Numerous proteins require a metallic cofactor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. We focused on Arabidopsis thaliana NFU1, NFU2 and NFU3, which participate in the late maturation steps. According to the strong photosynthesis defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream from HCF101 for the maturation of [Fe₄S₄]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe₂S₂] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in nfu2 roots and the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific of [Fe₄S₄] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe₂S₂] and [Fe₄S₄] proteins either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.