-LHC 2.02 ± 0.34 Synechocystis sp. PCC6803 (Kubota et al., 2010) PSI monomer 0.73 ± 0.03 PSI trimer 1.24 ± 0.02 Species . Preparation . O2 Absorption Rate . mmol O 2 mg Chl −1 h −1 B. minutum (this study) PSI-LHC 2.02 ± 0.34 Synechocystis sp. PCC6803 (Kubota et al., 2010) PSI monomer 0.73 ± 0.03 PSI trimer 1.24 ± 0.02 Open in new tab Table 1. Photosynthetic activity of PSI-LHC in B. minutum compared with PSI monomer and trimer in Synechocystis sp. PCC 6803 Values are represented as means ± sd (n = 3). Species . Preparation . O2 Absorption Rate . mmol O 2 mg Chl −1 h −1 B. minutum (this study) PSI-LHC 2.02 ± 0.34 Synechocystis sp. PCC6803 (Kubota et al., 2010) PSI monomer 0.73 ± 0.03 PSI trimer 1.24 ± 0.02 Species . Preparation . O2 Absorption Rate . mmol O 2 mg Chl −1 h −1 B. minutum (this study) PSI-LHC 2.02 ± 0.34 Synechocystis sp. PCC6803 (Kubota et al., 2010) PSI monomer 0.73 ± 0.03 PSI trimer 1.24 ± 0.02 Open in new tab Protein Compositions We further determined the protein composition of the PSI-LHC supercomplex in B. minutum. Proteins of the purified PSI-LHC supercomplex were separated through SDS-PAGE, and the silver-stained polypeptides were subjected to in-gel trypsin digestion followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. SDS-PAGE analysis demonstrated that B. minutum possessed smaller masses of PsaA and PsaB subunits and larger masses of the other subunits than those counterparts in C. reinhardtii (Fig. 1C), which is consistent with their genome information (Shoguchi et al., 2013). Notably, the PsaF and PsaL subunits in B. minutum were much larger than those in the green alga (Supplemental Figs. S2 and S3). These size differences in the PSI core subunits might contribute to the M r differences in the PSI-LHC supercomplexes between C. reinhardtii and B. minutum (Fig. 1A). Moreover, we found that there were many polypeptide bands in the gel positions between PsaF and PsaJ, which probably corresponded to LHCs (Fig. 1C). To classify the types of LHCs that were associated with PSI-LHC, we also compared the LHCs in the thylakoid membranes, those in the purified PSI-LHC supercomplexes, and the free LHC fractions from the SDG ultracentrifugation (Table 2). In summary, of the 145 LHCs encoded by the B. minutum genome, 79 and 25 were detected in the thylakoids and PSI-LHC supercomplex, respectively. The LHCs uniquely detected in the thylakoids and not in the PSI-LHC supercomplex were either not associated with PSI or were associated but lost during the SDG/Fd affinity-purification processes. The LHC classified in the LHCF5 group and the two unclassified LHCs (Supplemental Table S1, “other”) were not detected in the thylakoids, implying that these LHCs were not expressed under the culture conditions in the this study. Further comprehensive proteomic analyses of B. minutum grown under various other environmental conditions would provide more insights into these undetected LHCs. LHC types contained in isolated thylakoid, purified PSI-LHC supercomplex, and free LHC fraction after SDG ultracentrifugation Table 2. LHC types contained in isolated thylakoid, purified PSI-LHC supercomplex, and free LHC fraction after SDG ultracentrifugation The LHC grouping in the first column is based on the work of Maruyama et al. (2015). LHC Group . Thylakoid . PSI-LHC . Free LHC Fraction . F1 51 7 45 F2 2 2 1 F3 3 1 0 F4 6 2 3 F5 0 0 0 R1 3 3 0 R2 1 0 1 R3 7 6 2 R4 3 2 0 R5 3 2 0 Other 0 0 0 Total 79 25 52 LHC Group . Thylakoid . PSI-LHC . Free LHC Fraction . F1 51 7 45 F2 2 2 1 F3 3 1 0 F4 6 2 3 F5 0 0 0 R1 3 3 0 R2 1 0 1 R3 7 6 2 R4 3 2 0 R5 3 2 0 Other 0 0 0 Total 79 25 52 Open in new tab Table 2. LHC types contained in isolated thylakoid, purified PSI-LHC supercomplex, and free LHC fraction after SDG ultracentrifugation The LHC grouping in the first column is based on the work of Maruyama et al. (2015). LHC Group . Thylakoid . PSI-LHC . Free LHC Fraction . F1 51 7 45 F2 2 2 1 F3 3 1 0 F4 6 2 3 F5 0 0 0 R1 3 3 0 R2 1 0 1 R3 7 