Mosses are one of the earliest land plants that diverged from freshwater green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI-PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI-PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI-PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI-PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella.

Native polyacrylamide gel electrophoresis (PAGE) is a powerful technique for protein complex separation that retains both their activity and structure. In photosynthetic research, native-PAGE is particularly useful given that photosynthetic complexes are generally large in size, ranging from 200 kD to 1 MD or more. Recently, it has been reported that the addition of amphipol A8-35 to solubilized protein samples improved protein complex stability. In a previous study, we found that amphipol A8-35 could substitute sodium deoxycholate (DOC), a conventional electrophoretic carrier, in clear-native (CN)-PAGE. In this study, we present the optimization of amphipol-based CN-PAGE. We found that the ratio of amphipol A8-35 to α-dodecyl maltoside, a detergent commonly used to solubilize photosynthetic complexes, was critical for resolving photosynthetic machinery in CN-PAGE. In addition, LHCII dissociation from PSII–LHCII was effectively prevented by amphipol-based CN-PAGE compared with that of DOC-based CN-PAGE. Our data strongly suggest that majority of the PSII–LHCII in vivo forms C2S2M2 at least in Arabidopsis and Physcomitrella. The other forms might appear owing to the dissociation of LHCII from PSII during sample preparation and electrophoresis, which could be prevented by the addition of amphipol A8-35 after solubilization from thylakoid membranes. These results suggest that amphipol-based CN-PAGE may be a better alternative to DOC-based CN-PAGE for the study of labile protein complexes.

Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification in the peripheral antenna systems of photosystems (PSs). Here, the PSs of Mesostigma viride, one of the earliest-branching streptophyte algae, were analyzed to gain insight into their evolution. Isoform sequencing and phylogenetic analyses of light-harvesting complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9 and LHCP, while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-polyacrylamide gel electrophoresis (PAGE) resolved the photosynthetic complexes, including the PSI–PSII megacomplex, PSII–LHCII, two PSI–LHCI–LHCIIs, PSI–LHCI and the LHCII trimer. Results indicated that the higher-molecular weight PSI–LHCI–LHCII likely had more LHCII than the lower-molecular weight one, a unique feature of M. viride PSs. CN-PAGE coupled with mass spectrometry strongly suggested that the LHCP was bound to PSII–LHCII, while the algae-type LHCA2 and LHCA9 were bound to PSI–LHCI, both of which are different from those in land plants. Results of the present study strongly suggest that M. viride PSs possess unique features that were inherited from a common ancestor of streptophyte and chlorophyte algae.

Land plants evolved from a single group of streptophyte algae. One of the key factors needed for adaptation to a land environment is the modification of the peripheral antenna systems of photosystems. Here, the photosystems of Mesostigma viride, an earliest-branched streptophyte alga, were analyzed to gain insight into their evolution.Iso-seq and phylogenetic analyses of Light-Harvesting Complexes (LHCs) revealed that M. viride possesses three algae-specific LHCs, including algae-type LHCA2, LHCA9, and LHCP; while the streptophyte-specific LHCB6 was not identified. These data suggest that the acquisition of LHCB6 and the loss of algae-type LHCs occurred after the M. viride lineage branched off from other streptophytes. Clear-native (CN)-PAGE [Results]Separation of photosystems by ClearNative (CN)-PAGE Clear-Native (CN)-PAGE is a powerful technique that enables one to separate protein complexes while retaining their structure. Here, the M. viride photosystems were separated using amphipol-based CN-PAGE (Furukawa et al., 2019) after solubilization with a mild detergent, dodecyl maltoside (α-DDM). As a result, the PSI-PSII megacomplex, the PSII-LHCII supercomplexes, the PSI-LHCI-LHCII bands, the PSI-LHCI, and the LHCII trimer were resolved (Fig. 1A). The identification of the separated bands was accomplished using 2D-CN/SDS-PAGE followed by immunoblot analysis (Fig. 1B) and silver-staining (Fig. 1C), as described in previous studies (Järvi et al., 2011; Takabayashi et al., 2011). The identification of two PSI-LHCI-LHCII bands were confirmed by further analysis described in a later section of this report. The overall band profile (Fig. 1A) was similar to the profile for P. patens presented by Furukawa et al. ( 2019), however, a substantial difference was evident for PSI-LHCI-LHCII. Two PSI-LHCI-LHCII bands were found in M. viride (Fig. 1A), whereas only one PSI-LHCI-LHCII band was found in the profile of P. patens presented by Furukawa et al.