During PSII assembly and repair, PSII intermediate complexes are prone to photooxidative damage, requiring mechanisms to minimize this damage. Here, we report the functional characterization of RBD1, a PSII assembly factor that interacts with PSII intermediate complexes to ensure their functional assembly and repair. We propose that the redox activity of RBD1 participates together with the cytochrome b559 to protect PSII from photooxidation. This work not only improves our understanding of cellular protection mechanisms for the vital PSII complex but also informs genetic engineering strategies for protection of PSII repair to increase agricultural productivity.
Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.
Figure 1: 2pac is a nonphotosynthetic mutant with impaired assembly of PSII monomers and abnormal chloroplast architecture. (A) Growth phenotype and PSII efficiency (Fv/Fm) of wild-type (WT) and mutant spotted cells grown mixotrophically (TAP) under low light (5 μmol photons⋅m−2⋅s−1) or photoautotrophically (HS) under normal light (80 μmol photons⋅m−2⋅s−1) conditions. (B) Transmission electron microscopy analyses of WT and mutant cells grown heterotrophically. (C) BN-PAGE analyses of WT and 2pac solubilized thylakoid membrane (TM) protein complexes treated with 2 different detergents, α-DM and β-DM. (D) The 2D BN/SDS/PAGE and immunoblot analyses from WT and 2pac solubilized TM protein complexes against PSII subunits D2 and CP43, and RBD1. Immunodetection against D2 and CP43 varies. In WT samples, films were recorded after 5-s exposure compared with 1 min in 2pac samples. Lanes are labeled as follows: PSI, photosystem I; PSII-D, PSII dimer; PSII-M, PSII monomer; PSII-SC, PSII–LHCII supercomplexes; uCP43, unassembled CP43. (E) Immunoblots analyses against D2, RBD1, and acetylated tubulin proteins from greening experiment with the chlL mutant, as well as Coomassie-stained SDS/PAGE. Whole-cell samples were collected and subjected to denaturing SDS/PAGE and immunoblot analysis after 0, 6, and 24 h following the shift to normal growth light conditions. One hundred percent loading corresponds to about 1 × 106 cells.