Materials and Methods Plant Material & Growth Conditions Arabidopsis thaliana ecotype Col-0 was used as wild type and genetic background for all transgenic lines. Seeds were cold- treated for 2–3 days, sown on 1/2 strength Murashige & Skoog media, and then transferred to a growth chamber for germination at 20–24uC under long day conditions (16 h light/8 h dark cycle). Seedlings were further grown either in medium or soil pots. Prior to sample collection for mRNA analysis, developmental stages of the plant were observed and compared with the timeline indicated in Genevestigator using 20 replicates in each of the 3 trials. RNA Extraction & RT-PCR Gene expression was assessed using semi-quantitative RT-PCR. Total RNA was extracted from plant samples weighing 0.25 to 0.3 g using TRIZOL reagent (Invitrogen). Oligo(dT) primed reverse transcription of first strand cDNA synthesis was carried out with 7 mg total RNA using SuperScriptTM III (Invitrogen). Equal volumes of each first strand reaction were amplified with gene- specific primer pairs. Thermocycling conditions were 94uC for 4 mins followed by 30 cycles of 94uC for 30 s, 55–60uC for 30 s, and 74uC for 1–2 min. Primer sequences are available upon request. HDA-YFP/GFP fusion constructs Coding sequences of Class II histone deacetylase were amplified by PCR from expressed sequence tags (RIKEN) and subsequently cloned into the entry vector, pENTR/SD/D-TOPO or PCR8/ GW/TOPO, with spectromycin as bacterial marker. An LR clonase enzyme mix (Invitrogen) was used to transfer the insert from entry clones to its destination vectors, p2FGW7 with the GFP tag positioned at the N-terminal of the insert, and p2YGW7 which contains the YFP tag at the C-terminal of the gene. Both destination vectors are high copy vectors driven by a double 35S cauliflower mosaic virus promoter with ampicillin as bacterial marker. Purified plasmids were then analyzed and sequenced to confirm successful fusion constructs. Plant Transformation Class II HDAs in pENTR vectors were used for recombination to a binary vector, pK7WGF2, which contains a 35S CaMV promoter and a GFP tag at the carboxyl end of the insert. Spectromycin was used as bacterial marker. Purified plasmids were sequenced to confirm successful insertion and integrated into the Arabidopsis genome by Agrobacterium tumefaciens-mediated floral dipping method [80,81]. Seeds from transformed plants were germinated on 1/2 strength MS media with kanamycin for plant selection. Seven-day-old kanamycin resistant seedlings were then screened under fluorescence microscope for GFP signals then transplanted into soil pots for 3 weeks for seed production. T2 and T3 seedlings were used for the studies described herein. Protoplast Isolation and Transient Expression Leaves of 3-week old T2 transgenic lines were used to determine subcellular localization in protoplasts. Transient expression assays were subscribed from the methods of Yoo et al. [82] with some modifications. Mesophyll protoplasts were isolated from 3-week old Col-0 plants and Arabidopsis PSB-D cell lines [83]. Twenty mg of HDA-YFP/GFP fusion plasmid, VirD2-NLS as nuclear marker [84] and organelle markers [85] were co-transfected into 46104 protoplasts using polyethylene glycol (PEG) solution (0.4 g/ml PEG 4000, 0.8 M mannitol, 125 mM CaCl2), incubated for 5– 15 min at room temperature, washed and resuspended in W5 Subcellular Localization of Class II HDAs solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES at pH5.7), then incubated under white light for 16–24 h prior to imaging using Leica SP5 confocal microscope. For mitochondria staining, protoplasts were immersed in 0.2 mM MitoTracker Orange CMTMRos (Invitrogen, M7510) for 15 mins then mounted on slide with W5 solution. For the localization of HDA15 in different light treatments, transfected protoplasts were incubated under white light for 18 h then transferred to E30LEDL3 growth chambers (Percival Scientific) with far red, red, and blue light-emitting diode sources for 3 h. Low light intensities used as treatment were measured at 2.77 mmol m22 s21 (FR), 1.77 mmol m22 s21(R), and 3.84 mmol m22 s21(B). For trans- genic plants, seeds were grown in 1/2 MS media inside a growth chamber with 16 h light–8 h night cycle for white light treated seedlings while dark treated seeds were wrapped in foil and grown inside a dark growth chamber for 3–4 days. Transient Expression in Onion Epidermal Tissues Two mg of HDA-GFP fusion plasmids were coated on 20 ml of 50 mg ml21 gold particles with 2.5 M CaCl2 and 0.1 M spermidine then mixed rigorously using a vortex for 2 min. Plasmid-coated particles were dehydrated with 75% and 95% ethanol prior to bombardment. Single layer epidermal sheaths peeled from onion bulbs were placed on K MS plates then subjected to particle bombardment using the standard procedure provided by the manufacturer. Plasmid-coated gold particles were accelerated with a helium burst at 1100 psi in a PDS-1000/He instrument (BioRad). Plates containing transfected onion tissues were wrapped in foil and kept in the dark overnight (16–20 h) at room temp. Cell Fractionation and Immunodetection Nuclear and cytosolic fractions in protoplasts expressing HDA- GFP proteins were separated following the methods previously described by Ryu et al. [86] and Yanagisawa et al. [87]. Immunoblotting was carried out using 15–20 mg of proteins from transfected protoplasts, nuclear and cytosolic fractions were resolved by 10% SDS-PAGE, and detected with horseradish peroxidase- conjugated anti-GFP (Clontech), anti-RHA1 (Upstate), and anti- histone H3 (Upstate) antibodies [88]. Protein-Protein Interaction Assays For the biflourescence complementation (BiFC) assay, coding sequences of HDA5, HDA15, and 14-3-3 cloned into PCR8/ GW/TOPO were used for recombination into the destination vectors, pEarleyGate201-N-YFP and pEarleyGate202-C-YFP [89] using LR recombination mix. Kanamycin was used for bacterial selection. Purified plasmids were then analyzed for DNA sequencing for confirmation and further used for PEG transfection and imaging. Negative controls with empty vectors and test constructs and positive controls with BZR1 and 14-3-3s were initially tested to assess the efficiency of the BiFC assay (Figure S4). Coimmunoprecipitation (CoIP) was carried out using the methods of Ryu et al. [86] with some modifications. HDA-GFP plasmids with co-transfected with myc-tagged 14-3-3 K or E and then incubated for 6–8 h for protein expression. Total protein was extracted from transfected protoplasts using immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 75 mM NaCl, 5 uM EDTA, 1 mM DTT, protease inhibitor cocktail [Roche Applied Science], and 1% TritonX-100). Protein complex was precipitated with monoclonal anti-c-myc antibody (Cell Signaling) and protein A/G plus-agarose beads (Calbiochem). Precipitated proteins were detected with horseradish peroxidase-conjugated anti-GFP anti- body (Clontech). For yeast-two-hybrid assay, yeast strain AH109 PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e30846