6 2 R4 3 2 0 R5 3 2 0 Other 0 0 0 Total 79 25 52 LHC Group . Thylakoid . PSI-LHC . Free LHC Fraction . F1 51 7 45 F2 2 2 1 F3 3 1 0 F4 6 2 3 F5 0 0 0 R1 3 3 0 R2 1 0 1 R3 7 6 2 R4 3 2 0 R5 3 2 0 Other 0 0 0 Total 79 25 52 Open in new tab Most LHCs classified as LHCR were detected in the thylakoids and in the purified PSI-LHC supercomplexes, but not in the free LHC fractions (Table 2). Because LHCs dissociated from the photosystems during biochemical preparations should be detected in the free LHC fractions, those only detected in the purified PSI-LHC were presumed to be strongly associated with PSI. Our results therefore suggest that the LHCRs expressed in B. minutum are tightly associated with PSI and function as its light-harvesting antennas, similar to the LHCR proteins in red algae (Wolfe et al., 1994). Some of the LHCRs were not detected in the PSI-LHC but were detected in the thylakoids and the free LHC fraction, implying that those LHCRs were loosely associated with either PSI or PSII. A few LHCFs were also detected in the PSI-LHC supercomplex, suggesting that both LHCRs and some LHCFs function as light-harvesting antennae for PSI in B. minutum (Table 2). Both diatoms and Symbiodiniaceae are secondary symbionts derived from red algae and possess two types of LHC, namely LHCF and LHCR (Büchel, 2015). Previous studies in diatoms have also revealed that their PSI is associated not only with LHCR but also with LHCF (Veith et al., 2009; Lepetit et al., 2010). Therefore, the association of both LHCRs and LHCFs with PSI may be a common feature of the secondary symbionts derived from red algae. Spectroscopic Properties To investigate whether the detected LHC proteins were energetically coupled with the PSI core or not, we analyzed the absorption and fluorescence spectra of the PSI-LHC. The absorption spectra of the PSI-LHC showed a large absorption peak at 440 nm, likely originating from Chl a, and a shoulder at ∼460 nm, likely originating from Chl c 2 and Per (Fig. 2A; Supplemental Fig. S4; Mantoura and Llewellyn, 1983). The difference between the absorption spectra of the thylakoids and the purified PSI-LHC showed a clear peak at 461 nm, implying that large amounts of Chl c 2 and Per, possibly derived from LHCF proteins, were not associated with PSI-LHC in the thylakoid (Fig. 2B). To evaluate energetic coupling of LHC proteins with the PSI core, we measured fluorescence emission and excitation spectra of PSI-LHC at 77 K. The fluorescence emission spectra, obtained by excitation at 440 nm (Chl a), or at 460 nm (Chl c 2 and Per), showed a peak at 687 nm and a large shoulder at 705 nm (Fig. 2C; Supplemental Fig. S5) which likely originated from the LHC and the PSI core, respectively (Reynolds et al., 2008). This result indicates that Chl a, in addition to Chl c 2 and Per in the LHC, transfers excitation energy to the PSI core. The fluorescence excitation spectrum monitored at 705 nm (PSI core) showed that the amplitude of shoulders near 464 and 492 nm (Chl c 2 and Per; Fig. 2D; Supplemental Fig. S6) relative to the peak amplitude near 440 nm (Chl a) was larger than that in the absorption spectrum (Fig. 2A, red), indicating that Chl c 2 or Per more efficiently transfers excitation energy to the PSI core than Chl a. These spectral features are consistent with the LC-MS/MS results (Supplemental Table S1) showing that PSI was associated with LHCRs and also a small number of LHCFs that potentially bound Chl c 2 and Per. In summary, these spectroscopic data demonstrate the light-harvesting capability of both LHCR and LHCF in the PSI-LHC supercomplex purified from B. minutum. Figure 2. Open in new tabDownload slide Spectroscopic characterization of B. minutum PSI-LHC. A, Absorption spectra of the thylakoid membranes and PSI-LHC. The spectra were normalized to the peak in the Chl a Qy region (at ∼680 nm). B, Difference between the absorption spectra of thylakoids and PSI-LHC shown in A. C, Fluorescence emission spectra at 77 K of