Histone deacetylation assay in vitro and in vivo

The in vitro histone deacetylation assay was performed following the manufacturer's instructions for the Epigenase HDAC Activity/Inhibition Direct Assay Kit (Epigentek). Briefly, the full-length CDS of MdHDA6 was cloned into pET-32(+) and pGEX-4 T vectors, which contained 10× HIS tag and 1× GST tag, respectively. The resulting constructs (MdHDA6-HIS and MdHDA6-GST) were transformed into E. coli strain BL21. Production and purification of MdHDA6-HIS and MdHDA6-GST fusion protein were performed by using Ni-NTA (Nitrilotriacetic Acid) agarose (Smart-Lifesciences) and Glutathione agarose (Thermo), respectively. The purified MHDA6 protein needed to be incubated with acetylated substrate and assay buffer for 60 min, then capture antibody and detection antibody were added. Finally, color-developing solution was added for color development, and the absorbance of the reactive solution was measured at 450 nm. The HDAC activity of MdHDA6 was calculated using a standard curve following the manufacturer's instructions and expressed as nanograms/minute/milligram of protein. (HO3: HDAC Assay Standard as a positive control, HI: HDAC Inhibitor TSA, were provided by Kit; BBX7 and GST were set as the negative controls.)

For detecting the global histone acetylation status in vivo, total histones were extracted as described by (Lu et al. 2011) with minor modifications. Briefly, fresh leaf tissues (0.1 g) of 2-mo-old GL-3 and MdHDA6-OE plants were ground to a fine powder in liquid nitrogen and suspended in 300 μl 3×SDS loading buffer (250 mM pH 6.8 Tris–HCl; 10% sodium dodecyl sulfate (w/v), 0.5% bromophenol blue (w/v), 50% glycerin (v/v)5% β-mercaptoethanol, (v/v)). The mixture was fully oscillated and then boiled at 100 °C for 8 min. Total histones were extracted from the supernatant after 10 min of centrifugation at 15,000 × g. Then the histones were used for western blot using the antibodies of anti-H3 (PTM1002, PTM Bio), anti-H3ac (Millipore, 17-615), anti-H3K9ac (Millipore, 07-352), and anti-H3K14ac (Millipore, 07-353). Among them, the immunoblot signal of anti-H3 was used as the loading control.