ABSTRACT In plants and green algae, light-harvesting complexes I and II (LHCI and LHCII) constitute the antennae of photosystem I (PSI), thus effectively increasing the cross-section of the

PSI core. The moss _Physcomitrium patens_ (_P. patens_) represents a well-studied primary land-dwelling photosynthetic autotroph branching from the common ancestor of green algae and land

plants at the early stage of evolution. _P. patens_ possesses at least three types of PSI with different antenna sizes. The largest PSI form (_Pp_PSI-L) exhibits a unique organization found

neither in flowering plants nor in algae. Its formation is mediated by the _P. patens_-specific LHC protein, Lhcb9. While previous studies have revealed the overall architecture of

LHCII in stabilizing the complex. In addition, our results suggest that _Pp_PSI switches between different types, which share identical modules. This feature may contribute to the dynamic

adjustment of the light-harvesting capability of PSI under different light conditions. Access through your institution Buy or subscribe This is a preview of subscription content, access via

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL INSIGHTS INTO A UNIQUE PSI–LHCI–LHCII–LHCB9 SUPERCOMPLEX FROM MOSS

_PHYSCOMITRIUM PATENS_ Article 24 April 2023 UNCOVERING THE PHOTOSYSTEM I ASSEMBLY PATHWAY IN LAND PLANTS Article 19 March 2024 ANTENNA ARRANGEMENT AND ENERGY-TRANSFER PATHWAYS OF PSI–LHCI

FROM THE MOSS _PHYSCOMITRELLA PATENS_ Article Open access 16 February 2021 DATA AVAILABILITY The atomic coordinate of the _Pp_PSI-L complex has been deposited in the Protein Data Bank with

the accession code 8HTU. The composite overall and overall cryo-EM maps of the complex have been deposited in the Electron Microscopy Data Bank with accession codes EMDB-35018 and

EMDB-35026. In addition, locally refined cryo-EM maps of PSI-LHCI, LHCII trimers plus PsaH-PsaL-PsaO, outer LHCIs plus Lhcb9 and outer LHCIs plus Lhcb9 processed with deepEMhancer have been

deposited in the Electron Microscopy Data Bank with accession codes EMDB-35027, EMDB-35028, EMDB-35033 and EMDB-35034, respectively. All other data generated or analysed are available from

the corresponding authors on reasonable request. Source data are provided with this paper. CODE AVAILABILITY The Python script used for FRET rate calculation is available at

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Y. K. He, F. Bao and C. L. Ju from the College of Life Science, Capital Normal University, for providing the _P. patens_ strain; L. H. Chen, X. J. Huang, B. L. Zhu and F. Sun at the Center

for Biological Imaging (IBP, CAS) for support in cryo-EM data collection; C. Y. Zhang and Y. Yin from the Institute of Botany, CAS, for technical assistance in sample characterization; T.

Juelich (University of Chinese Academy of Sciences) for linguistic assistance during the preparation of the article. The project was funded by the Strategic Priority Research Program of CAS

(XDB37020101, XDB27020106), the National Natural Science Foundation of China (31930064 and 31970264) and the National Key R&D Program of China (2022YFC2804400) and was supported by the

National Laboratory of Biomacromolecules (2022kf07). AUTHOR INFORMATION Author notes * These authors contributed equally: Haiyu Sun, Hui Shang. AUTHORS AND AFFILIATIONS * National Laboratory

of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Haiyu Sun & Mei Li * University of Chinese

Academy of Sciences, Beijing, China Haiyu Sun * Beijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental Improvement, College of Life Science,

Capital Normal University, Beijing, China Hui Shang & Xiaowei Pan Authors * Haiyu Sun View author publications You can also search for this author inPubMed Google Scholar * Hui Shang

View author publications You can also search for this author inPubMed Google Scholar * Xiaowei Pan View author publications You can also search for this author inPubMed Google Scholar * Mei

Li View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS X.P. and M.L. conceived and coordinated the project. H. Sun and H. Shang performed the

purification and characterization of the _Pp_PSI-L sample; H. Sun and X.P. processed the cryo-EM data, built and refined the structural model. H. Sun performed the multi-body refinement. H.