PSI-LHC excited at 440 and 460 nm. The spectra were normalized to the maximum. D, Fluorescence excitation spectrum at 77 K of PSI-LHC monitored at 705 nm. Samples were diluted to 0.5 μg Chl mL−1 for each measurement. Figure 2. Open in new tabDownload slide Spectroscopic characterization of B. minutum PSI-LHC. A, Absorption spectra of the thylakoid membranes and PSI-LHC. The spectra were normalized to the peak in the Chl a Qy region (at ∼680 nm). B, Difference between the absorption spectra of thylakoids and PSI-LHC shown in A. C, Fluorescence emission spectra at 77 K of PSI-LHC excited at 440 and 460 nm. The spectra were normalized to the maximum. D, Fluorescence excitation spectrum at 77 K of PSI-LHC monitored at 705 nm. Samples were diluted to 0.5 μg Chl mL−1 for each measurement. Supramolecular Organization To elucidate the supramolecular organization of the PSI-LHC supercomplex in B. minutum, we visualized the supercomplex particles by negative-staining EM followed by single-particle analysis. After classification of the averaged PSI particles picked from the electron micrographs (Supplemental Figs. S7 and S8), we obtained two supercomplex structures. The supercomplex shown in Figure 3A and Supplemental Figure S9 was the dominant larger form with maximum dimensions of ∼250 Å (length) × 200 Å (width). Figure 3B depicts the second dominant and the smaller supercomplex with dimensions of ∼210 Å × 160 Å. We also found a small population (2.2%) of small particles, which were approximately the size of a PSI core complex with no bound LHCs, although they lacked definition for further analysis (Supplemental Fig. S10). To estimate the number of LHCs associated with these PSI-LHC supercomplexes, we superimposed the C. merolae PSI-LHCR structure determined by cryo-EM (Pi et al., 2018) with additional replicated LHCRs on the two-dimensional (2D) projection maps. In total, 18 LHCs were assigned to the larger PSI-LHC structure from B. minutum, and these LHCs were surrounding the PSI core (Fig. 3C). The smaller supercomplex lacked the larger structure's semicircular monolayer of LHCs covering the PsaO side of the PSI core (Fig. 3, B and D). The remaining eight LHCs were associated with the PsaF/PsaJ side as a crescent-shape LHCI belt reported in plants (Nelson and Junge, 2015), which extended to the PsaK side with two additional LHCs attached as a partial outer layer, much like the “LHCI dimer 1” in P. patens (Iwai et al., 2018). The larger supercomplex (Fig. 3, A and C) was much larger than the PSI-LHCI supercomplex in C. reinhardtii (200 Å × 170 Å), which binds 10 LHCs (Kubota-Kawai et al., 2019). These EM results are compatible with the result of SDG, where the B. minutum PSI-LHC was located at a lower position than the C. reinhardtii PSI-LHCI (Fig. 1A). Our results suggest that B. minutum possesses at least two unique PSI-LHC supercomplexes, where 8 to 18 LHCs are associated with the core. Figure 3. Open in new tabDownload slide Negative-stained 2D-structure of B. minutum PSI-LHC supercomplex. A, Two-dimensional reprojection of the larger B. minutum PSI-LHC supercomplex revealed by single-particle analysis of negative-stain EM. B, Two-dimensional reprojection of a low-resolution 3D reconstruction of the smaller PSI-LHC complex in B. minutum. C, The PSI core and LHCR structures of C. merolae were fitted to the projection map shown in A; PsaA, PsaB, PsaF, PsaJ, PsaK, and PsaO are depicted in blue, PsaC, PsaD, and PsaE in yellow, PsaL in orange, and LHCR proteins in green. LHCRs were replicated as much as necessary. The area outlined by the dashed red line corresponds to the complex shown in D. D, The image in B with Protein Data Bank models fitted. Scale bars = 10 nm. Figure 3. Open in new tabDownload slide Negative-stained 2D-structure of B. minutum PSI-LHC supercomplex. A, Two-dimensional reprojection of the larger B. minutum PSI-LHC supercomplex revealed by single-particle analysis of negative-stain EM. B, Two-dimensional reprojection