Abstract
Photosystem I (PSI) is one of two large pigment–protein complexes responsible for converting solar energy into chemical energy in all oxygenic photosynthetic organisms. The PSI supercomplex consists of the PSI core complex and peripheral light-harvesting complex I (LHCI) in eukaryotic photosynthetic organisms. However, how the PSI complex assembles in land plants is unknown. Here we describe PHOTOSYSTEM I BIOGENESIS FACTOR 8 (PBF8), a thylakoid-anchored protein in Arabidopsis thaliana that is required for PSI assembly. PBF8 regulates two key consecutive steps in this process, the building of two assembly intermediates comprising eight or nine subunits, by interacting with PSI core subunits. We identified putative PBF8 orthologues in charophytic algae and land plants but not in Cyanobacteria or Chlorophyta. Our data reveal the major PSI assembly pathway in land plants. Our findings suggest that novel assembly mechanisms evolved during plant terrestrialization to regulate PSI assembly, perhaps as a means to cope with terrestrial environments.
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Data availability
The raw data of RNA-seq and the reads per kilobase per million mapped reads (RPKM) of chloroplast genes calculated from RNA-seq have been deposited into the Gene Expression Omnibus under accession number GSE239827 and are publicly accessible at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE239827. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (2020YFA0907600 to C.L.) and the National Natural Science Foundation of China (31730102 to C.L. and 32000184 to Y.Z.). We thank R. Bock (Max Planck Institute of Molecular Plant Physiology) for generously providing the Nicotiana tabacum cv. Petit Havana seeds.
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A.Z., L.T., Y.Z. and C.L. designed the project. A.Z., L.T., T.Z., M.L., M.S. and Y.Z. performed the experiments. A.Z., Y.F., Y.Z. and C.L. analysed the data. Y.Z. and C.L. wrote the paper.
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Nature Plants thanks Jian-Ren Shen, Mei Li and Toshiharu Shikanai for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Spectroscopy characterization of the wild type (WT), the pbf8 mutant, and pbf8 com, and identification of the pbf8 mutant.
a, Chlorophyll contents in WT, pbf8, and pbf8 com. Chlorophyll a (green) and chlorophyll b (yellow) contents were normalized to fresh weight (FW), with the chlorophyll a/b ratio above each bar. Statistical significance was determined by one-way ANOVA. P-values were adjusted by Tukey’s post-hoc test; **P < 0.01; ns, not significantly different. Data are means ± SD (n = 3 independent biological replicates). b, 77 K chlorophyll a fluorescence emission spectra of thylakoid membranes isolated from WT, pbf8, and pbf8 com upon excitation at 436 nm. Signals were normalized to the PSII emission maximum at 687 nm. c, Chlorophyll a fluorescence induction kinetics in WT, pbf8, and pbf8 com. AL, actinic light; FR, far-red light; SL, saturating light; Fm, maximal fluorescence; \({F}_{{\rm{m}}}^{{\prime} }\), maximal fluorescence in the light-adapted state; Fo, minimal fluorescence; \({F}_{{\rm{o}}}^{{\prime} }\), minimal fluorescence in the light-adapted state; Fv/Fm, maximum efficiency of PSII photochemistry. Data are means ± SD (n = 3 independent biological replicates). d, Chloroplast ultrastructure. (i–iii), Chloroplast ultrastructure of WT, pbf8, and pbf8 com. (iv–vi), Magnified images of the regions indicated by red frames in (i–iii). GT, grana thylakoids; SL, stromal lamellae. Bars, 1 μm (i–iii), 0.2 μm (iv–vi). e, Diagram of the T-DNA insertion in PBF8 (At3g56010). The T-DNA is inserted into the second exon. f, Identification of homozygous pbf8 plants. Genomic DNA was used as a template for PCR analysis with specific primers. LP, left border primer; RP, right border primer; o8474, specific T-DNA primer for GABI-Kat lines. Primer sequences are listed in Supplementary Table 2. g, Reverse-transcription PCR (RT-PCR) analysis of PBF8 transcript levels in WT, pbf8, and pbf8 com plants using specific primers listed in Supplementary Table 2. ACTIN2 was used as a reference transcript. h, Immunoblot analysis of PBF8 protein levels in WT, pbf8, and pbf8 com plants. Equal amounts of total leaf protein (10 μg) extracted from plants were separated by SDS-PAGE and immunodetected using the anti-PBF8 antibody generated in this study. β-Actin was used as a loading control. In b–d, f–h, three independent biological replicates were performed, and a representative replicate is shown.
