The maize CMS-C fertility restore gene ZmRf4b encodes a SPL transcription factor that represses ZmMAP1D-mediated co-translational turnover of mitochondrial toxic ATP6C protein.

The promising CMS-C/Rf system has not been applied on a large scale in maize hybrid breeding due to the unclear restoration mechanism for the primary restorer ZmRf4b. In CMS-C maize, the mitochondrial chimeric gene atp6c encodes a cytotoxic variant of ATP6 that over-accumulates and disrupts the assembly of mitochondrial complex V, leading to male sterility. Here, we report the map-based cloning and functional characterization of ZmRf4b, which encodes a SQUAMOSA promoter-binding protein-like (SPL) transcription factor. Through DAP-seq and yeast two-hybrid screening, we identified ZmMAP1D, a mitochondrion-targeted methionine aminopeptidase, as a direct downstream target of ZmRf4b. ZmRf4b functions as a transcriptional repressor that directly binds the ZmMAP1D promoter; in the restorer line, the relatively low expression level of ZmRf4b weakens its repression of ZmMAP1D, permitting moderate ZmMAP1D accumulation. ZmMAP1D is then imported to mitochondria, where it co-translationally removes the N-terminal methionine of ATP6C, thereby targeting excess ATP6C for degradation. The reduction of ATP6C to normal levels restores mitochondrial complex V assembly, alleviates reactive oxygen species accumulation and tapetal programmed cell death, and ultimately restores male fertility. This study reveals a previously unrecognized fertility restoration mechanism operating through co-translational protein turnover-rather than transcriptional or translational suppression of the CMS gene-providing new insights into nucleus-mitochondria communication and mitochondrial protein homeostasis.