of a low-resolution 3D reconstruction of the smaller PSI-LHC complex in B. minutum. C, The PSI core and LHCR structures of C. merolae were fitted to the projection map shown in A; PsaA, PsaB, PsaF, PsaJ, PsaK, and PsaO are depicted in blue, PsaC, PsaD, and PsaE in yellow, PsaL in orange, and LHCR proteins in green. LHCRs were replicated as much as necessary. The area outlined by the dashed red line corresponds to the complex shown in D. D, The image in B with Protein Data Bank models fitted. Scale bars = 10 nm. Pigment Composition HPLC analysis was conducted to characterize the pigment composition of the supercomplexes. Four pigment species (Chl a, Chl c 2, Per, and Ddx) were detected in both the thylakoids and PSI-LHC samples (Fig. 4). When the amount of each pigment was normalized by that of Chl a, it was revealed that the PSI-LHC supercomplex fraction contained remarkably lower contents of Chl c 2 and Per than the thylakoids. Because LHCF-type antennae are presumed to bind more Chl c 2 and Per than LHCR-type antennae (Maruyama et al., 2015), this difference in pigment composition between the thylakoids and PSI-LHC supercomplex may reflect a lower content of LHCF in the PSI-LHC supercomplex than in the thylakoids. It is of note that Ddx was the second most abundant pigment in the PSI-LHC supercomplex (the most abundant is Chl a; Fig. 4). Ddx is a xanthophyll that could serve as a substrate for the Ddx cycle contributing to excitation energy dissipation (Goss and Jakob, 2010). Localization of Ddx in the thylakoid membranes in diatoms has been reported previously (Kuczynska et al., 2015). It was reported that large amounts of Ddx and its de-epoxidated form, Dtx, were detected in the light-harvesting fucoxanthin-Chl proteins (Beer et al., 2006), which are similar to LHCF in Symbiodiniaceae, and small amounts of those pigments were also detected in the isolated PSI fucoxanthin-Chl protein supercomplex in the diatom P. tricornutum (Lepetit et al., 2008). Similarly, other xanthophyll cycle pigments, including violaxanthin and zeaxanthin, were also detected in the PSI-LHC in red algae (Haniewicz et al., 2018), suggesting that the PSI-LHC in the organisms derived from red algae bind those photoprotective xanthophylls. Our quantitative pigment analysis also supports that the PSI-LHC supercomplex in B. minutum bound an amount of Ddx comparable to that in the thylakoids (Fig. 4), which suggests that in B. minutum, both LHCF and LHCR types of LHCs, and/or the PSI core complex itself, contain Ddx. Figure 4. Open in new tabDownload slide Pigment analysis of the thylakoid membrane and the purified PSI-LHC supercomplex. The amounts of Per, Chl c 2, and Ddx were normalized to the amount of Chl a. Data are means ± sd of n = 3 independent biological replicates. Asterisks indicated significant difference by Student's t test (*P < 0.05 and **P < 0.01). Figure 4. Open in new tabDownload slide Pigment analysis of the thylakoid membrane and the purified PSI-LHC supercomplex. The amounts of Per, Chl c 2, and Ddx were normalized to the amount of Chl a. Data are means ± sd of n = 3 independent biological replicates. Asterisks indicated significant difference by Student's t test (*P < 0.05 and **P < 0.01). Since we found that the B. minutum PSI-LHC possesses a large amount of Ddx, we investigated whether those Ddx pigments are converted into Dtx by HL treatment. We observed obvious accumulation of Dtx after HL treatment, which was not detected in cells without HL treatment (Fig. 5A). To investigate the locations of those Dtxs, we purified the PSI-LHC supercomplex from the thylakoids obtained from the HL-treated cells. Whereas the PSI-LHC supercomplex prepared from cells without HL treatment only contained Ddx, that prepared from cells with HL treatment contained both Ddx and Dtx (Fig. 5A), indicating that Ddx in the B. minutum PSI-LHC supercomplex is converted into Dtx under HL conditions. Moreover, the purified PSI-LHC possessed a higher Dtx/Ddx