Western blot assay demonstrates the change in histone acetylation levels during cold acclimation.

A. Protein samples were extracted from 7-day-old seedlings grown under 25°C (0 day), from seedlings that were further cold-treated at for 1–4 days, and from seedlings returned to 25°C for 1–4 days after cold treatment. Global acetylation levels of histones H3 and H4 were decreased after cold treatment and were recovered when seedlings were returned to 25°C. B. Reference cultivation without cold treatment was performed in parallel, showing that the histone acetylation levels underwent little change. Beta actin and total histone H3 were used as equal loading controls, and representative blots are shown. C. Deacetylation of histones H3 and H4 is revealed by immunostaining representative interphase nuclei with specific antibodies. DAPI was used as a counterstain. The ‘DAPI’ panel shows DAPI-stained DNA images, the ‘H3K9Ac’, ‘H44Ac’ and ‘H4K5Ac’ panels show immunostained images, and the ‘Merge’ panel shows a combination of blue and green signals. Under normal growth conditions, the strongly acetylated histones H3 and H4 signals were evenly distributed in the nucleus, and nucleoli were barely acetylated. When deacetylation of histones H3 and H4 occurred, weak acetylation signals were observed during cold acclimation. The histone acetylation levels were recovered after the seedlings were returned to 25°C (Bar = 10 µm). D. Histogram showing the mean gray values of the immunostaining signals for histone acetylation. Error bars represent the standard error of the mean. More than 500 nuclei were analyzed.