Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

翻译后修饰在精细调节真核生物钟系统中起着至关重要的作用。在植物中，O-糖基化对昼夜节律的影响和潜在的机制仍然是未知的。所述Ô -fucosyltransferase SPINDLY（SPY）和ø -GlcNAc转移SECRET AGENT（SEC）是两个突出ö -glycosylation在高等植物中的酶，在植物生长和发育两者重叠的和独特的功能。不同于O- GlcNAc在调节动物昼夜节律中的关键作用，这里我们报道核定位的SPY而不是SEC专门调节拟南芥的步伐。昼夜节律时钟。通过鉴定SPY的相互作用基因组，我们鉴定了PSEUDO-RESPONSE REGULATOR 5（PRR5）（一种昼夜节律的核心组成部分）作为一种新的SPY相互作用蛋白。PRR5可以被植物体内的SPY进行O-岩藻糖基化，而SPY催化域中的点突变消除了PRR5的O-岩藻糖基化。间谍突变体中PRR5的蛋白质​​丰度大大提高，而PRR5的降解率大大降低，这表明SPY介导的O促进了PRR5的蛋白水解。-岩藻糖基化。此外，多行遗传证据表明，PRR5是SPY的主要下游靶标，可以专门介导其对生物钟的调节。总的来说，我们的发现为SPY的O-岩藻糖基转移酶活性在调节昼夜节律中的特定作用提供了新颖的见解，并暗示O-糖基化可能通过哺乳动物中的O- GlcNAcylation在调节昼夜节律系统中发挥进化上保守的作用，但通过高等植物中的O-岩藻糖基化。