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Brassinosteroid nuclear signaling recruits HSP90 activity Despina Samakovli, Theoni Margaritopoulou, Constantinos Prassinos, Dimitra Milioni and Polydefkis Hatzopoulos Laboratory of Molecular Biology, Agricultural University of Athens, Iera Odos 75, 118 55 Athens, Greece

Summary Author for correspondence: Polydefkis Hatzopoulos Tel: +30 210 5294321 Email: [email protected] Received: 6 December 2013 Accepted: 6 April 2014

New Phytologist (2014) 203: 743–757 doi: 10.1111/nph.12843

Key words: Arabidopsis thaliana, BIN2, brassinosteroid signaling, gene expression, heat shock protein 90 (HSP90).

 Heat shock protein 90 (HSP90) controls a number of developmental circuits, and serves a sophisticated and highly regulatory function in signaling pathways. Brassinosteroids (BRs) control many aspects of plant development.  Genetic, physiological, cytological, gene expression, live cell imaging, and pharmacological approaches provide conclusive evidence for HSP90 involvement in Arabidopsis thaliana BR signaling.  Nuclear-localized HSP90s translocate to cytoplasm when their activity is blocked by the HSP90 inhibitor geldanamycin (GDA). GDA treatment promoted the export of BIN2, a regulator of BR signaling, from the nucleus into the cytoplasm, indicating that active HSP90 is required to sustain BIN2 in the nucleus. HSP90 nuclear localization was inhibited by brassinolide (BL). HSP90s interact with BIN2 in the nucleus of untreated cells and in the cytoplasm of BL-treated cells, showing that the site-specific action of HSP90 on BIN2 is controlled by BRs. GDA and BL treatments change the expression of a common set of previously identified BRresponsive genes. This highlights the effect of active HSP90s on the regulation of BR-responsive genes. Our observations reveal that HSP90s have a central role in sustaining BIN2 nuclear function.  We propose that BR signaling is mediated by HSP90 activity and via trafficking of BIN2– HSP90 complexes into the cytoplasm.

Introduction Molecular chaperones enable organisms to cope with environmental stress and are engaged in regulating physiological processes in all kingdoms. Heat shock protein 90 (HSP90) is an indispensable molecular chaperone present in high abundance in unstressed eukaryotic cells. As such, HSP90 interacts with diverse proteins such as steroid receptors, kinases and transcription factors, promoting their folding and function (Taipale et al., 2010). As a result of the perplexity of client proteins, the complexity of the chaperone cycle, and the association with auxiliary proteins, the HSP90 substrate recognition and binding remain poorly understood. Through transient interactions with structurally labile areas of client proteins, HSP90s regulate the conformational competence and activity of a specific set of metastable proteins which are key regulatory players in biological circuitries (Pearl & Prodromou, 2006; Echeverria & Picard, 2010). At the expense of ATP, HSP90 complexes with and separates from additional factors and co-chaperones to affect a kinetically dynamic process of client protein maturation. Specific inhibitors of HSP90-dependent ATPase activity, such as geldanamycin (GDA), have been successfully used in understanding the binding specificity (Echeverria & Picard, 2010; Krukenberg et al., 2011). The conceptual Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

buffering capacity of the HSP90 system, a mechanism dampening internal and external perturbations, thus canalizing canonical development, has been extensively supported by experimental and theoretical approaches (Rutherford & Lindquist, 1998; Queitsch et al., 2002; Samakovli et al., 2007; Hsieh et al., 2013). In plants, HSP90 is associated with the co-chaperone SGT1 in the stabilization of NLR proteins, which mediate plant defense mechanisms (Kadota et al., 2010). Recent studies have shown that HSP90s regulate the maturation of ZTL, an essential component of the Arabidopsis circadian clock, revealing that HSP90 could play a central role in protein homeostasis through conformational competence of F-box proteins (Kim et al., 2011). In both animals and plants, steroid hormones regulate many of the same physiological and developmental processes, including cell division/expansion, growth, and development. Apart from cell elongation and photomorphogenesis, brassinosteroid (BR)deficient mutants also indicated important roles of BRs in seed germination, vascular differentiation, plant architecture, flowering, and senescence (Clouse, 2011). In metazoans, the HSP90 chaperone system serves a sophisticated and highly regulatory function in steroid and Wnt signaling pathways (St€adeli et al., 2006; Cooper et al., 2011). Steroid hormones bind to receptors, which are retained competent in the cytoplasm via HSP90 interaction. Ligand binding disrupts such interaction and receptors New Phytologist (2014) 203: 743–757 743 www.newphytologist.com

