Induction of global changes in Nicotiana tabacum chromatin following nutritional stress

Abstract

Cultures of tobacco (Nicotiana tabacum) BY-2 cells provide a good model for studying plant cell growth under different nutrient conditions. A previous study reported changes in apparent DNA content, as measured with flow cytometry of propidium iodide (PI) stained cell nuclei, during the course of culture. The basis of these changes was the main impetus for this investigation. While fluorescence of BY-2 nuclei stained with PI (which binds DNA by intercalation) increased then decreased with culture time, that of cells stained with Hoechst 33342 (which binds DNA without intercalation) did not fluctuate, suggesting that changes in cellular DNA content were not occurring. Addition of PI, but not of Hoechst 33342, caused swelling of nuclei, implying that intercalation induced a change in chromatin conformation. Since decreased PI staining accompanied depletion of sucrose in the culture medium, the effects of nutritional stress were also investigated. Deprivation of sucrose caused severe reduction of growth, cell shrinkage, vacuolation and formation of monodansylcadaverine positive acidic vesicles, indicative of autophagy. Nitrate deprivation caused very different morphological changes, with cellular swelling and formation of starch granules. Interestingly, starch granules were packaged within a double membrane, usually indicative of autophagosomes. These changes were not related to alterations in PI staining. The mechanism for induced alteration in PI staining was investigated by examination of chromatin. Modifications in DNA methylation were observed both through differences in the DNA fragmentation pattern following treatment with methylationsensitive restriction endonucleases and by a decrease in total 5-methylcytosine content. Changes in histone H1 staining were also measured using fluorescent antibodies and mirrored PI staining by decreasing then increasing with culture time. It is hypothesised that cellular energy metabolism controls a process that partially removes histone H1, a nucleosome linking histone, from DNA. This allows a DNA glycosylase, an enzyme found in plants but not animals, to cut the N-glycosidic link between the DNA backbone and 5-methylcytosine, effectively acting as a demethylase. The chromatin alteration allows access to PI, explaining the increased staining. Depletion of an energy source at later stages of culture allows reattachment of histone H1 and restoration of DNA methylation.