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Cell dedifferentiation is a cell fate switching process in which differentiated cells undergo genome reprogramming to regain the competency of cell division and organ regeneration. The molecular mechanism underlying the cell dedifferentiation process remains obscure. In this report, we investigate the cell dedifferentiation process in Arabidopsis using a shotgun proteomics approach. A total of 758 proteins are identified by two or more matched peptides. Comparative analyses at four time points using two label-free methods reveal that 193 proteins display up-regulation and 183 proteins display down-regulation within 48 h. While the results of the two label-free quantification methods match well with each other, comparison with previously published 2-DE gel results reveal that label-free quantification results differ substantially from those of the 2-DE method for proteins with peptides common to multiple proteins, suggesting a limitation of the label-free methods in quantifying proteins with closely related family members in complex samples. Our results show that the shotgun approach and the traditional 2-DE gel approach complement each other in both protein identification and quantification. An interesting observation is that core histones and histone variants are subjected to extensive down-regulation, indicating that there is a dramatic change in the chromatin during cell differentiation.

The eukaryotic chromatin/chromosome stores genomic information, controls genetic material distribution, and plays an essential role in the establishment and maintenance of spatial and temporal gene expression profile. Despite over a century of research, the protein composition and higher level structure of chromatin still remain obscure, particularly in plants. In this report, we have developed a protocol for chromatin purification from rice suspension cells and examined proteins copurified with chromatin using both 2-DE gel and shotgun approaches. Nine hundred seventy-two distinct protein spots have been resolved on 2-DE gels and 509 proteins have been identified by MALDI-MS/MS following gel excision, which correspond to 269 unique proteins. When the chromatin copurified proteins are examined using shotgun method, a large number of histone variants in addition to the four common core histones have been identified. Other proteins identified include nucleosome assembly proteins, high mobility group proteins, histone modification proteins, transcription factors, and a large number of hypothetical and function unknown proteins. Furthermore, putative phosphoproteins copurified with chromatin have been examined using Pro-Q Diamond phosphoprotein stain and followed by MALDI-MS/MS. Our studies have provided valued new insight into chromatin composition in plants.

Cell dedifferentiation is a cell fate switching process in which a differentiated cell reverts to a status with competence for cell division and organ regeneration like an embryonic stem cell. Although the phenomenon of cell dedifferentiation has been known for over two and a half centuries in plants, little is known of the underlying mechanisms. Here, we have established the proteome map of Arabidopsis cotyledons and investigated the dynamic change of the cotyledon proteome in the time course of cell dedifferentiation. Among the 353 distinct genes, corresponding to 500 2-DE gel protein spots identified with high confidence, 12% have over twofold differential regulations within the first 48 h of induction of cell dedifferentiation. The distributions of these genes among different Gene Ontology categories and gene differential regulations within each of the categories have been examined. In addition, we have investigated the cotyledon phosphoproteome using Pro-Q Diamond Phosphoprotein in Gel Stain followed by mass spectrometry analyses. Among the 53 identified putative phosphoproteins, nine are differentially regulated during cell dedifferentiation. These studies have provided significant new insight into protein and phosphoprotein differential expression during cell dedifferentiation in plants.