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enter the nucleus, altering gene expression (Pratt et al., 2004). HSP90 regulates the glycogen synthase kinase 3 (GSK3), a key component in insulin and Wnt signaling pathways. Although most effects of HSP90 were manifested in cytosolic processes, its role in the nucleus is only beginning to be appreciated. In yeast, HSP90 has been involved in a signal-dependent disassembly of transcriptional regulatory complex (Freeman & Yamamoto, 2002). In Drosophila, HSP90 was shown to interact and stabilize Trx, an essential element of the epigenetic memory (Tariq et al., 2009). Brassinosteroid signaling in plants proceeds via a multitude of kinase/phosphatase cascade. BRs bind to the extracellular domain of the plasma membrane BRASSINOSTEROID INSENSITIVE 1 (BRI1)/BRI1 KINASE INHIBITOR 1 (BKI1) receptor complex, causing the release of the negative regulator BKI1 (He et al., 2000; Kinoshita et al., 2005; Wang & Chory, 2006). The subsequent dimerization of BRI1 and BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) forms a more active BR receptor complex (Wang et al., 2008), and conceivably inactivates BRASSINOSTEROID INSENSITIVE 2 (BIN2) through BR-SIGNALING KINASES (BSKs) and BRI1 SUPPRESSORS 1 (BSU1; Li & Nam, 2002; Mora-Garcıa et al., 2004; Vert & Chory, 2006; Tang et al., 2008). The downstream signaling pathway leads to the activation of BRI1 EMS SUPPRESSOR1 (BES1) and BRASSINAZOLE RESISTANT1 (BZR1), key transcriptional factors controlling BR-responsive gene expression (Wang et al., 2002; Yin et al., 2002; Kim et al., 2009; Sun et al., 2010; Tang et al., 2011). In the absence of BR, BZR1 and BES1 are phosphorylated and inactivated by the GSK3/SHAGGY-like protein kinase BIN2 (He et al., 2002; Wang et al., 2002; Yin et al., 2002). Thus, BR signaling is ultimately mediated by the phosphorylation status of BZR1 and BZR2/BES1, which are activated by BRs via Protein Phosphatase 2A (PP2A) move to the nucleus, and facilitate signal transduction (He et al., 2002, 2005; Vert et al., 2005; Sun et al., 2010; Tang et al., 2011). GSK3b plays an essential role in the Wnt signaling pathway that is crucial for animal development. Hsp90a/b modulates the phosphorylation of b-catenin by interaction in common complex with GSK3b/ axin1/b-catenin (Cooper et al., 2011). Taken together, the BR signaling pathway shares similarities with the metazoan Wnt signaling pathway and it is distinct from steroid signaling pathways. Although it was thought for a long time that HSP90 might be involved in BR-mediated signal transduction in plants, in a similar manner to steroid-dependent signaling in animal systems (Sangster & Queitsch, 2005), it was only recently shown that HSP90s interact with BES1 (Lachowiec et al., 2013; Shigeta et al., 2014). In this study, we investigate the central role of HSP90 specifically in regulating the nuclear BR signaling network. We have identified BIN2 as an HSP90-interacting protein. Furthermore, we show that active HSP90s have a prominent role in sustaining BIN2 nuclear localization and in the downstream BR-responsive gene expression. HSP90s regulate BIN2 subcellular localization and therefore its spatially kinase activity, ultimately affecting BR signaling progression. We propose that BR signaling is mediated through the HSP90 activity and via trafficking of BIN2–HSP90 complexes. New Phytologist (2014) 203: 743–757 www.newphytologist.com

Materials and Methods Plant material and transformation Arabidopsis thaliana (L.) Heynh ecotype Columbia-0 (Col), transgenic Arabidopsis, and Nicotiana benthamiana L. (tobacco) plants were grown under 16 : 8 h, light : dark cycles at 22°C. Arabidopsis seeds were germinated on Murashige–Skoog (MS) medium and Arabidopsis transgenic plants were selected on MS medium containing 50 mg l 1 kanamycin and 200 mg l 1 cefotaxime, under the same growth conditions. The T-DNA insertion lines SALK_007614 (hsp90.1, At5g52640) and SALK_ 038646 (hsp90.3, At5g56030) were obtained from the European Arabidopsis Center, Nottingham, UK. The homozygous Arabidopsis lines HSP90.1::GUS (Haralampidis et al., 2002) and HSP90.3::GUS (Prasinos et al., 2005) were crossed with ucu-1 mutant. The resulting F1 plants were self-fertilized, and double mutants in segregating F2 populations were identified. Sevenday-old transgenic seedlings harboring the HSP90.1–GFP, the HSP90.3–GFP, or the BIN2–GFP construct were treated with dimethyl sulfoxide (DMSO, mock), 2 or 5 lM geldanamycin, 1 lM 1-naphthaleneacetic acid (NAA), or with different concentrations (10 7 M, 10 6 M, 10 5 M) of epi-brassinolide (22(S), 23(S)- Homobrassinolide; Sigma) for 1–5 d. Green fluorescent protein (GFP) reporter gene constructs The smGFP cDNA was PCR-amplified using gene-specific primers (Supporting Information, Table S1) and cloned into the pGEM-T vector (Promega). The AtHSP90.1 cDNA and the AtHSP90.3 cDNA were amplified using the specific set of primers (Table S1). The amplified fragments were subsequently fused in-frame upstream of the smGFP sequence and cloned into the pGreen0029T binary vector under the control of the AtHSP90.1 and the AtHSP90.3 promoter, respectively. The constructs obtained were named HSP90.1::HSP90.1–GFP and HSP90.3:: HSP90.3–GFP. All constructs were sequenced to check the accuracy of amplification and translational fusions and used to transform the Agrobacterium tumefaciens strain GV3101::pGV2260 by the direct transfer method. Transgenic Arabidopsis plants were obtained by the floral dip method. Histochemical and fluorometric assays for GUS activity Histochemical b-glucuronidase (GUS) activity assays were performed in flowering tissues of at least 10 independent HSP90.1:: GUS and HSP90.3::GUS F1 Arabidopsis lines, and repeated in several F2 progeny plants under normal and heat shock conditions. Tissues were incubated at 37°C overnight in X-gluc reaction buffer (50 mM sodium phosphate buffer, pH 7.2, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide and 2 mM X-gluc). Quantitative GUS assays were carried out on HSP90.3:: GUS and HSP90.3::GUS/ucu1-1 Arabidopsis lines as previously described (Haralampidis et al., 2002). Fluorescence was measured with an L550B luminescence spectrometer. Standard curves were Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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prepared with 4-methylumbelliferone (4-MU; Sigma). All measurements were repeated five times on 10 independently transformed plants from each line.

