Data_Sheet_1_Proteogenic Dipeptides Are Characterized by Diel Fluctuations and Target of Rapamycin Complex-Signaling Dependency in the Model Plant Arabidopsis thaliana.ZIP

As autotrophic organisms, plants capture light energy to convert carbon dioxide into ATP, nicotinamide adenine dinucleotide phosphate (NADPH), and sugars, which are essential for the biosynthesis of building blocks, storage, and growth. At night, metabolism and growth can be sustained by mobilizing carbon (C) reserves. In response to changing environmental conditions, such as light-dark cycles, the small-molecule regulation of enzymatic activities is critical for reprogramming cellular metabolism. We have recently demonstrated that proteogenic dipeptides, protein degradation products, act as metabolic switches at the interface of proteostasis and central metabolism in both plants and yeast. Dipeptides accumulate in response to the environmental changes and act via direct binding and regulation of critical enzymatic activities, enabling C flux distribution. Here, we provide evidence pointing to the involvement of dipeptides in the metabolic rewiring characteristics for the day-night cycle in plants. Specifically, we measured the abundance of 13 amino acids and 179 dipeptides over short- (SD) and long-day (LD) diel cycles, each with different light intensities. Of the measured dipeptides, 38 and eight were characterized by day-night oscillation in SD and LD, respectively, reaching maximum accumulation at the end of the day and then gradually falling in the night. Not only the number of dipeptides, but also the amplitude of the oscillation was higher in SD compared with LD conditions. Notably, rhythmic dipeptides were enriched in the glucogenic amino acids that can be converted into glucose. Considering the known role of Target of Rapamycin (TOR) signaling in regulating both autophagy and metabolism, we subsequently investigated whether diurnal fluctuations of dipeptides levels are dependent on the TOR Complex (TORC). The Raptor1b mutant (raptor1b), known for the substantial reduction of TOR kinase activity, was characterized by the augmented accumulation of dipeptides, which is especially pronounced under LD conditions. We were particularly intrigued by the group of 16 dipeptides, which, based on their oscillation under SD conditions and accumulation in raptor1b, can be associated with limited C availability or photoperiod. By mining existing protein-metabolite interaction data, we delineated putative protein interactors for a representative dipeptide Pro-Gln. The obtained list included enzymes of C and amino acid metabolism, which are also linked to the TORC-mediated metabolic network. Based on the obtained results, we speculate that the diurnal accumulation of dipeptides contributes to its metabolic adaptation in response to changes in C availability. We hypothesize that dipeptides would act as alternative respiratory substrates and by directly modulating the activity of the focal enzymes.

作为自养生物（autotrophic organisms），植物捕获光能以将二氧化碳转化为腺苷三磷酸（ATP，adenosine triphosphate）、烟酰胺腺嘌呤二核苷酸磷酸（NADPH，nicotinamide adenine dinucleotide phosphate）以及糖类，这些物质对于生物合成结构单元、储能与生长至关重要。夜间，植物可通过动员碳（C）储备维持代谢与生长。面对昼夜循环等环境变化，酶活性的小分子调控对于重编程细胞代谢至关重要。本课题组近期证实，蛋白质降解产物——蛋白源性二肽（proteogenic dipeptides），在植物与酵母中均可作为蛋白稳态与中心代谢交叉界面的代谢开关。二肽会随环境变化积累，并通过直接结合并调控关键酶活性来调控碳通量分配。本研究提供证据表明，二肽参与了植物昼夜周期的代谢重编程过程。具体而言，我们在短日照（SD，short-day）与长日照（LD，long-day）昼夜循环（二者光照强度存在差异）下，检测了13种氨基酸与179种二肽的丰度。在所检测的二肽中，分别有38种与8种在短日照与长日照条件下呈现昼夜振荡现象，且均在日间结束时达到积累峰值，随后在夜间逐渐下降。相较于长日照条件，短日照下不仅呈现振荡的二肽数量更多，其振荡幅度也更高。值得注意的是，呈现节律性的二肽富含可转化为葡萄糖的生糖氨基酸。鉴于雷帕霉素靶蛋白（TOR，Target of Rapamycin）信号通路在调控自噬与代谢中的已知作用，我们进一步探究了二肽水平的日间波动是否依赖于TOR复合物（TORC，TOR Complex）。已知雷帕霉素靶蛋白激酶活性显著降低的Raptor1b突变体（raptor1b），其体内二肽的积累水平出现上调，这一现象在长日照条件下尤为显著。我们尤其关注16种二肽组成的类群：基于它们在短日照下的振荡特征以及在raptor1b突变体中的积累模式，这类二肽可能与碳有效性受限或光周期相关。通过挖掘已公开的蛋白质-代谢物相互作用数据，我们为代表性二肽脯氨酰谷氨酰胺（Pro-Gln）确定了潜在的蛋白质相互作用因子。所得到的相互作用因子列表涵盖了碳代谢与氨基酸代谢相关的酶类，这些酶同时也与TOR复合物介导的代谢网络存在关联。基于上述结果，我们推测二肽的日间积累有助于植物响应碳有效性变化实现代谢适应。我们提出假说：二肽可作为替代呼吸底物，并通过直接调控关键酶的活性发挥作用。