Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function, yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich ubiquitinated peptides coupled with isobaric labeling to enable quantification of up to 18-multiplexed samples. This approach identified 17,940 ubiquitinated lysine sites arising from 6,453 proteins from Arabidopsis (Arabidopsis thaliana) primary roots, seedlings, and rosette leaves. Gene ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and metabolism. We determined ubiquitinated lysine residues that directly regulate the stability of three transcription factors, CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) using in vivo degradation assays. Furthermore, codon mutation of CIB1 to create a K166R conversion to prevent ubiquitination, via CRISPR/Cas9-derived adenosine base editing, led to an early flowering phenotype and increased expression of FLOWERING LOCUS T (FT). These comprehensive site-level ubiquitinome profiles provide a wealth of data for future functional studies related to modulation of biological processes mediated by this posttranslational modification in plants.

中文翻译：

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定量蛋白质组学揭示了拟南芥中广泛的赖氨酸泛素化和转录因子稳定状态


蛋白质活性、丰度和稳定性可以通过翻译后修饰（包括泛素化）进行调节。泛素化在真核生物中是保守的，并且在调节细胞功能中起着核心作用，但我们缺乏植物中泛素修饰的蛋白质的全面目录。在本研究中，我们描述了一种基于抗体的方法，该方法富集泛素化肽，并与同量异位标记相结合，能够定量多达 18 个多重样品。这种方法鉴定了 17,940 个泛素化赖氨酸位点，这些位点来自拟南芥 （Arabidopsis thaliana） 初级根、幼苗和莲座叶的 6,453 种蛋白质。基因本体分析表明，泛素化蛋白与许多生物过程有关，包括激素信号传导、植物防御、蛋白质稳态和代谢。我们确定了直接调节三种转录因子稳定性的泛素化赖氨酸残基，隐色素相互作用碱性螺旋环螺旋 1 （CIB1）、CIB1 样蛋白 2 （CIL2） 和对质子RHIZOTOXICITY1敏感 （STOP1） 使用体内降解测定。此外，CIB1 的密码子突变通过 CRISPR/Cas9 衍生的腺苷碱基编辑产生 K166R 转换以防止泛素化，导致早期开花表型和开花基因座 T （FT） 的表达增加。这些全面的位点水平泛素组图谱为未来与植物翻译后修饰介导的生物过程调节相关的功能研究提供了大量数据。