ratio than the thylakoids (Fig. 5B), which suggests that de-epoxidation of Ddx is more efficient in the PSI-LHC supercomplex than in the PSII-LHC or in the membrane as a free form, if it occurs at all in the latter two entities. Figure 5. Open in new tabDownload slide Accumulation of xanthophyll pigments in the purified PSI-LHC supercomplex under HL conditions. A, Molar ratios of Per, Chl c 2, Ddx, and Dtx to Chl a in the thylakoids and PSI-LHC purified from cells either with HL treatment or without (GL) for 3 h. B, Molar ratio of Dtx normalized to the Ddx amount in the cells with HL treatment. Data are means ± sd of n = 3 independent biological replicates. Figure 5. Open in new tabDownload slide Accumulation of xanthophyll pigments in the purified PSI-LHC supercomplex under HL conditions. A, Molar ratios of Per, Chl c 2, Ddx, and Dtx to Chl a in the thylakoids and PSI-LHC purified from cells either with HL treatment or without (GL) for 3 h. B, Molar ratio of Dtx normalized to the Ddx amount in the cells with HL treatment. Data are means ± sd of n = 3 independent biological replicates. Finally, NPQ of B. minutum cells with or without HL treatment was determined. The results show that the cells with HL treatment had higher NPQ values than those without (Fig. 6). If the HL-induced NPQ was dependent upon the elevated Dtx, the PSI-LHC could be involved in this photoprotective process because Dtx was preferentially bound to it. We therefore tested whether the absorption cross section of P700 was affected by HL treatment. When the light-induced P700 oxidation kinetics were compared in the presence of PSII blocker 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the HL-treated cells indeed showed a slower rate of P700+ formation than the cells without HL treatment (Supplemental Fig. S11). At least three mechanisms could account for the slower rate of P700+ formation: (1) a decreased light-harvesting efficiency due to the xanthophyll-dependent energy dissipation in LHC (Ballottari et al., 2014); (2) a decreased light-harvesting efficiency due to the P700+-dependent energy dissipation in the PSI core (Slavov et al., 2016); and (3) an increased re-reduction of P700+ due to an elevated cyclic electron flow around PSI (Aihara et al., 2016). The former two mechanisms require PSII-to-PSI spillover to exert NPQ. It is anticipated that further analyses will clarify which of the three mechanisms is more appropriate and how Dtx is involved directly or indirectly in the mechanism. Figure 6. Open in new tabDownload slide Fluorescence quenching analysis of B. minutum cells. NPQ light-response curves of cells either with HL treatment or without (GL) for 3 h. The cells were dark adapted for 15 min before the measurements. Chl fluorescence was measured with increasing actinic light irradiances by light phases. Data are means ± sd of n = 3 independent biological replicates. Figure 6. Open in new tabDownload slide Fluorescence quenching analysis of B. minutum cells. NPQ light-response curves of cells either with HL treatment or without (GL) for 3 h. The cells were dark adapted for 15 min before the measurements. Chl fluorescence was measured with increasing actinic light irradiances by light phases. Data are means ± sd of n = 3 independent biological replicates. CONCLUSION In this study, we isolated and characterized the PSI-LHC supercomplex from a dinoflagellate B. minutum. The results of mass-spectroscopic and EM structural analyses revealed that the PSI-LHC supercomplex in B. minutum is associated with the largest number of light-harvesting antennae reported so far, which may enable B. minutum to reside in corals at low ocean depths. We also localized the xanthophyll cycle pigment Ddx in the PSI-LHC complex. The Ddx in PSI-LHC was converted to Dtx under HL conditions, which was correlated to NPQ capacity. The PSI-LHC is thus likely to be involved in photoprotection in B. minutum. MATERIALS AND METHODS Culture Conditions Breviolum minutum