Mitochondrial C1-FDX Depletion Perturbs ROS Homeostasis and Iron Metabolism in Arabidopsis

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Mitochondrial complex I (CI) is a major source of reactive oxygen species (ROS) in plant cells, and iron–sulfur (Fe-S) cluster proteins are critical for maintaining CI structure and electron transport. C1-FDX, a mitochondrion-localized ferredoxin containing a [2Fe-2S] cluster, acts as a structural component and assembly factor of mitochondrial CI and complex V (CV) in Arabidopsis. In this study, we further characterized the physiological and molecular functions of C1-FDX in regulating ROS production, NADH/NAD+ homeostasis, and iron metabolism. Histochemical staining demonstrated that loss of function of C1-FDX in Arabidopsis c1-fdx mutants accumulated excessive superoxide anions (O2•−) but exhibited reduced hydrogen peroxide (H2O2) relative to the wild type (WT). Biochemical assays further confirmed that there was an elevated NADH/NAD+ ratio in mutant seedlings, which facilitated O2•− overproduction at mitochondrial CI. Loss of C1-FDX also led to Fe3+ accumulation in mutant tissues. Immunoblot and qRT-PCR results showed that the abundance of MnSOD markedly increased, while other antioxidant proteins, including FeSOD, CAT1, and GR, were downregulated in c1-fdx mutants of Arabidopsis. The enhanced MnSOD activity accelerated the conversion of O2•− into H2O2, and the subsequently accumulated Fe3+ may be derived from the Fenton reaction, which consumes H2O2, ultimately resulting in decreased H2O2 levels. Additionally, deficiency of C1-FDX disturbed the abundance of mitochondrial chaperones, proteases, and alternative respiratory pathway proteins. Collectively, our findings demonstrate that C1-FDX participates in the regulation of mitochondrial ROS balance and iron homeostasis and suggest there is a novel regulatory link between ferredoxin, the electron transport chain, and the Fenton reaction in plant mitochondria.