Extended Data Fig. 2 Phylogenetic analysis of PBF8.
a, Phylogenetic tree of Arabidopsis PBF8 and putative PBF8 orthologs from other plant species generated using MEGA11 software. UniProtKB protein entries and the corresponding species are given for each sequence. The protein encoded by At3g56010 is shown in blue. The phylogenetic tree was reconstructed using the neighbor-joining method with 1,000 bootstrap replicates. Support values as percentages are shown next to the related branches. Bar, 0.2 amino acid substitutions per site. b, A membrane-spanning region is predicted in PBF8. Bioinformatic analysis of the membrane-spanning region of PBF8 was performed using DeepTMHMM (https://dtu.biolib.com/DeepTMHMM). The x-axis shows the amino acid position; the y-axis gives the probability of the presence of a transmembrane segment.
Extended Data Fig. 3 PBF8 acts as an assembly factor for PSI–LHCI rather than as a factor modulating PSI–LHCI stability.
a, Relative transcript levels of chloroplast genes in the wild type (WT) and the pbf8 mutant determined by comparing the average RPKM values obtained by RNA-seq analysis (n = 2 each). The arrows point to plastid PSI subunit genes (PsaA–C, PsaI, and PsaJ) and plastid PSI assembly factor genes (ycf3 and ycf4). RPKM, reads per kilobase per million mapped reads. b, RNA gel blots of plastid PSI subunit genes (PsaA–C, PsaI, and PsaJ). c, RNA gel blots of plastid PSI assembly factor genes (ycf3 and ycf4). In b, c, ethidium bromide–stained 25 S rRNA is shown below each blot as a loading control. d, Association of mRNAs transcribed from chloroplast PSI subunit genes with polysomes in the wild type (WT) and pbf8. Translational efficiency of the plastid PSI subunit mRNAs PsaA/B, PsaC, PsaI, and PsaJ and the plastid PSI assembly factor mRNAs ycf3 and ycf4 was analyzed by polysome-loading assays. Total leaf lysates from the WT and pbf8 were fractioned on 15–55% sucrose gradients. Ten fractions of equal volume from the sucrose density gradients are numbered from left to right. Equal aliquots of extracted RNAs from each fraction were analyzed by northern blotting. Ribosomal RNA distribution in the gradients was revealed by staining the gels with ethidium bromide (EtBr). The arrows at the top indicate the sucrose density gradients (from low to high). Two independent biological replicates were performed, and a representative replicate is shown. e, Photograph of a wild-type plant grown for 3 weeks. Leaf numbers 1–6 represent leaves from the oldest to the youngest. Note that this shows no senescence. Bar, 1 cm. f, Levels of PsaA and PBF8 in the leaves of wild-type plants at different leaf ages. Coomassie brilliant blue (CBB) staining shows equal loading. The absence of senescence symptoms in the WT suggests that the decrease in PBF8 levels in older leaves is not caused by leaf senescence. g, h, Maximum level of photo-oxidizable P700 (ΔAmax; normalized to initial values at time zero, t0). Detached leaves from the WT and pbf8 in the presence of lincomycin and cycloheximide were exposed to 80 (g) or 200 (h) μmol photons m–2 s–1 for up to 24 h. Data are means ± SD (n = 3 independent biological replicates). Statistical significance compared with the WT was based on a two-tailed Student’s t-test; ns, not significantly different. i, j, Immunodetection of specific thylakoid membrane proteins. Detached leaves from the WT and pbf8 grown in the presence of lincomycin and cycloheximide were exposed to 80 (i) or 200 (j) μmol photons m–2 s–1 for up to 24 h. In b–c, e–f, i–j, three independent biological replicates were performed, and a representative replicate is shown.
