SlSEC1- and SlSPY-mediated O-glycosylation stabilizes the transcription factor SlNOR to promote tomato fruit ripening

O-glycosylation is a critical post-translational modification (PTM) that regulates protein function, yet its role in regulating plant transcription factors (TFs) remains poorly understood. Here, we report that O-glycosylation regulates NON-RIPENING (SlNOR), the master NAC TF controlling tomato (Solanum lycopersicum) fruit ripening. Using proteomic and interaction assays, we identified SlNOR as a substrate of 2 conserved nucleocytoplasmic O-glycosyltransferases: the O-GlcNAc transferase SlSEC1 and the O-fucosyltransferase SlSPY. We mapped 3 O-glycosylation sites (Thr93, Thr134, and Ser165) within the NAC domain of SlNOR. Biochemical assays suggested that O-glycosylation protects SlNOR from protein degradation. Accordingly, simultaneous mutagenesis of the 3 O-glycosylation sites reduced SlNOR protein stability and nuclear accumulation. Functionally, O-glycosylated SlNOR exhibited enhanced transcriptional activation of the ethylene biosynthesis genes (SlACS2 and SlACO1), which was corroborated by its increased DNA-binding affinity in electrophoretic mobility shift assays. Genetic evidence from CRISPR/Cas9-generated mutants revealed that loss of SlSEC1 or SlSPY reduces ethylene production and delays ripening, while the Slsec1-1 Slspy double mutant displayed a cooperative ripening delay and severe growth defects. Collectively, our findings uncover a key PTM-based regulatory mechanism in which SlSEC1/SlSPY-mediated O-glycosylation enhances SlNOR stability and transcriptional activity, thereby coupling a master transcriptional regulator to ethylene biosynthesis for the control of fruit ripening.