Subcellular Localization of Class II HDAs Figure 9. Nucleocytoplasmic shuttling of HDA15. To illustrate if HDA15-GFP undergoes nucleocytoplasmic shuttling, transfected protoplasts were incubated under white light overnight then covered with foil for 3 hours then re-exposed to white light. HDA15-GFP transfected protoplasts exhibited nuclear localization after 18 h of white light incubation. Further dark treatment for 3 h elicited partial cytoplasmic translocation of HDA15- GFP. Re-exposition of these protoplasts to white light after one hour lead to its complete nuclear import clearly demonstrating that light drives the nucleocytoplasmic shuttling of HDA15. VirD2NLS was co-transfected as nuclear marker (blue). Scale bars were calibrated to 10 mm. doi:10.1371/journal.pone.0030846.g009 It is worth noting though that HDA8 and HDA14 are uniquely conserved only in plants. The potential function of HDA8 in the cytoplasm and HDA14 in the mitochondria and chloroplasts may involve regulatory mechanisms in energy homeostasis similar with the human NAD-dependent sirtuin family [73]. HsSirt3, in particular, translocates into the mitochondria upon stress [74]. In this case, however, it could be the reverse such that HDA14 will only translocate into the nucleus given the proper environmental signals. Studies by Wallace and Fan have elaborated on the dynamic interplay between bioenergetics and epigenetics linking human epigenetic disorders with mitochondrial dysfunction [75,76]. In the context of plants, however, energy regulatory dynamics is complicated by light harvesting, inorganic chemo- synthesis, to carbon fixation. Moreover, its static nature exposes plants to a wider scope of stress implicating greater magnitude of epigenomic changes thus its survival tactics are more intricately complex than mobile organisms. Histone acetylation, in partic- ular, has been associated in regulating gene expression in developmental transitions [7,5], varying environmental signals such as light [77] and low temperature [78], and stress hormone signals [79]. In addition, the cytoplasmic localization of HDA8 and HDA14 in plants may entail crucial roles in deacetylating proteins essential in photosynthesis and other cytoplasmic metabolic pathways. Recent studies by Finkemier et al. [20] and Wu et al. [21] have pioneered a systematic study on non-histone proteins targeted for Lys acetylation in Arabidopsis. Based on their results, a significant proportion of Lys- acetylated proteins are involved in photosyn- thesis, TCA cycle, glycolysis, structural proteins, cell signaling, and plant stress responses. Of particular interest is the deacetylation of multiple Lys groups in the large subunit of rubisco using the human Sirt3, which induced a 40% increase in its maximum catalytic activity. Assuming HDA8 and HDA14 directly deacety- late any of these photosynthetic proteins and metabolic enzymes, this would justify its evolutionary divergence from heterotrophs intricately accessorizing itself with a more efficient energy regulatory response to balance the dynamic interplay between the environment, chromatin modification, and bioenergetics. Light drives the nucleocytoplasmic shuttling of HDA15 The nuclear importation of HDA15 in the presence of light may implicate its functional activity within the light signaling pathway. More so, its expression is mostly abundant in the stems and leaves all throughout the developmental stages of the plant. Thus, we cannot exclude the possibility that HDA15 may perform active roles in photomorphogenesis. Moreover, the localization of HDA15 appears to be mostly restricted inside a confined spot inside the nucleus, possibly nucleolus suggesting a function in deacetylating nucleolar core histones. This study have paved the baseline evidence that plant Class II HDAs, HDA15 in particular, can undergo nucleocyto- plasmic shuttling with light signaling its complete nuclear transport. However, it is puzzling to note why and how HDA15 specifically drives into the center since differences in light exposure or wavelength do not influence its nuclear distribution. Further- more, it is yet to be determined whether its cytoplasmic export functionally regulates its inactivity or remains active targeting the deacetylation of cytoplasmic proteins. PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e30846