Epigenetic regulations in cell wall degradation and regeneration in Oryza sativa
It is well known that chromatin components are key players in establishing and maintaining spatial and temporal gene expression in plants, however, little is known about the epigenetic regulation on cell wall degradation and regeneration. This study aimed to 1) investigate the global proteome and phosphoproteome of rice chromatin, and 2) characterize changes in chromatin components and chromatin structure associated with cell wall degradation and regeneration, and 3) characterize the differentially regulated proteins and eventually explore the mechanism. In this dissertation, we examine proteins copurified with chromatin using both 2-DE gel and shotgun approaches from rice (Oryza sativa) suspension cells. Nine hundred seventy-two distinct protein spots were resolved on 2-DE gels and 509 proteins were identified by MALDI-MS/MS following gel excision, these correspond to 269 unique proteins. When the chromatin copurified proteins are examined using shotgun proteomics, a large number of histone variants in addition to the four common core histones were identified. Furthermore, putative phosphoproteins copurified with chromatin were examined using Pro-Q Diamond phosphoprotein stain and followed by MALDI-MS/MS. Our studies provided new insights into the chromatin composition in plants. To study the epigenetic regulation of the cell wall degradation and regeneration, we examined cellular responses to the enzymatic removal of the cell wall in rice suspension cells using proteomic approaches. We found that removal of cell wall stimulates cell wall synthesis from multiple sites in protoplasts instead of from a single site as in cytokinesis. Microscopy examination and chromatin decondensation assay further showed that removal of the cell wall is concomitant with substantial chromatin reorganization. Histone post-translational modification studies using both Western blots and isotope labeling assisted quantitative mass spectrometry analyses revealed substantial histone modification changes, particularly H3K18AC and H3K23AC, are associated with the degradation and regeneration of the cell wall. Label-free comparative proteome analyses further revealed that chromatin associated proteins undergo dramatic changes upon removal of the cell wall, particularly cytoskeleton, cell wall metabolism, and stress-response proteins. This study demonstrates that cell wall removal is associated with substantial chromatin change and may lead to stimulation of cell wall synthesis using a novel mechanism.