OsSirt2b was found to localize in the mitochondria, OsHDAC6 in chloroplasts, and OsHDAC10 in both chloroplast and mitochon- dria although the localization of their respective homologues in Arabidopsis namely, SRT2, HDA2, and HDA14, still remains to be elucidated [22]. Class II histone deacetylases in humans and other metazoans have been well described to undergo nucleocytoplasmic shuttling [23–26]. This functional regulatory mechanism renders Class II HDACs to be active as histone deacetylases while inside the nucleus and inactive when exported out into the cytoplasm. Class II HDAs generally contain a nuclear localization signal (NLS) at the amino terminal and a nuclear export signal (NES) near the carboxyl end. However, mammalian Class II HDACs are classified further into Class IIa (HsHDA504, HsHDA505, HsHDA507, and HsHDA509) and Class IIb (HsHDA506 and HsHDA510) [24]. Class IIa HDACs are dependent on 14-3-3 binding to translocate into the cytoplasm while Class IIb HDACs rely on their strong NES and NLS for nuclear import and export. Although most of these Class II HDACs remain inactive in the cytoplasm, HDAC7 and HDAC6 continue to play versatile roles such as mitochondrial proteins implicated in apoptosis, SUMO E3 ligases, and target several cytosolic substrates such as tubulin, cortactin, Hsp90, b- catenin, and peroxiredoxin [27–38]. While inside the nucleus, Class IIa HDACs form complexes with MEF2, a subfamily of MADS-box transcription factors, which generally function as transcriptional repressors. They are likewise phosphorylated by Ca2+ dependent calmodulin kinase prior to binding with 14-3-3 proteins. This phosphorylation- dependent binding of 14-3- 3 to the N terminus of Class II HDACs masks the arginine/lysine-rich motif NLS which simul- taneously unmasks a latent NES near its C terminus. Once phosphorylated, they are inactivated as a histone deacetylase and chaperoned out of the nucleus [26,39]. When proper environ- mental signals are cued, these HDACs translocate back into the nucleus, dephosphorylated via binding with MEF2 transcription factors, and activated as histone deacetylases [23,28]. In general, 14-3-3 proteins are highly conserved, multifunction- al regulatory proteins, which have been implicated in the modulation of distinct biological processes by phosphorylation- dependent protein binding interactions [40]. There are 13 known isoforms in Arabidopsis, which are divided into two phylogenetic groups [41,42]. Functional diversity and redundancy among these isoforms remain an open debate [43,44]. Comparative inter- actomic studies by Paul and his colleagues [45] reveal highly conserved 14-3-3 interactions between humans and plants. Proteomic profiling using tandem affinity purified 14-3-3 com- plexes in Arabidopsis suggested the high potential of 14-3-3s to heterodimerize in vivo [46]. To date, there are more than 300 known client targets of plant 14-3-3s and most these interacting clients are involved in primary metabolism, ion homeostasis, and hormone signaling including ABA, BR, and GA [45,46,40,47–54]. On the other hand, Class IIb HDACs in humans generally contain double domains, manifest tissue specificity, predominate in the cytoplasm, and target the deacetylation of histones, tubulin, Hsp90, cortactin, b-catenin, and peroxiredoxin [24,23,55– 57,34,32,37,38]. In human HDAC6, it has been shown that its two catalytic domains function independently as proven by site directed mutagenesis of its two HDAC domains [58]. Conse- quently, the separation of these domains commences into the obliteration of its enzymatic activity [24]. On the other hand, the C-terminal domain of HDAC10 lacks an active residue required for its enzymatic activity. However, its interaction with the functional N-terminal domain renders it active as a histone deacetylase [59]. Subcellular Localization of Class II HDAs Based on sequence analyses of plant Class II HDAs in our previous study [4], not all of these five HDAs contain both NLS and NES. However, they all have conserved Ser and Thr residues, which can be potential phosphorylation sites for 14-3- 3 binding. Moreover, HDA18 contains a double HDAC domain, which is comparable to Class IIb HDACs but its conserved histone deacetylase domain is 54% similar to HDA5. In addition, human HDAC6 has a cysteine- and histidine-rich domain called ZnF- UBP which parallels with the ZnF-RanBP of HDA15. Still, the localization of plant Class II HDAs is unknown and their potential to undergo nucleocytoplasmic shuttling remains elusive. Studies on plant Class II HDAs are scarce and pertinent information on their subcellular compartmentalization and specific expression patterns would provide significant insights on their potential function and active roles in plant development. Based on our findings, RT-PCR analysis on the different vegetative parts and developmental stages of Arabidopsis plants reveals that Class II HDAs are ubiquitously expressed throughout all tissues with minimal developmental specificity. Moreover, stable and transient expression assays using HDA-YFP/GFP fusion constructs indicate cytoplasmic localization of HDA5, HDA8, and HDA14 further suggesting their potential for nuclear transport and deacetylating organellar and cytosolic proteins. Organelle markers confirm HDA14 to abound in the mitochon- dria and chloroplasts while HDA5 localizes in the ER. HDA15, on the other hand, shuttles in and out of the nucleus upon light exposure. In the absence of light, it is exported out of the nucleus while further re-exposition into light treatments signals its nuclear import. Taken together, this provides the final piece of the puzzle indicating that nucleocytoplasmic shuttling is indeed a hallmark for all eukaryotic Class II histone deacetylases. Results 1. Expression patterns and localization of Class II HDAs mRNA expressions of Class II HDAs were assessed in Col-0 using different vegetative organs and whole plants at varying developmental stages. As illustrated in Figure 1, Class II HDA transcripts were broadly expressed in all the organs and developmental stages indicating that they may play an active role in the plant’s overall growth and development. In comparison to all the plant parts, the stems elicited the strongest expression in all the Class II HDAs. Moreover, only HDA18 was abundantly expressed in roots but remained minimal for HDA14, HDA8, and HDA5. Although HDA18 appears to be actively expressed in roots and stems, they were barely detected in the leaves and mature siliques. On the other hand, transcript levels of HDA15 were prominently detected at the upper shoot parts but not in the roots. At varying developmental stages, HDA5, HDA8, and HDA18 exhibited developmental specificity while HDA14 and HDA15 were homogenously yet strongly expressed all throughout the developmental stages. The expression of HDA5 is kept at a minimum during developed rosette leaf and young flower. HDA8 is weakly expressed from young rosette leaf to young flower but remains abundant thereafter. In addition, HDA18 is equally expressed throughout development but remains low during developed rosette leaves and undetected during young flower stage. 2. HDA5, HDA8, and HDA14 are localized in the cytoplasma To determine the subcellular localization of Class II HDAs, HDA-YFP constructs with the YFP fused at the C terminal of the HDAs were used for transient expression in protoplast. As PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30846