氧连氮乙酰葡萄糖胺(O-GlcNAc)修饰是一种重要的动态可逆的蛋白质翻译后修饰。SPINDLY(SPY)在赤霉素、细胞分裂素等激素信号转导以及生长发育的各个阶段发挥着重要作用。由于结构的相似性，长期以来SPY被认为是O-GlcNAc转移酶(OGT)，但最近被鉴定为氧连单岩藻糖转移酶。SPY的核质定位对其功能具有重要影响，但具体调节机制不详。本实验室鉴定到SPY被O-GlcNAc修饰，暗示植物中可能存在类似动物O-GlcNAc修饰调控OGT核质定位的机制。本项目希望利用免疫沉淀结合电子转移解离质谱鉴定SPY蛋白中O-GlcNAc修饰的精确位置，寻找催化该修饰的OGT酶，并构建修饰位点突变的转基因植株，观察该修饰对SPY的亚细胞定位以及生物功能的影响，揭示O-GlcNAc修饰对氧连岩藻糖修饰的调节机制，为理解O-GlcNAc修饰对植物生长发育的调控机制以及氧连糖基化之间交叉作用提供新的认识。

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a widespread dynamic and reversible posttranslational modification, which plays important roles in multiple developmental and growth processes. SPINDLY acts as a repressor in gibberellin response pathway but a positive regulator in cytokinin response, and takes parts in almost all development and growth processes, such as root, shoot, leaf growth, embryo development and abiotic stress responses. Because of high similarity with animal O-GlcNAc transferase (OGT), SPY has long been hypothesized as an OGT. However, SPY was recently report as a novel O-fucosyltransferase, which attaches monofucose to specific serine and threonine residues. SPY is localized in both the nucleus and the cytosol, and this nucleus/cytosol localization of SPY affects its bio-function. However, the regulation of SPY sub-cellular localization is still unclear. We identified an O-GlcNAc modification on SPY protein in our previous study, which supports a hypothesis that a similar post-translational regulation on OGT exists in plant as that in mammal, where self-O-GlcNAc modification regulates subcellular localization and catalytic activity of OGT. This project will identify the exact O-GlcNAc modified site by immuno-precipitation following by Electron Transfer Dissociation (ETD) mass spectrometry analysis, find whether plant OGT, SEC, catalyzes this modification, and analyze the effect on SPY subcellular localization and functional activity by site-directed mutagenesis. This work will elucidate the mechanism by which O-GlcNAc modification regulate plant O-fucosyltransferase, SPY, and the crosstalk between two O-linked glycan modifications in plant development and growth regulation.