strain Mf1.05b was cultured essentially as described in Aihara et al. (2016), except that artificial seawater containing f/2 medium was used as a growth medium and white fluorescent light at 150 μmol photons m−2 s−1 was given as growth light (GL) for B. minutum. Cells were cultured in a 2-gallon polycarbonate bottle with air bubbling and stirring. Chlamydomonas reinhardtii cells were grown in Tris acetate-phosphate medium (Gorman and Levine, 1965) with air bubbling at 23°C in low light (10–30 μmol photons m−2 s−1). The clade of the B. minutum strain used in this study (Mf1.05b) was confirmed as “B” by RFLP assay (Santos et al., 2003). The ITS2 region was amplified by PCR with primers ss5 (5′-GGTGATCCTGCCAGTAGTCATATGCTTG-3′) and ss3z (5′-AGCACTGCGTCAGTCGGAATATTCACCGG-3′; Rowan and Powers, 1991) using the MightyAmp Genotyping Kit (Takara Bio; Supplemental Fig. S12). Isolation of Thylakoid Membranes Cultured B. minutum cells at the midlog phase were harvested for thylakoid membrane isolation. Cells were harvested at 6 h after the beginning of light period by centrifugation at 5,000g and resuspended in isolation buffer containing 0.33 m Suc, 25 mm HEPES, 1.0 m betaine, 5 mm MgCl2, and 1.5 mm NaCl (pH 7.5). Cells were disrupted with mixed glass beads (0.1 and 0.5 mm) using a bead beater (BioSpec Products; 20 cycles of 10 s homogenization at 170-s intervals). Broken cells were loaded onto two-step SDG ultracentrifugation tubes with 1.3 and 1.8 m Suc stacking and subjected to ultracentrifugation at 90,000g on a P28ST rotor (Hitachi-koki) for 25 min at 4°C. A fraction of 1.3 m Suc was harvested and resuspended with a buffer containing 25 mm HEPES and 1.0 m betaine (pH 7.5). Thylakoid membranes from C. reinhardtii were isolated according to the study by Iwai et al. (2008). The total concentrations of Chl a and c 2 were measured as described previously (Jeffrey and Humphrey, 1975; Takahashi et al., 2008). For the HL treatment, cultured cells were illuminated with white LED light at 600 μmol photons m−2 s−1 for 3 h. Cells were disrupted with a bead beater under white LED illumination at 1,000 µmol photons m−2 s−1. Thylakoids were isolated as described in this section. SDG Fractionation of Photosynthetic Protein Complexes in Thylakoid Membranes Thylakoid membranes were adjusted to 0.5 mg total Chl mL−1 in 25 mm HEPES and 1.0 m betaine (pH 7.5) and solubilized for 15 min on ice in the dark with 1.0% (w/v) α-DDM. The unsolubilized thylakoids were separated by centrifugation at 20,000g for 5 min, and solubilized membranes (500 μL) were loaded onto a discontinuous SDG tube (0.1, 0.4, 0.7, 1, and 1.3 m Suc in a buffer containing 25 mm HEPES and 1.0 m betaine with 0.05% (w/v) α-DDM (pH 7.5) and subjected to ultracentrifugation at 90,000g on a P40ST rotor (Hitachi-koki) for 20 h at 4°C. PSI Affinity Purification Isolated PSI-LHC supercomplexes were further purified with a Fd affinity column according to a previous study (Kubota-Kawai et al., 2018), with one modification: C. reinhardtii Fd-1 protein was bound to the resin. The PSI-LHC fraction of the SDG was collected, and the buffer was replaced with 25 mm HEPES and 0.05% (w/v) α-DDM (pH 7.5). The collected PSI-LHC samples were loaded onto the Fd column. The column was washed with 10 column volumes of the buffer and eluted with a buffer containing 500 mm NaCl. LC-MS/MS Analysis The purified PSI-LHC supercomplexes were subjected to SDS-PAGE with a 16% to 22% polyacrylamide gradient gel containing 7 m urea. Proteins were stained with the Silver Stain MS Kit (Wako). Each protein band was cut out and in-gel digested with trypsin according to Shevchenko et al. (1996). The in-gel digested samples were analyzed by an ultraperformance liquid chromatography (UPLC) system (EASY-nLC 1000) coupled to an Orbitrap Elite mass spectrometer (both from Thermo Fisher Scientific). The analyzed data were submitted for protein database searching using Proteome Discoverer software (Thermo Fisher Scientific) and Mascot