Sun, X.P. and M.L. analysed the data and wrote the manuscript; all authors discussed and commented on the results and the manuscript. CORRESPONDING AUTHORS Correspondence to Xiaowei Pan or

Mei Li. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Plants_ thanks Jun Minagawa, Tomas Morosinotto and the

other, anonymous, reviewer(s) for their contribution to the peer review of this work. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional

claims in published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 SAMPLE PREPARATION AND PROTEIN COMPOSITION ANALYSIS OF _PP_PSI-L COMPLEX. A, Sucrose density

gradient of solubilized thylakoid membranes of _P. patens_ cultured in the media with and without glucose, respectively. The green bands corresponding to _Pp_PSI-L and _Pp_PSI-S are

indicated and labeled. The negative stained image of _Pp_PSI-L and _Pp_PSI-S are also shown. B, SDS-PAGE analysis of the purified _Pp_PSI-S and _Pp_PSI-L complexes. The protein composition

of each Coomassie band was indicated based on the mass spectrometry and proteomics data analysis. C, Sucrose density gradient of solubilized thylakoid membranes isolated from _P. patens_

treated with high light (HL; 1000 µmol photons m−2 s−1 for 1 h) or low light (LL; 17 µmol photons m−2 s−1 for 1 h). The green bands corresponding to _Pp_PSI-L and _Pp_PSI-S are indicated and

labeled. D, Immunoblot analysis of subunits of thylakoid membranes, _Pp_PSI-S and _Pp_PSI-L samples. PsaA was used as control to show that _Pp_PSI-S and _Pp_PSI-L samples contain PsaA with

similar amount. Both pThr and Lhcb9 are present in the _Pp_PSI-L complex, but are absent in the _Pp_PSI-S sample. The band detected by Anti-Thr-P is identified as LhcbM proteins based on its

molecular weight. Data presented in this figure were repeated at least three times, and the same results were obtained. Source data EXTENDED DATA FIG. 2 CHARACTERIZATION OF _PP_PSI-S AND

_PP_PSI-L SAMPLES. A, Light-induced P700 oxidation kinetics of _Pp_PSI-S and _Pp_PSI-L samples. The mean values and standard deviations (represented by shaded areas) were calculated from two

independent measurements. B, HPLC analysis of pigment content in _Pp_PSI-S and _Pp_PSI-L samples. Based on the characteristic absorption spectrum of each peak fraction, the six major

pigment peaks separated from the sample are identified as neoxanthin (Neo), violaxanthin (Vio), lutein (Lut), chlorophyll _b_ (Chl _b_), chlorophyll _a_ (Chl _a_) and β-carotene (BCR). C,

Room-temperature absorption spectra of _Pp_PSI-S and _Pp_PSI-L samples. The _Pp_PSI-L sample showed higher peaks around 470 and 660 nm (indicated by arrows), demonstrating that the Chl _b_

(from LHCII) content of this fraction is higher than that of _Pp_PSI-S complex. The spectra were normalized to the maximum in the red region. D, 77K steady-state fluorescence spectrum of

_Pp_PSI-S (black line) and _Pp_PSI-L (red line). The blue shift of _Pp_PSI-L compared with _Pp_PSI-S indicates that _Pp_PSI-L contains more Chl _b_ than _Pp_PSI-S. Data in this figure

(b,c,d) were repeated more than three times, and all resulted in the same results. EXTENDED DATA FIG. 3 SINGLE PARTICLE CRYO-EM ANALYSIS AND EVALUATION OF _PP_PSI-L COMPLEX. A, Single

particle cryo-EM data processing procedure. Three datasets are combined. B, The gold standard Fourier shell correlation (FSC) curves of the final density maps with criterion of 0.143. C,

Angular distribution of particles included in the final 3D reconstruction. D, Local resolution of the cryo-EM map estimated by ResMap. EXTENDED DATA FIG. 4 STRUCTURE OF LHCII IN _PP_PSI-L.

