Quantitative glycoproteomics identifies target N-glycoproteins of the Golgi-class I α-mannosidase SlMNSI1 that is important for tomato fruit development.

Protein N-glycosylation is a fundamental post-translational modification, yet its role in regulating fruit development remains poorly understood. Here, we investigated the function of the class I α-mannosidase SlMNSI1 in tomato (Solanum lycopersicum) using chemical genetics, CRISPR-Cas9-mediated gene editing, quantitative site-specific glycoproteomics, and genetic complementation analyses. Inhibition of SlMNSI by kifunensine or SlMNSI1 knockout caused severe pleiotropic defects and impaired fruit development, leading to the accumulation of Man9GlcNAc2 or Man8GlcNAc2, respectively, on glycoprotein substrates. Analysis of the tomato fruit site-specific N-glycoproteome identified 3091 intact N-glycopeptides containing 873 N-glycosites and 158 N-glycans within 573 N-glycoproteins. Among these, 97 N-glycoproteins containing 127 N-glycosites were identified as candidate targets of SlMNSI1. Strikingly, SlMNSI1 itself is N-glycosylated at Asn288 and Asn334, and N-glycosylation is essential for Golgi localization and protein stability. Genetic complementation assays verified this in vivo: overexpression of wild-type SlMNSI1 fully restored normal fruit development in the mutant, whereas overexpression of a non-glycosylatable SlMNSI1 variant only partially complemented these phenotypes. Collectively, our findings reveal the critical role of SlMNSI1 and mannosidic N-glycans in fruit development.