Table_1_Proteome Analysis of Condensed Barley Mitotic Chromosomes.XLSX

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

蛋白质在核基因组的三维结构和功能中发挥着至关重要的作用。尽管组蛋白负责将 DNA 组织成核小体纤维，但其他蛋白质则参与间期核和有丝分裂/减数分裂染色体的更高阶染色质结构构建。尽管蛋白质在维持基因组完整性和将遗传信息传递至子细胞和后代的过程中发挥着关键作用，但其功能知识仍相对零散。这一情况在植物染色体蛋白，尤其是凝缩染色体蛋白方面尤为显著。在本研究中，我们通过流式细胞术分离纯化了大麦有丝分裂中期染色体，并对其蛋白质进行了表征。来自肽酶消化产物中的肽段在液相色谱串联质谱联用前，分别通过阳离子交换或反相梯度系统进行分离。基于软件预测、现有数据库定位信息、序列同源性和结构域表征的组合，对包含近 900 个鉴定结果的染色体蛋白进行了分类。生物环境评估揭示了包括组蛋白、拓扑异构酶 2、多聚酶 2、凝缩素亚单位和许多与染色质相关的功能蛋白在内的几组丰富蛋白的存在。发现了参与 DNA 复制、转录和修复等过程以及核仁蛋白。我们通过实验验证了核仁蛋白 FIBRILLARIN 1 在中期染色体上的存在，这表明植物染色体在有丝分裂期间被蛋白质覆盖，类似于人类和动物的染色体。这些结果显著提升了我们对植物染色体蛋白的认识，并为它们的功能特征化和比较系统发育分析提供了基础。