version 2.5.1 (Matrix Science). The protein database was generated from the polypeptide sequences obtained from the genome sequence data of the B. minutum strain Mf1.05b.01 (http://marinegenomics.oist.jp/gallery/). Pigment Analysis Pigments in thylakoids and the PSI-LHCI supercomplex were extracted with 50% (v/v) acetone and 50% (v/v) methanol. Extracted pigments were subjected to UPLC with an UPLC BEH C18 column (130 Å, 1.7 µm, 2.1 mm × 50 mm; 186002350, Acquity Group) and eluted with gradient from 70% (v/v) acetonitrile and 10% (v/v) 2-propanol to 50% (v/v) acetonitrile and 50% (v/v) 2-propanol (Tokutsu and Minagawa, 2013). Data were acquired and processed using Empower 3 chromatography data software (Waters). Measurement of Photosynthetic Activity The photochemical activity of the PSI-LHC supercomplexes was measured by O2 consumption assay according to Kubota et al. (2010), with minor modifications. PSI-LHC supercomplexes were suspended in 25 mm HEPES (pH 7.5; including 1.0 m Suc, 1.0 m betaine, 1 mm diaminodurene, 1.5 mm methylviologen, 1 mm ascorbate, and 2 mm NaN3), and then O2 absorption activity was recorded at 25°C with a Witrox 4 oxygen meter (Loligo Systems) under white light illumination with a metal halide lamp (NPI) at 10,000 μmol photons m−2 s−1. Averaged oxygen consumption values were recorded every 30 s Spectroscopic Analysis Absorption spectra were recorded using the V-650 ultraviolet-visible spectrometer with an integrating sphere (JASCO) at room temperature. Absorption spectra were obtained at 0.5-nm intervals in the range 350 to 800 nm. Fluorescence emission and excitation spectra were measured using a FluoroMax-4 spectrofluorometer (HORIBA) at 77K. Emission spectra were measured at 1-nm intervals in the range 650 to 800 nm by excitation at 440 and 460 nm (slit width = 5 nm). The obtained spectra were normalized to the maximum peak of each spectrum. The excitation spectrum was measured at 1-nm intervals in the range 400 to 650 nm with fixed emission at 705 nm (slit width = 2 nm). The oxidation kinetics of P700 was analyzed by monitoring the difference between two transmission pulse signals at 820 and 870 nm using a Dual/KLAS-NIR spectrophotometer (Heinz Walz) according to previously reported methods (Klughammer and Schreiber, 2016). Prior to the measurement, cell concentrations were set to 1.0 × 106 cells mL−1 and incubated in the dark for 2 min to get P700 fully reduced. Dark-light induction kinetics were obtained in the presence of 10 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea with actinic light by red LED (33 μE, 635 nm) with 1-ms pulse width. Measurements were repeated three times and averaged. NPQ Measurement Cells grown under GL with or without HL treatment were adapted to the dark conditions for 10 min. Maximum fluorescence yields under dark (Fm) and light treatment (Fm′) were measured using PAM-2500 (Heinz Walz) with increasing actinic light irradiance by light phase (0, 12, 36, 74, 111, 151, 208, 281, 373, 484, 629, and 795 μmol photons m−2 s−1). NPQ values were calculated using the equation NPQ = (Fm − Fm′)/Fm′. Each light phase continued for 20 s. All measurements were done at 25°C. EM and Single-Particle Analysis Purified PSI-LHC supercomplexes were adjusted to 3.0 μg total Chl mL−1, applied to glow-discharged carbon-coated copper grids for 30 s, and negatively stained three times for 30 s with 2% (w/v) uranyl acetate. Micrographs were collected with a JEOL JEM-1010 electron microscope at 80 kV and 150,000× magnification with an Olympus Veleta CCD camera (2,000 × 2,000 pixels) at a pixel size of 5 Å. The resolution of the micrographs was estimated using CTFFIND4 (Rohou and Grigorieff, 2015). The data were processed with RELION 2.1 (Kimanius et al., 2016). A selection of particles was manually picked and subjected to 2D classification before being used for automated picking, resulting in a total of 19,922 particles. Further 2D classification was carried out with empty, aggregated, small