was transformed with pGBKT7 vector expressing HDA5 and pGADT7 vector expressing 14-3-3 K or E. Transformed cells were grown on synthetic media without Leu, Trp, and His containing 3 mM 3-aminotriazole or medium lacking Leu and Trp. Supporting Information Figure S1 Protoplast transient expression using GFP- HDA fusion constructs. Subcellular localization of Class II HDAs was determined via protoplast PEG transfection using GFP-HDA fusion constructs. HDA5, HDA8, and HDA14 were cytoplasmic while HDA15 was restricted inside the nucleus. VirD2NLS fused with mCherry was used as a nuclear marker. Scale bars were calibrated to 10 mm. (TIF) Figure S2 Subcellular localization in transgenic proto- plasts. Protoplasts from transgenic lines of Class II HDAs were also used to determine their corresponding subcellular localization. Although GFP signals were relatively weak, HDA5, HDA8, and HDA14 were found to abound in the cytoplasm while HDA15 emanated strong nuclear signals. (TIF) Figure S3 Particle bombardment in onion tissues. Overlay pictures reveal the nuclear and cytoplasmic localization of HDA8-YFP and the nuclear concentration of HDA15. (TIF) Figure S4 BiFC negative and positive controls. Empty vectors and YN/YC constructs were tested as negative controls. References 1. Pandey R, Muller A, Napoli C, Selinger D, Pikaard C, et al. (2002) Analysis of histone acetyltransferases and histone deacetylase families in Arabidopsis thaliana suggests functional diversification of chromatin modifications among multicel- lular eukaryotes. Nuc Acid Res 30: 5036–5055. 2. Wu K, Tian L, Malik K, Brown D, Miki B (2000) Functional analysis of HD2 histone deacetylase homologues in Arabidopsis thaliana. Plant J 22: 19–27. 3. Hollender C, Lui Z (2008) Histone deacetylase genes in Arabidopsis development. J Integ Plant Biol 50: 875–885. 4. Alinsug MV, Yu CW, Wu K (2009) Phylogenetic analysis, subcellular localization, and expression patterns of RPD3/HDA1 family histone deacety- lases in plants. BMC Plant Biology 9: 37. 5. Tian L, Chen ZL (2001) Blocking histone deacetylation in Arabidopsis induces pleitropic effects on plant gene regulation and development. Proc Natl Acad Sci USA 98: 200–205. 6. Tian L, Wang JJ, Fong MP, Chen M, Cao H, et al. (2003) Genetic control of developmental changes induced by disruption of Arabidopsis histone deacetylase 1 (AtHD1) expression. Genetics 165: 399–409. 7. Tian L, Fong MP, Wang JJ, Wei NE, Jiang H, et al. (2005) Reversible histone acetylation and deacetylation mediate genome-wide, promoter-dependent and locus- specific changes in gene expression during plant development. Genetics 169: 337–345. 8. Benhamed M, Bertrand C, Servet C, Zhou DX (2006) Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression. Plant Cell 18: 2893–903. 9. Long JA, Ohno C, Smith ZR, Meyerowitz EM (2002) TOPLESS regulates apical embryonic fate in Arabidopsis. Science 312: 1520–1523. 10. Tanaka M, Kikuchi A, Kamada H (2008) The Arabidopsis histone deacetylases HDA6 and HDA19 contribute to the repression of embryonic properties after germination. Plant Physiol 146: 149–161. 11. Kim KC, Lai Z, Fan B, Chen Z (2008) Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense. Plant Cell 20: 2357–71. 12. Yu CW, Liu X, Luo M, Chen C, Lin X, et al. (2011) HDA6 interacts with FLD and regulates flowering in Arabidopsis. Plant Physiol 156: 173–184. 13. To TK, Nakaminami K, Kim JM, Morosawa T, Ishida J, et al. (2011) Arabidopsis HDA6 is required for freezing tolerance. Biochem Biophys Res Commun 406: 414–419. 