Phosphoproteomic Analysis of Cytoplasmic Male Sterility in Sugar Beet (Beta vulgaris L.)

Sugar beet cytoplasmic male sterility (CMS) underpins hybrid seed production, yet the contribution of protein phosphorylation remains unexplored. Using 4D label-free phosphoproteomics on anthers of the Owen-type CMS line MS327 and its maintainer OT328 at the 2–3 mm bud stage, we identified 1241 phosphoproteins, 1975 phosphopeptides and 3357 phosphosites, of which 469 proteins and 848 sites were quantifiable. Twenty-one phosphopeptides exhibited significant differential abundance (fold change > 2.0, p < 0.05; 3 up, 18 down), while 69 and 141 peptides were detected exclusively in the maintainer and the sterile line, respectively. Pathway enrichment implicated the spliceosome and fatty acid elongation pathways in male sterility. Integrating KEGG, GO and predicted protein–protein interaction networks analyses, we identified nuclear cap-binding protein subunit 2 (NCBP2), the RRM-containing splicing factor SR45a and 3-ketoacyl-CoA synthase as candidate phosphoproteins associated with male sterility. All six core proteins were deeply conserved across the examined species. Although transcript levels of the six corresponding genes did not differ significantly between the two lines in either leaf or anthers, anther phosphorylation levels did, pointing to post-translational rather than transcriptional control. This first phosphoproteomic survey of sugar beet CMS provides a valuable readout of post-translational reprogramming and offers promising candidate targets for future functional validation.