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 SIMNSI1 ...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 SIMNSI1 in tomato(Solanum lycopersicum)using chemical genetics,CRISPR-Cas9-mediated gene editing,quantitative site-specific glycoproteomics,and genetic complementation analyses.Inhibition of SIMNSI by kifunensine or SIMNSI1 knockout caused severe pleiotropic defects and impaired fruit development,leading to the accumulation of Man 9 GlcNAc 2 or Man 8 GlcNAc 2,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 SIMNSI1.Strikingly,SIMNSI1 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 SIMNSI1 fully restored normal fruit development in the mutant,whereas overexpression of a non-glycosylatable SIMNSI1 variant only partially complemented these phenotypes.Collectively,our findings reveal the critical role of SIMNSI1 and mannosidic N-glycans in fruit development.展开更多
基金supported by the National Key Research and Development Program of China(2022YFD2100100)the Natural Science Foundation of Zhejiang Province(LY23B020008)+1 种基金the 111 Project(B17039)the National Natural Science Foundation of China(22336004).
文摘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 SIMNSI1 in tomato(Solanum lycopersicum)using chemical genetics,CRISPR-Cas9-mediated gene editing,quantitative site-specific glycoproteomics,and genetic complementation analyses.Inhibition of SIMNSI by kifunensine or SIMNSI1 knockout caused severe pleiotropic defects and impaired fruit development,leading to the accumulation of Man 9 GlcNAc 2 or Man 8 GlcNAc 2,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 SIMNSI1.Strikingly,SIMNSI1 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 SIMNSI1 fully restored normal fruit development in the mutant,whereas overexpression of a non-glycosylatable SIMNSI1 variant only partially complemented these phenotypes.Collectively,our findings reveal the critical role of SIMNSI1 and mannosidic N-glycans in fruit development.
