Identification of the F’ composition by LC-MS/MS.

C1-FDX (Complex I-ferredoxin) has been defined as a component of CI in a ferredoxin bridge in Arabidopsis mitochondria. However, its full function remains to be addressed. We created two c1-fdx mutants in Arabidopsis using the CRISPR-Cas9 methodology. The mutants show delayed seed germination. Over-expression of C1-FDX rescues the phenotype. Molecular analyses showed that loss of the C1-FDX function decreases the abundance and activity of both CI and subcomplexes of CV. In contrast, the over-expression of C1-FDX-GFP enhances the CI* (a sub-complex of CI) and CV assembly. Immunodetection reveals that the stoichiometric ratio of the α:β subunits in the F1 module of CV is altered in the c1-fdx mutant. In the complemented mutants, C1-FDX-GFP was found to be associated with the F’ and α/β sub-complexes of CV. Protein interaction assays showed that C1-FDX could interact with the β, γ, δ, and ε subunits of the F1 module, indicating that C1-FDX, a structural component of CI, also functions as an assembly factor in the assembly of F’ and α/β sub-complexes of CV. These results reveal a new role of C1-FDX in the CI and CV assembly and seed germination in Arabidopsis.

C1-FDX（复合物I-铁氧还蛋白，Complex I-ferredoxin）最初被定义为拟南芥线粒体中铁氧还蛋白桥联结构中复合物CI（Complex I）的组成成分。然而，其完整的生物学功能仍有待阐明。本研究借助CRISPR-Cas9技术构建了两株拟南芥c1-fdx突变体，该突变体呈现种子萌发延迟的表型。过表达C1-FDX可挽救该突变表型。分子生物学分析结果显示，C1-FDX功能缺失会降低复合物CI以及复合物CV（Complex V）各亚复合物的丰度与活性。与之相反，过表达C1-FDX-GFP则可促进CI*（复合物CI的一种亚复合物）及复合物CV的组装。免疫检测结果表明，c1-fdx突变体中复合物CV的F1结构域内α与β亚基的化学计量比发生改变。在互补突变体中，C1-FDX-GFP被检测到可与复合物CV的F’亚复合物及α/β亚复合物相结合。蛋白质相互作用实验结果显示，C1-FDX可与F1结构域的β、γ、δ及ε亚基发生相互作用，这表明作为复合物CI结构组成成分的C1-FDX，同时还可作为组装因子参与复合物CV的F’亚复合物与α/β亚复合物的组装过程。上述研究结果揭示了C1-FDX在拟南芥复合物CI与CV组装以及种子萌发过程中的全新生物学功能。