Proteomic Analysis of Plant Mitotic Chromosomes

Transmission of hereditary information to daughter cells depends critically on the formation of chromosomes and their ordered behavior. Condensation of nuclear chromosome domains into compact mitotic chromosomes involves interaction of DNA with histone and non-histone proteins, some of which are modified during the process. Chromosome condensation is accompanied by formation of proteinaceous kinetochore domain, which binds to mitotic spindle, controls separation of sister chromatids and their movement towards mitotic spindle poles. Despite their obvious significance, apart for small basic histone proteins, the knowledge of chromosome proteins in plants remains limited. As the first step towards obtaining a more complete picture of plant chromosome protein composition, we performed proteomic analysis of barley mitotic metaphase chromosomes. The work involved chromosome purification by flow cytometric sorting followed by protein extraction, separation and digestion with the subsequent mass spectrometry of purified peptides. This approach allowed identification of a large number of proteins that form mitotic chromosomes. The protocol does not affect chromosome protein composition and minimizes the risk of contamination by non-chromosomal (e.g. cytoplasmic remnants) proteins. Thus, the approach should contribute towards understanding the macromolecular organization of plant mitotic chromosomes and revealing the processes that accompany their formation and influence their behavior. Subsequent work will focus on specific roles of each of the newly identified proteins in chromosome structure and function. This research was supported by grant awards from the Czech Science Foundation (14-28443S) and the National Program of Sustainability I (LO1204) from the Ministry of Education, Youth and Sports of the Czech Republic.