Functional Analysis of Protein O-Glycosylation in Regulating Nuclear Growth Repressor DELLA and Plant Development in Arabidopsis

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Project Summary Plant development requires strict coordination among complex internal signaling networks to enhance adaptation to changing environments. The conserved transcription regulators DELLA proteins play a central role in this process via direct protein-protein interactions with key transcription factors. Recent studies using genetic and physiological analyses together with chemical biology methods indicate that DELLA's binding affinity to interacting proteins are oppositely regulated by two types of O-linked glycosylation on specific Ser/Thr residues: O-linked N-acetylglucosamine (O-GlcNAc) modification, and O-fucosylation (O-Fuc). These two distinct O-glycosyl modifications on DELLA are catalyzed by two paralogs in Arabidopsis: SECRET AGENT (SEC), an O-GlcNAc transferase (OGT) that reduces DELLA activity, and SPINDLY (SPY), a novel protein O-fucosyltransferase (POFUT) that enhances DELLA activity. These studies uncovered direct roles of OGT (SEC) and POFUT (SPY) in fine-tuning plant development by modulating DELLA interactions with key regulators in multiple signaling pathways. OGT-mediated protein O-GlcNAcylation has been studied extensively in animals, and is known to play a key role in regulating a plethora of intracellular signaling events in response to nutrient status. In contrast, the physiological functions of OGT in plants are largely unknown. Moreover, SPY is the first POFUT identified for O-fucosylation of nuclear proteins, uncovering a novel mechanism for transcriptional regulation. The dynamic interplay between O-GlcNAc/O-Fuc modifications in regulating the nuclear growth repressor DELLA activity may provide a new paradigm in linking metabolic status to gene expression and cell growth in response to internal and external cues. This hypothesis will be tested using targeted metabolomics and chemical biology approaches (Specific Aim 1). In addition, structure analysis of SPY and SPY/substrate complexes will identify key residues that contribute to the substrate and enzymatic specificity of SPY. The interplay between O-GlcNAc and O-Fuc is likely to modulate diverse cellular activities beyond DELLA function. The pleiotropic phenotypes of spy and sec mutants, recently published Arabidopsis O-GlcNAc proteome and preliminary results in this lab suggest that many Arabidopsis proteins involved in transcriptional control are common targets of SEC and SPY. The global roles of SPY and SEC in plant development will be elucidated by genetic studies using inducible knockdown/overexpression SPY and SEC lines in conjunction with genomic and proteomic approaches (Specific Aim 2). This study will have broader implications. SPY orthologs, although absent in animals, are highly conserved in diverse organisms, including plants, bacteria, and parasitic protists, suggesting that intracellular O-fucosylation regulates a wide range of biological processes in diverse organisms.