|Law, R - FORM USDA EMPLOYEE|
Submitted to: Plant Physiology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 9, 2005
Publication Date: May 13, 2005
Citation: Law, R.D., Suttle, J.C. 2005. Chromatin remodeling in plant cell culture: patterns of DNA methylation and histone H3 and H4 acetylation vary during growth of asynchronous potato cell suspensions. Plant Physiology and Biochemistry. 43:527-534. Interpretive Summary: For an indeterminate period of time following harvest, potatoes will not sprout and are physiologically dormant. Dormancy is gradually lost during postharvest storage and the resultant sprouting is detrimental to the nutritional and processing qualities of potatoes. Because of this sprouting results in severe financial loss to producers. Currently sprouting is controlled through the use of synthetic sprout inhibitors. The research being conducted in this lab is directed towards 1) identifying key physiological processes that naturally regulate tuber dormancy and, ultimately, 2) modifying these processes genetically thereby eliminating the need for artificial sprout suppression. At the cellular level, current research is directed toward identifying the molecular bases for low rates of RNA synthesis that accompany and possibly regulate tuber dormancy. Two processes (DNA methylation and histone acetylation) have been shown to regulate RNA synthesis in other organisms. Due to their small size and limited protein content, characterization of these regulatory molecules in potato tuber meristems (eyes) is difficult. In this sutdy, the feasibility of using potato cell cultures as a convenient source of these compounds for preliminary study was determined. Rapidly growing cell cultures proved to be an excellent source of these materials and provided suficient quantites to permit determination of the levels and modifications of these molecules during growth. THese studies will facilitate future research on similar changes occurring during tuber dormancy in potatoes and will shed new light on molecular mechnisms regulating gene expression.
Technical Abstract: Changes in DNA cytosine methylation and core histone multi-acetylation were determined in cell suspension cultures of potato (Solanum tuberosum L. cv. Russet Burbank) during 15 days of in vitro culture. Cell subculture induced a transient 2-fold increase in 5-methylcytosine levels within 5'-CCGG-3' sequences and a transient 3-fold increase in transcription rates which peaked at 6 and 9 days after subculture, respectively. Similarly, multi-acetylation of histones H3.1, H3.2 and H4 rose 2-, 1.5- and 3-fold by 9, 9 and 12 days after subculture, respectively. In contrast, subculture also resulted in a temporary 33% decline in genome-wide 5-methylcytosine levels by 6 days after subculture, and no change in 5'-CG-3' methylation. All observed epigenetic changes were reset during aging of cell cultures. Inclusion of the histone deacetylase inhibitor trichostatin A (TSA) and/or the cytosine methylation inhibitor 5-azacytidine (5AC) in culture sequentially decreased genome-wide 5mC levels by ~25% at day 9, then decreased 5'-mCmCGG-3' by 30 to 50% and increased H3 and H4 multi-acetylation by 30 to 60% at day 15, compared to controls. Treatment with 5AC or TSA alone or in combination had no effect on RNA synthesis at day 9. At day 15, 5AC treatment remained ineffective, while de novo RNA synthesis was ~2-fold higher in cells grown in both inhibitors or in TSA alone. Collectively, these results demonstrate that in potato suspension culture, rapid reversible changes in 5-methylcytosine levels precede regulatory post-translational acetylation of core histones, and suggest that interactions between these epigenetic processes appear to be necessary to power transcription and growth induction in potato cells.