Extended Data Fig. 4 Characterization of pbf8 PBF8-HA plants.
a, Photographs of plants of the wild type (WT) and pbf8 PBF8-HA grown for 3 weeks. Bars, 1 cm. b, Chlorophyll a fluorescence induction kinetics in the WT and pbf8 PBF8-HA. AL, actinic light; FR, far-red light; SL, saturating light; Fm, maximal fluorescence; \({F}_{{\rm{m}}}^{{\prime} }\), maximal fluorescence in the light-adapted state; Fo, minimal fluorescence; \({F}_{{\rm{o}}}^{{\prime} }\), minimal fluorescence in the light-adapted state; Fv/Fm, maximum efficiency of PSII photochemistry. Data are means ± SD (n = 3 independent biological replicates). c, P700 redox kinetics in the WT and pbf8 PBF8-HA. P700 redox state was investigated by P700 absorbance at 820 nm induced by far-red light (720 nm). ox., P700 oxidation; red., P700 reduction; ΔAmax, maximum photo-oxidizable P700. d, Immunoblot analysis of thylakoid membrane proteins from the leaves of the WT and pbf8 PBF8-HA; equal amounts of total leaf proteins were loaded (10 μg). Thylakoid membrane protein complexes and their diagnostic components are labeled on the right. In a–d, three independent biological replicates were performed, and a representative replicate is shown. e, Chlorophyll contents of leaves from the WT and pbf8 PBF8-HA. Chlorophyll a (green) and chlorophyll b (yellow) contents were normalized to fresh weight (FW). The chlorophyll a/b ratio is given above each bar. Statistical significance compared with the WT was determined by one-way ANOVA. P-values were adjusted by Tukey’s post-hoc test; ns, not significantly different. Data are means ± SD of five biological replicates.
Extended Data Fig. 5 Polypeptide composition of the affinity-purified PBF8-HA preparation.
a, Separation of chlorophyll–protein complexes of thylakoid membranes from the wild type (WT) and pbf8 PBF8-HA#1 by SDG ultracentrifugation. The purified thylakoid membranes were subjected to SDG ultracentrifugation; the resulting fractions were analyzed by immunoblotting using specific antibodies against PsaA, HA, and PBF8. Four chlorophyll–protein complexes were separated by SDG centrifugation in the WT and pbf8 PBF8-HA; left to right: LHCII monomer (LHCII-M), LHCII trimer (LHCII-T), PSII, and PSI–LHCI. b, Separation of chlorophyll–protein complexes of thylakoids from the WT and pbf8 PBF8-HA by BN-PAGE, and immunoblot analysis of chlorophyll–protein complexes separated by 2D BN-PAGE/SDS-PAGE using specific antibodies against PsaA and PBF8. Thylakoids isolated from the WT and pbf8 PBF8-HA#1 were solubilized using 1% (w/v) β-DM and resolved by BN-PAGE. NDH–PSI, NDH–PSI supercomplex; PSII SC, PSII supercomplexes; PSI-LHCI/PSII-D, PSI-LHCI and dimeric PSII; PSII-M, monomeric PSII; CP43 free PSII, monomeric PSII without CP43; LHCII-T, trimeric LHCII. Thylakoids from the WT and pbf8 PBF8-HA contained 10 μg chlorophyll. c, Polypeptide composition of the affinity-purified PBF8-HA preparation separated by SDS-PAGE and visualized by staining with Coomassie brilliant blue. PSI–LHCI, purified mature PSI–LHCI from the WT used as a reference. PBF8-HA (IP), affinity-purified PBF8-HA preparation from pbf8 PBF8-HA#1 plants. Control sample, protein sample used as a negative control that was obtained from the WT following the same procedure used to purify the PBF8-HA preparation from pbf8 PBF8-HA#1 plants. The arrow indicates the band corresponding to PBF8-HA. d, Polypeptide composition of the affinity-purified PBF8-HA preparation, as detected by immunoblotting. PSI–LHCI, purified mature PSI–LHCI from the WT used as a reference. PBF8-HA (IP), affinity-purified PBF8-HA preparation from pbf8 PBF8-HA#1 plants. Control sample, protein sample used as a negative control that was obtained from the WT following the same procedure used to purify the PBF8-HA preparation from pbf8 PBF8-HA#1 plants. e, Immunoblot analysis of the fractions of the affinity-purified PBF8-HA preparation after SDG ultracentrifugation (related to Fig. 4b). The purified PBF8-HA preparation was concentrated and subjected to SDG ultracentrifugation; the gradient fractions were analyzed by immunoblotting using specific antibodies against PSI subunits (PsaG, PsaJ, PsaK, PsaN, PsaO, and Lhca1–4). In a–e, three independent biological replicates were performed, and a representative replicate is shown.
