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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