particles or low-occupancy classes discarded, resulting in 4,614 large particles, including “top” views, tilts, and one side-view class contained within 10 classes (Supplemental Fig. S7) and 5,507 small particles contained within 10 classes. These large-particle classes were taken forward to initial model generation with a resolution limit of 25 Å imposed using the RELION 3 initial model algorithm (Zivanov et al., 2018). This initial model was used as a reference for 3D refinement after 3D classification. The final large-particle reconstruction contained 4,036 particles. Examples of the particles contained within the class used for model fitting are shown in Supplemental Figure S8. Protein Data Bank models based upon the cryo-EM structures of the PSI core and LHCR in C. merolae (Pi et al., 2018) were superimposed on the 2D reprojection of the low-resolution 3D reconstruction according to the PSI-LHCR structure in red algae (Fig. 3, A and C). Similarly, for the smaller supercomplex particles, a subset was used for initial model generation. Finally, 2,490 particles were taken forward into the smaller reconstruction. A 2D reprojection of the 3D reconstruction (Fig. 3B) was used to estimate positions for PDB models in the same fashion as for the larger complex (Fig. 3D). Supplemental Data The following supplemental materials are available. Supplemental Figure S1. Polypeptide profiles in SDG fractions following solubilization by 1.0% α-DDM. Supplemental Figure S2. Amino acid sequence alignment of the PsaF subunit among photosynthetic organisms. Supplemental Figure S3. Amino acid sequence alignment of the PsaL subunit among photosynthetic organisms. Supplemental Figure S4. Absorption spectra of the thylakoid membrane and the PSI-LHC supercomplex. Supplemental Figure S5. Fluorescence emission spectra at 77 K of the thylakoid and the PSI-LHC supercomplex excited at 440 or 460 nm. Supplemental Figure S6. Fluorescence excitation spectra at 77 K of the thylakoid and the PSI-LHC supercomplex. Supplemental Figure S7. Ten classes of large B. minutum PSI-LHC supercomplex particles. Supplemental Figure S8. Example particles contained within the class in the red box in Supplemental Figure S7. Supplemental Figure S9. Two-dimensional reprojection of the larger B. minutum PSI-LHC supercomplex revealed by single-particle analysis of negative-stain EM. Supplemental Figure S10. Negative-stained 2D structure of PSI core particles in B. minutum Supplemental Figure S11. Light-induced P700 oxidation kinetics in GL- and HL-treated cells. Supplemental Figure S12. An RFLP assay of the strain used in this study. Supplemental Table S1. 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JP15H05599 and JP16H06553), the Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, and the Gordon and Betty Moore Foundation Marine Microbiology Initiative Experimental Model Systems (grant no. 4985). 2 Present address: Faculty of Science, Yamagata University, Yamagata 990–8560, Japan. 3 Present address: National Institute for Physiological Sciences, Okazaki 444–8585, Japan. 4 Author for contact: minagawa@nibb.ac.jp. 5 Senior author. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jun Minagawa (minagawa@nibb.ac.jp). H.K., R.T., and J.M. designed the research; H.K., R.T., H.K.-K., R.N.B.-S., and E.K. performed the experiments; R.T. supervised the experiments; all authors analyzed the data; H.K. and R.T. wrote the first draft of the article; J.M. supervised and completed the writing; and all authors reviewed the manuscript. www.plantphysiol.org/cgi/doi/10.1104/pp.20.00726 © 2020 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - Characterization of a Giant PSI Supercomplex in the Symbiotic Dinoflagellate Symbiodiniaceae
JF - Plant Physiology
DO - 10.1104/pp.20.00726
DA - 2020-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/characterization-of-a-giant-psi-supercomplex-in-the-symbiotic-b37rWduymq
SP - 1725
EP - 1734
VL - 183
IS - 4
DP - DeepDyve
ER -