Extended Data Fig. 6 Identification of PSI subunits that interact with PBF8 by bimolecular fluorescence complementation (BiFC) and split-luciferase assays.
a, BiFC assays with the cYFP-PBF8 fusion protein and the nYFP-PBF8 fusion protein showing that PBF8 does not dimerize. b, BiFC assays with the cYFP-PBF8 fusion protein and various nYFP-PSI subunits. The constructs encoding nYFP-tagged PSI subunits and the cYFP-tagged PBF8 were transiently co-transfected in Arabidopsis protoplasts. nYFP was fused to the C termini of each PSI subunit individually. cYFP was fused to the C terminus of PBF8 or the N terminus of PBF8 to assess the interactions between PBF8 and PsaA, PsaB, PsaG, PsaH, PsaJ, PsaK, PsaN, PsaO, and Lhca1–4 or the interactions between PBF8 and PsaC–F and PsaI. The endogenous transit peptide was retained for nucleus-encoded proteins (PBF8, PsaD–H, PsaK, PsaL, PsaN, PsaO, and Lhca1–4), and the Arabidopsis RbcS 1a chloroplast transit peptide was employed for chloroplast-encoded PSI subunits (PsaA–C, PsaI, and PsaJ). Bright field, YFP (yellow), chlorophyll autofluorescence (magenta), and the merged images are shown. In a–b, representative images were selected based on the observation of 150 protoplasts in two independent experiments for each interaction. Scale bars, 10 μm. c, Split-luciferase assays with the Cluc-tagged PBF8 fusion protein and the Nluc-tagged PSI subunit fusion protein. The constructs encoding Cluc-tagged PBF8 and Nluc-tagged PSI subunits were transiently co-expressed from the constitutive CaMV 35 S promoter in the leaves of Nicotiana benthamiana plants; luciferase activity (LUC) was measured 2 d later. Nluc was fused to the C termini of each PSI subunit. Cluc was fused to the C terminus of PBF8 or the N terminus of PBF8 to examine the interactions of PBF8 with PsaA, PsaB, PsaG, PsaH, PsaJ, PsaK, PsaN, PsaO, and Lhca1–4 or the interactions of PBF8 with PsaC–F and PsaI. The endogenous transit peptide was retained for nucleus-encoded proteins (PBF8, PsaD–H, PsaK, PsaL, PsaN, PsaO, and Lhca1–4), and the Arabidopsis RbcS 1a chloroplast transit peptide was added for chloroplast-encoded PSI subunits (PsaA–C, PsaI, and PsaJ). In addition, the unrelated Nluc-tagged protein MPH1 (MAINTENANCE OF PSII UNDER HIGH LIGHT 1) and Cluc-tagged VTE6 (VITAMIN E DEFICIENT6) were used as negative controls. Data were analyzed via one-way ANOVA. P-values were adjusted by Tukey’s post-hoc test; ****P < 0.0001; ns, not significantly different. Data are means ± SD of eight biological replicates.
