Subcellular Localization of Class II HDAs in Arabidopsis thaliana : Nucleocytoplasmic Shuttling of HDA15 Is Driven by Light Malona V. Alinsug1, Fang Fang Chen1, Ming Luo1,2, Ready Tai1, Liwen Jiang 3 , Keqiang Wu 1 * 1 Institute of Plant Biology, College of Life Science, National Taiwan University, Taipei, Taiwan, 2 Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China, 3 School of Life Sciences, Centre for Cell and Developmental Biology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Abstract Class II histone deacetylases in humans and other model organisms undergo nucleocytoplasmic shuttling. This unique functional regulatory mechanism has been well elucidated in eukaryotic organisms except in plant systems. In this study, we have paved the baseline evidence for the cytoplasmic and nuclear localization of Class II HDAs as well as their mRNA expression patterns. RT-PCR analysis on the different vegetative parts and developmental stages reveal that Class II HDAs are ubiquitously expressed in 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 cytoplasmic proteins. Organelle markers and stains confirm HDA14 to abound in the mitochondria 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 where further re-exposition to light treatments signals its nuclear import. Unlike HDA5 which binds with 14-3-3 proteins, HDA15 fails to interact with these chaperones. Instead, HDA15 relies on its own nuclear localization and export signals to navigate its subcellular compartmentalization classifying it as a Class IIb HDA. Our study indicates that nucleocytoplasmic shuttling is indeed a hallmark for all eukaryotic Class II histone deacetylases. Citation: Alinsug MV, Chen FF, Luo M, Tai R, Jiang L, et al. (2012) Subcellular Localization of Class II HDAs in Arabidopsis thaliana: Nucleocytoplasmic Shuttling of HDA15 Is Driven by Light. PLoS ONE 7(2): e30846. doi:10.1371/journal.pone.0030846 Editor: Qiang Wu, National University of Singapore, Singapore Received July 3, 2011; Accepted December 23, 2011; Published February 17, 2012 Copyright: 2012 Alinsug et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by grants from the National Science Council of Taiwan (97-2311-B-002-004-MY3, 98-2628-B-002-016-MY3 and 99-2321-B-002- 027-MY3) and National Taiwan University (98R0066-36 and 99R80840). MVA is supported by a grant from the Democratic Pacific Union graduate studies program and research fellowship from NSC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kewu@ntu.edu.tw Introduction Histone acetylation has been known to induce an open chromatin configuration leading to transcriptional activation while deacetylation stimulates chromatin condensation triggering transcriptional quies- cence. Plant histone deacetylases (HDA or HDACs) are classified into three distinct families namely RPD3/HDA1 superfamily, Sirtuin family, and the HD2 family which is unique in plants [1,2,3]. Twelve out of the eighteen known HDAs in Arabidopsis belong to the RPD3/HDA1-like histone deacetylase superfamily, which is further subdivided into three classes namely Class I, II, and IV. Recent phylogenetic studies by Alinsug et al. [4] have identified HDA10 and HDA17 in addition to HDA6, HDA7, HDA9, and HDA19 as Class I HDA based on sequence homology. Although HDA8 and HDA14 are only represented in plants, they exhibit higher sequence similarity with the conserved histone deacetylase domains of HDA5, HDA15, and HDA18, members of the Class II HDAs while HDA2 is classified as Class IV. Among the RPD3/HDA1-like superfamily HDAs in Arabidop- sis, HDA6, HDA19, and HDA18 have been well elucidated to play crucial roles in plant development and environmental stress response and display tissue-specific expression [5–11,3]. HDA6 is the most extensively studied plant histone deacetylase acting as a global repressor involved in flowering, freezing tolerance, ABA and salt stress response, senescence, repression of embryonic properties, JA pathway, and establishment of nucleolar dominance [12–16,10]. Similarly, HDA19 is a global repressor in embryonic and flower development, ABA and abiotic stress response, light- responsive gene expression, JA and ethylene signaling, and regulates basal defense via interaction with WRKY transcription factors [10,5,6,17,8,18,11]. HDA18, on the other hand, had been implicated in root epidermal patterning [19]. Previous studies by Finkemeier et al. [20] and Wu et al. [21] elaborated on the reversible acetylation of cytoplasmic proteins in Arabidopsis indicating that histones are not the only proteins being acetylated & deacetylated. A substantial proportion of these cytoplasmic proteins are involved in photosynthesis and central metabolism where the deacetylation of rubisco and phosphoglyc- erate kinase using human Sirt3 lead to a significant increase in their catalytic activity. However, the use of human Sirt3 as a deacetylase may exhibit certain specificities towards plant Lys acetylation sites. Thus, the observed potency may have been underestimated had a plant specific deacetylase been used. Unfortunately, none of the 18 known histone deacetylases in Arabidopsis have been identified to be cytoplasmic since HDA6 and HDA19 were established to be exclusively nuclear. In rice, PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30846