Mitochondrial Genome Comparison and Functional Analysis of atp8 Underlying Cytoplasmic Male Sterility in Medicago sativa L.

Cytoplasmic male sterility (CMS) represents a crucial biological tool for the effective utilization of heterosis in plant breeding. Despite its importance, the molecular mechanisms underlying cytoplasmic male sterility in M. sativa have not yet been fully elucidated. In this study, the mitochondrial genomes of the cytoplasmic male-sterile line MS-JN1A and the maintainer line MS-JN1B of M. sativa were assembled and sequenced. Comparative analysis of the mitochondrial genomes revealed that the mitochondrial genome of the sterile line has undergone pronounced structural rearrangements. Moreover, the differentially expressed mitochondrial gene atp8, together with its associated open reading frames (ORFs), was identified as a key candidate underlying CMS. In the anthers of MS-JN1A, ATP levels and reactive oxygen species (ROS) accumulation were concomitantly elevated, indicating a disruption of mitochondrial energy-redox homeostasis. Further functional analyses demonstrated that anther-specific overexpression of atp8 directly induced premature programmed cell death (PCD) in the tapetum, accompanied by extensive transcriptional reprogramming of pathways associated with energy metabolism and ROS responses.. Collectively, our findings propose that atp8-mediated disruption of mitochondrial energy-ROS homeostasis represents a central molecular mechanism driving cytoplasmic male sterility in M. sativa, and they provide new insights into mitochondrial regulation of male fertility in plants. Overall design: RNA-seq of Medicago sativa L. in Wild plant and Overexpression atp8 group