Advances in Plant Nuclear and Chromosomal Proteomics

A variety of eukaryotic proteins is involved in maintaining nuclear organization, gene expression and nuclear and cell division. However, except for histones and a few other nuclear proteins, only a fraction of these proteins is known in plants. Proteomics is a powerful approach for large-scale protein identification and may contribute to better understanding of the main structural and functional components of plant cell nuclei and chromosomes. Biochemical composition of sub-cellular components may be altered during their isolation and during subsequent protein purification. Typically, a series of fractionation steps is employed to obtain purified nuclei and chromosomes. This is time consuming, laborious and the isolated fractions can be contaminated by cytoplasmic proteins. We have developed an alternative and efficient method for the isolation of nuclei and chromosomes, which does not affect their protein content and which comprises only a single step. The new protocol involves flow cytometric sorting of cell nuclei in G1, S, or G2 phases of the cell cycle, and plant mitotic metaphase chromosomes. It is important that this approach avoids contamination by non-nuclear proteins. Our preliminary results indicate that the flow cytometric sorting coupled with mass spectrometry will permit a comprehensive characterization of the proteome of plant cell nuclei in various phases of cell division cycle as well as proteins of mitotic chromosomes.