Figure 3. Cell fractionation and immunoblot detection HDA- GFP transfected protoplasts were separated into cytoplasmic and nuclear fractions then subjected to immunoblot analysis using anti-GFP antibody. Histone H3 and RHA1 were used as nuclear and cytoplasmic markers, respectively, on WT protoplasts. P protoplast extract, N nuclear fraction, C cytosolic fraction. doi:10.1371/journal.pone.0030846.g003 the other hand, HDA15 failed to interact with either 14-3-3 k or e which may suggest its potential to rely on its own NES and NLS signals. 4. NLS and NES signals of HDA15 navigate its subcellular compartmentalization HDA15 contains three NLS signals, one classical par4 type NLS near the N terminal end and two overlapping bipartite NLS near the C terminal end (Figure 7A). The predicted NES was stationed before the second and third NLS near the carboxyl end. Since the second and third NLS were overlapping, they were jointly omitted as NLS2. To test whether these NLS and NES functionally navigate the localization of HDA15, truncated constructs were generated deleting these predicted signals from HDA15-YFP. As shown in Figure 7B, the removal of either NLS1 or NLS2 still renders HDA15 nuclear indicating that both NLS are functional. In the absence of the other, one can still direct the nuclear localization of HDA15. However, the deletion of both NLS signals culminates into its nuclear export signifying the functionality of its NES. Taken together, these results demonstrate that HDA15 can translocate in and out of the nucleus whereby its nuclear localization and export signals navigate its subcellular compart- mentalization. This classifies HDA15 as a Class IIb histone deacetylase. 5. Nuclear localization of HDA15 is strongly expressed in stems and leaves Although HDA15 was clearly observed to be nuclear in both transgenic and transfected protoplasts, its mRNA expression patterns in the different vegetative organs varied with strong preference for the stems and leaves. To confirm these findings, HDA15-GFP transgenic lines were grown on 1/2 MS media to assess differences in its signal and localization. As demonstrated in Figure 8, whole plant localization of HDA15-GFP in four day old de-etiolated seedlings exhibited nuclear confinements of HDA15 in the hypocotyl and leaf protoplasts. However, weak GFP signals were observed in the root (Figure 8B). Although relatively weak, the strongest signal in the root appears to concentrate at the quiescent center. Light exposed organs, namely hypocotyl and cotyledons (Figure 8C–F), exhibited very strong nuclear GFP signals. On the other hand, etiolated seedlings grown in the dark for four days elicited predominantly cytoplasmic concentrations in the hypocotyl Subcellular Localization of Class II HDAs (Figure 8G). These results were further confirmed using transient expression of HDA15-GFP in protoplasts incubated under white light for 18 h displaying nuclear concentrations of HDA15 (Figure 8E inset) similar to transgenic protoplasts. Separating the white light spectrum into far red, red, and blue light as light treatment for 3 h after 18 h white light incubation similarly resulted into its nuclear localization suggesting that light quality does not affect its localization (Figure S5). On the contrary, transfected protoplasts treated in complete darkness for 3 h after 18 h of white light incubation generated cytoplasmic localization of HDA15 (Figure 8H). This indicates that the nuclear localization of HDA15 is signaled by light and is not influenced by any specific wavelength. 6. Light drives the nucleocytoplasmic shuttling of HDA15 The nuclear and cytoplasmic localizations of HDA15 in the presence and absence of light, respectively, unveil its strong potential to undergo nucleocytoplasmic shuttling. This functional regulatory mechanism may control its activation and inactivation as a histone deacetylase just like its corresponding orthologues in humans and other eukaryotes. To determine if plant Class II HDAs, HDA15 in particular, can also display nucleocytoplasmic shuttling, HDA15-GFP transfected protoplasts were incubated under white light overnight then covered with two layers of foil for three hours then re-exposed to white light. As shown in Figure 9, HDA15-GFP transfected protoplasts exhibited nuclear localization after 18 h of white light incubation. Further dark treatment elicited cytoplasmic translocation of HDA15. 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. Discussion Class II HDAs display minimal developmental specificity and are strongly expressed in stems Unlike human Class II HDACs which exhibit strict developmental specificity, Arabidopsis Class II HDAs display minimal developmen- tal specificity and are ubiquitously expressed in all tissues with stronger signals in stems. This affirms our previous bioinformatics data where Class II HDAs have been predicted to be prominently expressed in stems [4]. HDA5 and HDA8 may display abundant transcript levels in stems, leaves, and mature seeds, however, they may only be active at certain life stages of the plant. On the contrary, HDA14 and HDA15 are homogenously expressed all throughout its developmental stages but mostly in stems. In addition, HDA18 abounds in the roots and stems but inactive during young flower stage. Prior studies by Xu et al. [19] have implicated the role of HDA18 on root epidermal patterning such that reduced trichostatin A (TSA) treatment deregulates the expression of key patterning genes GLABRA (GL2), CAPRICE (CPC), and WEREWOLF (WER). Considering the same genes are relatively active in trichome development and patterning in leaves, the involvement of HDA18 on epidermal patterning maybe exclusive on the roots as its expression is kept at a minimum in the leaves. The strong expression profile of Class II HDAs in stem stirs baffling questions with the possibility of the involvement of this entire class of histone deacetylases in hypocotyl or stem development. Cytoplasmic localization of HDA5, HDA8, and HDA14 entails their potential for nuclear transport and versatility as deacetylases of non-histone proteins The translocation of human Class II HDACs into the cytoplasm is generally considered as a functional regulatory mechanism, PLoS ONE | www.plosone.org 5 February 2012 | Volume 7 | Issue 2 | e30846