Combined transcriptomics and metabolomics analysis reveals the mechanism behind the pollen abortion in early stage among male sterile lines of alfalfa

This study investigates early-stage anther development in cytoplasmic male sterile (CMS) alfalfa lines (MSJN1A) compared to their isotypic maintainer line (MSJN1B). Histological analyses revealed abnormal degradation of tapetal cells post-meiosis in the CMS line. Notably, during the early mononuclear stage, the central vacuole in the microspores was absent, leading to evident pollen abortion. These findings suggest that pollen abortion in the CMS line is associated with the delayed disintegration of the tapetum and structural anomalies in microspore vacuoles. Non-targeted metabolomic sequencing was employed to analyze the early anther metabolites of alfalfa, identifying four hundred and one and four hundred and five metabolites in the late tetrad and early mononuclear stages, respectively. Among these, thirty-nine metabolites were consistently up-regulated, while eighty-eight were down-regulated. Differential analysis revealed forty-five and thirty-seven unique metabolites in each respective stage. These metabolites primarily featured in pathways related to energy, phenylpropane, sucrose and starch, and fatty acid metabolism. Integrated analysis demonstrated that differentially expressed genes (DEGs) and differential metabolites (DMs) were co-enriched in these pathways. Additionally, quantitative real-time PCR and physiological index analysis confirmed the down-regulation of key genes during anther development, illustrating that changes in gene regulation upstream could significantly impact downstream metabolite levels, ultimately influencing pollen fertility.