Extended Data Fig. 7 Characterization of psaf PSAF-HA plants.
a, Photographs of wild type (WT) and psaf PSAF-HA plants after 3 weeks of growth. Bars, 1 cm. b, Chlorophyll a fluorescence induction kinetics in WT and PSAF-HA plants. AL, actinic light; FR, far-red light; SL, saturating light; Fm, maximal fluorescence; \({F}_{{\rm{m}}}^{{\prime} }\), maximal fluorescence in the light-adapted state; Fo, minimal fluorescence; \({F}_{{\rm{o}}}^{{\prime} }\), minimal fluorescence in the light-adapted state; Fv/Fm, maximum efficiency of PSII photochemistry. Data are means ± SD (n = 3 independent biological replicates). c, P700 redox kinetics in WT and psaf PSAF-HA plants. P700 redox state was investigated based on P700 absorbance at 820 nm induced by far-red light (720 nm). AL, actinic light (80 μmol m–2 s–1); FR, far-red light; SL, saturating light pulse; ox., P700 oxidation; red., P700 reduction; ΔAmax, maximum photo-oxidizable P700. d, Immunoblot analysis of thylakoid membrane proteins from the leaves of WT and psaf PSAF-HA plants; equal amounts of total leaf proteins were loaded (10 μg). Thylakoid membrane protein complexes and their diagnostic components are labeled on the right. In a–d, three independent biological replicates were performed, and a representative replicate is shown. e, Chlorophyll contents of leaves of WT and psaf PSAF-HA plants. Chlorophyll a (green) and chlorophyll b (yellow) contents were normalized to fresh weight (FW). Chlorophyll a/b ratio is given above each bar. Statistical significance compared to WT was determined by one-way ANOVA. P-values were adjusted by Tukey’s post-hoc test; ns, not significantly different. Data are means ± SD. n = 5 independent biological replicates.
Extended Data Fig. 8 A stable PSI assembly intermediate (PSI*) can be detected in the wild type (WT) but not in the pbf8 mutant.
a, Immunoblot detection of PSI* in thylakoids separated by BN-PAGE from fully expanded leaves of WT and pbf8 plants grown for 3 weeks. Thylakoids equivalent to 10 μg chlorophyll were solubilized by 1% (w/v) β-DM and resolved by BN-PAGE. The gel on the left was transferred to PVDF membranes, and the blots were probed with antibodies against PsaA, PsaD, and Lhca3. PSI* at ~500 kDa45 can be detected in WT but not in pbf8. b, Coomassie brilliant blue (CBB) staining of PSI* in thylakoids separated by BN-PAGE from the first primary leaves of 12-d-old WT and pbf8 seedlings. Thylakoids equivalent to 15 μg chlorophyll were solubilized by 1% (w/v) β-DM and resolved by BN-PAGE. The gel on the left was stained by CBB; a faint band representing PSI*45 can be seen in the WT but not in pbf8. c, d, PSI* is enriched in young leaves. PSI* was detected from mature and young leaf tissues of Arabidopsis (c) and tobacco (N. tabacum) (d). The mature and young leaf tissues were harvested according to Wittenberg et al.45. Thylakoids equivalent to 10 μg chlorophyll were solubilized by 1% (w/v) β-DM, resolved by BN-PAGE, and stained by CBB. In a–d, three independent biological replicates were performed, and a representative replicate is shown. e, f, Mass spectrometry of PSI* from young leaf tissues of Arabidopsis (e) and tobacco (N. tabacum) (f). The slice corresponding to PSI* was excised from the CBB stained 1D-Blue Native gel and subjected to mass spectrometry using the label-free quantification method. Average abundances of each identified protein from three biological replicates are plotted by the intensity-based absolute quantification (iBAQ) value (y axis) and peptide peak intensity (x axis). PsaA–E, PsaH, and PsaL were identified in PSI* subunits while PsaI was not reliably detected by MS due to its small size.
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Zhang, A., Tian, L., Zhu, T. et al. Uncovering the photosystem I assembly pathway in land plants. Nat. Plants 10, 645–660 (2024). https://doi.org/10.1038/s41477-024-01658-3
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DOI: https://doi.org/10.1038/s41477-024-01658-3
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