14. Chen LT, Luo M, Wang YY, Wu K (2010) Involvement of Arabidopsis histone deacetylase HDA6 in ABA and salt stress response. J Exp Bot 61: 3345– 3353. Subcellular Localization of Class II HDAs BZR1 was used as a positive control for the 14-3-3 kappa and epsilon interactions. (TIF) Figure S5 Nucleolar localization of HDA15-YFP in different light treatments. Transfected protoplasts were incubated under white light for 18 h then transferred to far red, red, and blue light treatments for 3 h at low light intensities (FR 2.77 mmol m22 s21, R 1.77 mmol m22 s21. B 3.84 mmol m22 s21). Similar with white light treated protoplasts, HDA15-YFP was restricted in a small spot inside the nucleus, potentially nucleolus. VirD2NLS-mCherry was co-transfected as a nuclear marker (blue). Scale bars were calibrated to 10 mm. (TIF) Acknowledgments The authors would like to extend their gratitude to the reviewers and editors for their comments and insights in fine-tuning our final paper. We are equally indebted to Yi-Chun Chuang and Cai-Ji Gao for their technical assistance in confocal microscopy, Dr. Shelly Cruz Duldulao of Academia Sinica Taiwan, Dr. Brian Wade Jamandre of Griffith University Australia, and Prof. Chien-Yuan Pan of National Taiwan University for the pertinent materials, instruments, and special techniques in trouble shooting some of the problems we have encountered in our experiments. Author Contributions Conceived and designed the experiments: MVA KW. Performed the experiments: MVA FFC LJ. Analyzed the data: MVA FFC LJ KW. Contributed reagents/materials/analysis tools: MVA LJ KW. Wrote the paper: MVA KW. Repeated BiFC experiment: ML RT. 15. Wu K, Zhang L, Zhou C, Yu CW, Chaikam V (2008) HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis. J Exp Bot 59: 225–34. 16. Earley K, Lawrence RJ, Pontes O, Reuther R, Enciso AJ, et al. (2006) Erasure of histone acetylation by Arabidopsis HDA6 mediates large-scale gene silencing in nucleolar dominance. Genes Dev 20: 1283–1293. 17. Chen LT, Wu K (2010) Role of histone deacetylases HDA6 and HDA19 in ABA and abiotic stress response. Plant Signal Behav 5: 1318–1320. 18. Zhou C, Zhang L, Duan J, Miki B, Wu K (2005) HISTONE DEACETY- LASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis. Plant Cell 17: 1196–204. 19. Xu CR, Liu C, Wang YL, Li LC, Chen WQ, et al. (2005) Histone acetylation affects expression of cellular patterning genes in the Arabidopsis root epidermis. Proc Natl Acad Sci USA 102: 14469–14474. 20. Finkemeier I, Laxa M, Miguet L, Howden AJM, Sweetlove LJ (2011) Proteins of diverse function and subcellular localization are lysine acetylated in Arabidopsis. Plant Physiol 155: 1779–1790. 21. Wu X, Oh MH, Schwarz EM, Larue CT, Sivaguru M, et al. (2011) Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis. Plant Physiol 155: 1769–1778. 22. Chung PJ, Kim YS, Park SH, Nam BH, Kim JK (2009) Subcellular localization of rice histone deacetylases in organelles. FEBS Letters 583: 2249–254. 23. Yang XJ, Gregoire S (2005) Class II histone deacetylases: from sequence to function, regulation, and clinical implication. Mol Cell Biol 25: 2873–2884. 24. Verdin E, Dequiedt F, Kasler HG (2003) Class II histone deacetylases: versatile regulators. Trends Genet 19: 286–293. 25. Nishino T, Miyazaki M, Hoshino H, Miwa Y, Horinouchi S, et al. (2008) 14- 3-3 regulates the nuclear import of class IIa histone deacetylases. Biochem Biophys Res Comm 377: 852–856. 26. Wang AH, Yang X (2001) Histone deacetylase 4 possesses intrinsic nuclear import and export signals. Mol Cell Biol 21: 5992–6005. 27. Bakin RE, Jung MO (2004) Cytoplasmic sequestration of HDAC7 from mitochondrial and nuclear compartments upon initation of apoptosis. J Biol Chem 279: 51218–51225. 28. Gregoire S, Yang XJ (2005) Association with Class IIa histone deacetylases upregulates sumoylation of MEF2 transcription factors. Mol Cell Biol 25: 2273–2287. 29. Hubbert C, Guardiola A, Shao R (2002) HDAC6 is a microtubule-associated deacetylase. Nature 417: 455–458. 30. Matsuyama A, Shimazu T, Sumida Y (2002) In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 21: 6820–6831. PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e30846