Integrated phenotypic, physiological, and comparative transcriptome analysis reveals the regulatory network of cytoplasmic male sterility in onion

Onion ( Allium cepa L.) is a globally important pungent vegetable. The application of male sterile lines has greatly improved the efficiency and purity of hybrid seed production. However, the molecular mechanisms underlying onion CMS remain unclear. In this study, an integrated approach combining phenotypic observation, physiological assay, and transcriptome analysis was employed to compare the differences in anther development between three pairs of onion CMS lines and their corresponding maintainer lines. Pollen staining results revealed that the pollen grains of CMS lines exhibited typical abortive phenotypes, including shrunken morphology, uneven orange-yellow staining and partial loss of intracellular contents. Physiological assays showed that anthers of CMS lines had significantly reduced soluble sugar content and lower activities of POD, CAT and SOD, while accumulating excessive ROS and MDA, thereby causing oxidative damage to pollen development. Comparative transcriptome analysis identified 2295 shared DEGs, and functional enrichment analysis revealed that these shared DEGs were mainly enriched in pathways related to pollen wall formation, carbohydrate metabolism, sugar transport, ROS metabolism, and plant hormone signal transduction. The expression patterns of some key DEGs were further validated qRT-PCR analysis. Meanwhile, the CMS-related mitochondrial gene orf725 was found to be abnormally highly expressed in the anther of CMS lines. Based on these findings and previous reports, we propose a potential regulatory network mediated by mitochondria-to-nucleus retrograde signaling to explain the molecular basis of male sterility in onion. This study provides novel insights into the molecular mechanism of S-type CMS and offers a valuable theoretical foundation for hybrid breeding in onion.