The sec mutants show no dramatic phenotype, but the double loss-of-function spy sec mutants are embryo lethal 9.

The spy mutant was identified as a gibberellin (GA) response mutant with phenotypes of enhanced seed germination, early flowering, increased stem elongation, and hyposensitivity to the stress hormone abscisic acid (ABA) 7, 8. The Arabidopsis genome encodes two OGT homologs: SPINDLY (SPY) and SECRET AGENT (SEC). As a sensor of primary metabolic status, O-GlcNAcylation plays key roles in cellular homeostasis and responses to nutritional and stress factors 1, 2, 4, 5, whereas dysregulation of O-GlcNAcylation has been implicated in many diseases including cancer, diabetes, cardiovascular and neurodegenerative diseases 5, 6. Extensive studies in animals have shown that thousands of proteins involved in diverse biological processes are modified on serine and threonine residues by O-GlcNAcylation, which is catalyzed by O-GlcNAc transferase (OGT) using UDP-GlcNAc as donor substrate 1, 3. Posttranslational modification (PTM) of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an important regulatory PTM that modulates protein activities and thereby controls cellular functions according to nutrient and energy status 1, 2.

Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing. Proteomic analyses show AtACINUS’s O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. O-GlcNAc modification plays important roles in metabolic regulation of cellular status.

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