Subcellular Localization of Class II HDAs Figure 8. Whole plant localization of HDA15-GFP in 4-day old seedlings. Four day old de-etiolated seedlings of HDA15-GFP were observed for subcellular localization with the projection confocal image of the entire seedling (A), root tip (B), hypocotyl (C), and cotyledon (D). A magnified view of the leaf (E) and hypocotyl cells (F) of long day grown seedlings reveal nuclear concentrations of HDA15-GFP. On the contrary, those grown in total darkness for 4 consecutive days exhibited cytoplasmic localization of HDA15-GFP (G). In concurrence with this, transgenic protoplasts revealed nuclear confinements of HDA15-GFP upon white light treatment (E) similar to transfected protoplasts (inset E). However, 3-hour dark treated transfected protoplasts after 18 h of white light incubation resulted to its cytoplasmic translocation (H). VirD2NLS was co-transfected as nuclear marker (blue). Red color indicates autofluorescence emitted by chloroplasts. Scale bars for A to D is 100 mm, F & G was set at 25 mm, and H at 10 mm. doi:10.1371/journal.pone.0030846.g008 cytoplasm targeting the deacetylation of non-histone proteins. The cytoplasmic localization of Class II HDAs may not necessarily entail inactivity but its enzymatic target clients may encompass beyond nuclear histones expanding its substrate repertoire. The speckled distribution of HDA14 was confirmed in the mitochondria and chloroplasts similar to its rice orthologue, OsHDAC10 [22]. Based on iPSORT and SignalP prediction programs, HDA14 contains a mitochondrial and chloroplast transit peptide encoded at amino acids 4 to 11, yet, still has the potential to translocate into the nucleus considering that a classical type, pat7 NLS is embedded in its sequence near the carboxyl end. Its similarity to HDA5, HDA15, and HDA18 could be this nuclear importation given the proper signals to pool it towards the nucleus. PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e30846