A, Cartoon representation of LHCII-a monomer. Transmembrane helices A-C and two short amphiphilic helices D-E are labelled. Chlorophylls are shown as sticks with the central-Mg atoms shown

as spheres. Chls _a_ (green) and Chls _b_ (blue) are assigned according to the conserved sites in spinach LHCII (PDB code 1RWT). Carotenoids at sites L1, L2, V1 and N1 are denoted by sticks.

B, Stromal side view of the LHCII trimer. The phosphorylated Thr in LHCII-a is highlighted in ball-and-stick mode, and pigment molecules are shown as sticks. For clarity, the phytol chains

of chlorophylls are omitted. C, D, Map features of characteristic residues around the N-terminal tail (C) and Y57 (D) in LhcbM2 and the corresponding sequence alignment result. EXTENDED DATA

FIG. 5 LHCI BELTS FROM _P. PATENS_ AND _C. REINHARDTII_. A, Superposition of inner LHCI belt (yellow) and outer LHCI belt (magenta) of _Pp_PSI-L complex aligned on Lhca1. B,C, Stromal (B)

and lumenal (C) side view of the outer LHC belts from _Pp_PSI-L and _Cr_PSI-LHCI-LHCII (PDB code 7DZ7). Two structures are superposed on the inner LHCIs, and the outer LHCIs of _Pp_PSI-L are

further aligned on the outer LHCIs of _Cr_PSI-LHCI-LHCII. The inner and outer LHCIs are shown in cartoon mode. Lhca proteins in _Pp_PSI-L are shown as the same colour as in Fig. 1a. Lhca

proteins in _Cr_PSI-LHCI-LHCII are coloured yellow. In (B), the PSI core and the inner LHCI belt of _Pp_PSI-L are shown in surface mode, and coloured differently. The clash regions between

Lhca1-o and Lhca2.1-i, and between Lhca3-o and Lhca3-i in the stromal side are highlighted by red boxes in (B). The long C-terminal regions of _Cr_Lhca5 and _Cr_Lhca6 in the lumenal side are

shown in ribbon mode and highlighted by elliptical circles in (C). EXTENDED DATA FIG. 6 STRUCTURE AND LOCATION OF LHCB9. A, Superposition of the Lhcb9, LhcbM2 and Lhca1 structures. The

conserved Chls are shown as spheres at their central-Mg positions. Chl 614 and carotenoid at V1 site found in LhcbM2 are shown as lines. Carotenoids located at L1, L2 and N1 binding sites

are shown as sticks. The unique carotenoid located at L3 site in Lhcb9 is shown in stick-ball mode. The N- and C-terminal tails of Lhcb9 are labelled. B. Stromal side view of the monomeric

Lhcb9 and LHCIs in _Pp_PSI-L. The inner belt, outer belt and Lhcb9 are displayed as ribbon, and their helix C and N-terminal region (Nter) of Lhcb9 are highlighted in cartoon mode. Other

subunits are shown in surface mode. The red arrow indicates that helix C of Lhcb9. Chl pairs 603-609(−617) are shown as sticks. EXTENDED DATA FIG. 7 CHLOROPHYLL ARRANGEMENT IN THE _PP_PSI-L

