Subcellular Localization of Class II HDAs Figure 1. Class II HDA mRNA expression. Expression patterns of Class II HDAs in different vegetative organs and developmental stages were assessed using RT-PCR with ubiquitin (UBQ) as loading control. Legend: R roots, S stem, L leaves, MS mature seeds, YRL young rosette leaf, DRL developed rosette leaf, I inflorescence, YF young flower, MF mature flower, FS flowers and siliques. doi:10.1371/journal.pone.0030846.g001 exhibited in Figure 2, HDA5 displayed prominently cytoplasmic concentrations with partial overlaps inside the nucleus. Strong HDA8- YFP signals were detected in the cytoplasm and embedded near the nucleus while HDA14 exhibited multiple small granular spots along the cytoplasma. On the other hand, HDA15 was exclusively nuclear. In concurrence with this, another set of constructs were made fusing the GFP at the N-terminal of the HDAs which may mask some of the organelle targeting sequences imprinted along the histone deacetylases. Apparently, these GFP-HDA fusion con- structs yielded similar results with the HDA-YFP set. HDA5 was prominently cytoplasmic lining through the core across the nucleus. HDA8 was found to abound in the cytoplasm enveloping the chloroplasts. HDA14 was evidently cytoplasmic with big and small granular spots floating along the cytoplasma. On the other hand, HDA15 was clearly restricted in the nucleus with strong signals emanating from the nucleolus (Figure S1). Moreover, transgenic lines expressing the HDA:GFP transgene under the control of the 35S promoter of the Cauliflower mosaic virus were generated. Protoplasts from 3-week old leaves were isolated and observed for subcellular localization. However, the GFP signals elicited by these transgenic protoplasts were relatively weak (Figure S2). Nevertheless, HDA5, HDA8, and HDA14 were evidently distributed along the cytoplasmic area. On the other hand, HDA15 was confined exclusively inside the nucleus with GFP signals emanating in the nucleolus. Based on these results, it is unclear whether HDA5, HDA8, and HDA14 are exclusively cytoplasmic or nuclear as well. To resolve this, cell fractionation and immunoblot detection was carried out. As illustrated in Figure 3, HDA5 and HDA8 were detected in both cytoplasma and nuclear fractions while HDA14 was exclusively cytoplasmic. On the other hand, HDA15 was restricted in the nucleus. To have a larger view of the HDA’s organelle localization and dynamics, transient expression of HDA-YFP/GFP in onion epidermal tissues using particle bombardment was conducted. As shown in Figure 4A, HDA5 exhibited nuclear concentrations with well-defined enrichments along the cytoskeletal region. HDA8 localizes both in the nucleus and cytoplasm while HDA14 remains exclusively cytoplasmic with speckled distribution and refined localization within organelles. Still, HDA15 remained nuclear (Figure S3). Although most of the GFP signals observed in all the Class II HDAs were static, we have discovered the dynamic movement of HDA5 along the cytoskeletal area (Figure 4B) suggesting a function potentially in tubulin deacetylation which may be similar to human HDAC6. This would explain the predominant HDA5 spots in transgenic protoplasts where the cytoskeletal area may be too thin or weak to exude GFP signals in comparison to the web- like signals in transient protoplasts, which strongly highlights the cytoskeleton obscuring the spots. Based on these results, HDA5 and HDA8 were consistently observed in the cytoplasm with partial enrichments along the nuclear vicinity. On the other hand, HDA14 was distinctly localized in specific cytoplasmic organelle/s. In an attempt to identify specific organellar localization of these HDAs, subcellular markers were employed and co-transfected together with the HDA-YFP constructs in Arabidopsis PSB-D cell lines. These cells are devoid of chloroplasts to avoid ambiguous signals emitted from autofluorescence and reveal a clearer view of the localization of HDAs. Co-transfection was likewise employed in Col-0 protoplasts to investigate potential chloroplast distribution. Cytoplasmic HDA5 shows striking co-localization with the cytoskeleton network. Since the endoplasmic reticulum is com- posed of an extensive network of cisternae held together by the cytoskeleton, an ER marker fused with mRFP was used. HDEL contains a targeting sequence with Lys-Asp-Glu-Leu residues found in the endoplasmic reticulum protein retention receptor1 first isolated in humans [60]. Overlay pictures bet YFP signals from HDA5 consistently matched the mRFP fluorescence from HDEL confirming the localization of HDA5 in the ER (Figure 5). It is probable HDA8 may generally be suspended in the cytosol as none of the organelle markers tested co-localized with its pertinent distribution. With the use of VirD2NLS as nuclear marker, HDA8 also occupies the peripheral areas of the nucleus in contrast to HDA15, which is strictly confined inside a small spot, potentially nucleolus. Moreover, HDA14-YFP was positively confirmed in the mitochondria with YFP signals co-localizing with the MitoTracker, a mitochondria specific stain. In addition, strong HDA14-YFP signals likewise overlapped with the chloroplasts. 3. Interaction of 14-3-3 with Class II HDAs Human Class II HDACs have been subdivided as Class IIa, which is dependent on 14-3-3 for nuclear export and cytoplasmic PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e30846