The Role of N-Glycosylation in Maintaining Self-Incompatibility Stability of Apple S-RNase

The plant self-incompatibility determinant S-RNase is a classical pistil-specific glycoprotein, yet it remained unclear whether the glycosylation modification of S-RNase is involved in inhibiting the elongation of pollen tubes within the style during self-pollination. In this study, we systematically characterized the N‑glycosylation profile of apple (Malus domestica) S-RNase in vivo using high-resolution mass spectrometry. Pollen tube growth inhibition assays demonstrated that glycosylated S-RNase exhibited a six-fold lower concentration threshold for suppressing pollen tube elongation compared to its non-glycosylated counterpart. Simulated pollen-tube microenvironment assays revealed that glycosylated S-RNase retained markedly higher catalytic activity than non-glycosylated S-RNase under acidic conditions typical of the pollen tube cytoplasm. Destruction of the sugar chains at key N-glycosylation sites of S₂- and S₅-RNases led to approximately 70 % loss of enzymatic activity, approaching the level of non-glycosylated S-RNase. Proteomic interaction analysis indicated that glycosylated S-RNase displayed a more extensive protein interaction profile than the non-glycosylated form. Glycosylated S-RNase tended to form complexes with ribosomal proteins, potentially enhancing its RNA-cleaving activity, whereas non-glycosylated S-RNase was likely subjected to ubiquitin-proteasome-mediated degradation in vivo. This study revealed the pivotal regulatory role of S-RNase N-glycosylation in the gametophytic self-incompatibility (GSI) response, established a mechanistic framework for glycosylation-mediated regulation in Rosaceae GSI systems, and provided a theoretical basis for manipulating pollination compatibility through glycoengineering.