Project description:SPY is an O-linked N-acetylglucosamine transferase involved in many signaling and developmental processes in Arabidopsis. SPY also has a role in root development. In the spy mutant, a middle cortex is produced prematurely. To understand how SPY regulates root cortex proliferation, we sought to identify the genes affected by the spy mutation in the root.
Project description:SPY is an O-linked N-acetylglucosamine transferase involved in many signaling and developmental processes in Arabidopsis. SPY also has a role in root development. In the spy mutant, a middle cortex is produced prematurely. To understand how SPY regulates root cortex proliferation, we sought to identify the genes affected by the spy mutation in the root. The transciptomes from the root of one-week-old seedlings, Col or spy-3 mutant, were compared. The seedlings were grown on a nylon membrane placed on 1X MS agar medium and the plates are positionally vertically in a Pecival growth chamber with 16 hr light and 8 hr dark. Three biological replicates were performed.
Project description:Both wild-type and SPY-3 OX A. thaliana plants (Arabidopsis thaliana L. ecotype-Columbia) were grown in GM plates for 3 weeks under a 16 h light/8 h dark regimen (40 M-1 10 umol photons/m2/s) at 22M-0C. Dehydration treatment: The 3-week-old plants were grown for 2 h without watering.
Project description:The recent discovery of SPY-catalyzed protein O-fucosylation reveals a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates important roles of SPY in diverse developmental and physiological processes, however, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrate that SPY mediates sugar-dependent growth. We further identify hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in proteomic analyses are undetectable or decreased in the spy mutants and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, hormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked-N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and hormonal responses. Our study identifies numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.
Project description:The recent discovery of SPY-catalyzed protein O-fucosylation reveals a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates important roles of SPY in diverse developmental and physiological processes, however, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrate that SPY mediates sugar-dependent growth. We further identify hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in proteomic analyses are undetectable or decreased in the spy mutants and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, hormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked-N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and hormonal responses. Our study identifies numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.