COMPLEX. A,B, Stromal-side view of chlorophylls within the _Pp_PSI-L complex at the stromal layer (A) and lumenal layer (B). Chlorophylls located in the interface of neighbouring LHCs and

between the core and LHCs are shown as stick-ball mode and labelled, other chlorophylls are shown as lines. Red Chls from Lhcb9 and Lhca3 are shown as spheres. The pigment cluster containing

two pairs of red Chls in Lhcb9 and Lhca3-o are highlight with red dashed circle. C, The detailed arrangement of the pigment cluster encircled in (A). The red Chls and three closely

associated carotenoid molecules are shown as stick-ball mode. The closest Mg-to-Mg distance between the two red Chl pairs is indicated by black line and the distance is labelled. For

clarity, the phytol chains of chlorophylls are omitted. EXTENDED DATA FIG. 8 MULTI-BODY REFINEMENT OF THE _PP_PSI-L. A, The three bodies corresponding to _Pp_PSI-S moiety, LHCII and outer

LHCIs plus Lhcb9 are defined by the transparent masks in yellow, cyan and magenta, respectively. B, The contributions of all 18 eigenvectors to the variance. C-E, The flexibility of LHCII

and Lhcb9-outLHCIs relative to the _Pp_PSI-S moiety in the principal components along the top three eigenvectors (#1-3). The models are fitted into the bin 1 and bin 10 maps in each

component and then superposed on the _Pp_PSI-S moiety. In (C-E), the left and right panels are viewed from the stromal side and from the membrane plane. The eye symbols in the left panels in

(D, E) indicate the viewing angles for the side views shown on the right panels. The Lhcb9-outLHCIs-LHCII moiety is shown in magenta and yellow for the two states (bin 1 and bin 10) in

(C,D). In (E), The Lhcb9-outLHCIs and LHCII are shown in pink and cyan, respectively, for one state (bin 1) and shown in limon in another state (bin 10). The dashed lines and dotted line

indicate the lumenal layer of the membrane spanning regions of these rigid bodies. EXTENDED DATA FIG. 9 COMPARISON OF FOUR (INNER) LHCA PROTEINS FROM _P. PATENS_, _Z. MAYS_ AND _C.

REINHARDTII_. A, Comparison of the inner LHCI belts from _Pp_PSI-L, _Cr_PSI-LHCI-LHCII and LHCI belt from _Zm_PSI-LHCI-LHCII. Each Lhca proteins in _Cr_PSI-LHCI-LHCII and _Zm_PSI-LHCI-LHCII

structures are separately aligned on the corresponding Lhca proteins in _Pp_PSI-L structure. The AC loops are highlighted as cartoon. B-E, Structural comparison of the corresponding Lhca

proteins (Lhcas located at the same positions in LHCI belt) in _Pp_PSI-L, _Cr_PSI-LHCI-LHCII and _Zm_PSI-LHCI-LHCII. The AC loop region is highlighted by red arrows. The conserved Chls are

show as spheres at their central-Mg position. The specific Chls and carotenoids are shown as sticks and labelled. The conserved carotenoids are shown as lines in (B-E). SUPPLEMENTARY

INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figs. 1–3 and Tables 1–3. REPORTING SUMMARY SUPPLEMENTARY VIDEO 1 Supplementary Video 1. SUPPLEMENTARY VIDEO 2 Supplementary Video 2.

SUPPLEMENTARY VIDEO 3 Supplementary Video 3. SOURCE DATA SOURCE DATA EXTENDED DATA FIG. 1 Unprocessed gel (ED_Fig. 1b) and western blots (ED_Fig. 1d). SOURCE DATA EXTENDED DATA FIG. 1 MS

data of _Pp_PSI-L (ED_Fig. 1b) and MS data of _Pp_PSI-S (ED_Fig. 1b). RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this

article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of

such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Sun, H., Shang, H., Pan, X. _et al._ Structural insights into the assembly and

energy transfer of the Lhcb9-dependent photosystem I from moss _Physcomitrium patens_. _Nat. Plants_ 9, 1347–1358 (2023). https://doi.org/10.1038/s41477-023-01463-4 Download citation *

Received: 22 December 2022 * Accepted: 21 June 2023 * Published: 20 July 2023 * Issue Date: August 2023 * DOI: https://doi.org/10.1038/s41477-023-01463-4 SHARE THIS ARTICLE Anyone you share

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