Dissect O-fucosylation- and O-GlcNAcylation-mediated modulation of EIN2 processing and activities in mediating ethylene signaling and sugar responses

Intracellular protein O-linked glycosylations such as O-linked β-N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) and O-fucose modification (Ofucosylation) are emerging as ubiquitous and essential PTMs in plants for modulating growth and development, signal transduction, as well as diverse cellular activities. Forty years of research on O-GlcNAcylation in animals has established this O-glycosylation pathway as a central nutrient sensing mechanism that modifies thousands of intracellular proteins and governs almost all aspects of cellular processes examined. Recent reports from us and other labs have revealed the ubiquitous presence of OGlcNAc and O-fucose modifications on intracellular proteins in plants. However, out of the hundreds of O-GlcNAc and O-fucose modified proteins identified, only a handful
have been characterized so far. Much work needs to be done to fully understand the functional spectrum and regulatory mechanisms of O-GlcNAc and O-fucose modifications in plants. Importantly, previous work on O-GlcNAc and O-fucose
modifications in plants has paid little attention to the potential nutrient sensing function of O-glycosylations. There has not been enough effort among the plant scientists to dissect the molecular mechanism underlying O-GlcNAcylation- and Ofucosylation-mediated regulation of protein functions.
Our proteomic analysis has identified ETHYLENE-INSENSITIVE 2 (EIN2), a master regulator of plant development, ethylene signaling, and sugar signaling, as an OGlcNAcylated and O-fucosylated protein. Ethylene signaling is tightly linked to sugar responses and previous studies in Arabidopsis support a general antagonism between ethylene signaling and the hexokinase-mediated sugar signaling pathway. Independent of ethylene signaling, EIN2 also mediates the newly discovered sugar-TARGET OF RAPAMYCIN (TOR)-EIN2 signaling axis to regulate plant growth. This makes EIN2 an interesting O-GlcNAc and O-fucose modified protein for in-depth analysis. Our preliminary analysis shows that O-fucosylation plays a positive role in ethylene signaling, suggesting that the O-glycosylation pathways, like other major energy sensing pathways, converge on the regulation of EIN2 and ethylene signaling to modulate important aspects of plant growth and sugar responses. We propose to (1) dissect Ofucosylation- and O-GlcNAcylation-mediated modulation of EIN2 processing and activities in mediating ethylene signaling and (2) explore potential roles of Ofucosylation and O-GlcNAcylation in modulating the TOR-EIN2 sugar signaling axis. Further, we also plan to employ proteomic and yeast three-hybrid approaches to (3) identify potential “reader” proteins that recognize O-fucosylated and O-GlcNAcylated
EIN2 and “eraser” proteins that remove O-fucose and O-GlcNAc modifications. Identifying these proteins is vital for a mechanistic understanding of the biochemical effects of protein O-glycosylations and their dynamic cycling.