(Olympus Soft Imaging Solutions, M€ unster, Germany). Final merging of images was performed using Adobe Photoshop CS2 (version 9.01; Adobe Systems, San Jose, CA, USA).

Yeast two-hybrid assay

Immunoblot and coimmunoprecipitation assays

Interaction studies were performed in yeast strain SG335 using the Matchmaker GAL4 two-hybrid system according to the manufacturer’s protocol (Clontech, Mountain View, CA, USA). The full-length cDNAs of HSP90.1 and HSP90.3 were cloned into the pGBKT7 vector (Clontech), which contains the Gal4 DNAbinding domain and AtBIN2 (At4g18710) was cloned into the pGADT7 vector (Clontech), which contains the Gal4 DNA activation domain. Yeast transformants were selected on synthetic dropout nutrient medium (SD/-Leu/-Trp) and interactions were assayed on selective medium (SD/-Leu/-Trp/-His supplemented with 15 mM 3-amino-1,2,4-triazole). To further test the specificity of the interactions, the b-galactosidase expression of the His+ colonies was analyzed by filter-lift assays.

Tobacco leaf cells transiently and singly expressing or coexpressing the HSP90.3–HA–YFPc and BIN2–cMyc–YFPn fusion proteins were harvested 3 d after infiltration. The tissue was ground in liquid nitrogen, and nuclear and cytoplasmic fractions were separated as previously described (Bowler et al., 2004). The protein extracts were incubated overnight with anti-hemagglutinin (antiHA) antibody and then with Protein G PLUS-Agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C for 4 h. The immunoprecipitated proteins and the corresponding protein extracts as inputs were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by anti-c-Myc antibody (Santa Cruz Biotechnology, Inc.), anti-HA antibody (Santa Cruz Biotechnology, Inc.), anti-phosphotyrosine (Cell Signaling) antibody and anti-acetyl-histone H3 antibody (Millipore).

RNA isolation and analysis Total RNA was isolated from 10-d-old seedlings that were exposed to 3 lM BRZ2001, 1 lM BL or 2 lM GDA for up to 72 h and from control seedlings using the phenol-sodium dodecyl sulfate (SDS) extraction method. RNA concentrations were determined spectrophotometrically and verified by ethidium bromide staining on agarose gels. DNA was eliminated with RQ1 RNase free DNase (Promega). Reverse transcription (RT) was performed on 3 lg of total DNA-free RNA using Superscript II Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol. PCR amplification for each transcript was performed using the pair of gene-specific primers (Table S1). The linear PCR amplification of each transcript was confirmed in preliminary experiments by comparing the relative amounts of PCR products under low and high RT-PCR cycles of amplification. RT-PCR reactions were normalized using the Arabidopsis adenine phosphoribosyl transferase 1 (APT1, At1g27450) or the housekeeping gene 18S rRNA (Table S1). The gene specificity of RT-PCR products was confirmed by sequencing. Bimolecular fluorescence complementation (BiFC) and fluorescent microscopy imaging The full-length cDNAs of HSP90.1, HSP90.3, and BIN2 were cloned into either the pSPYCE or the pSPYNE vector. Transient expression of the fusion proteins was carried out in 4-wk-old N. benthamiana plants. Infiltrations were done by injection on the bottom face of the leaf using a syringe without a needle. The plants were incubated under normal growing conditions and analyzed 3–4 d after infiltration. At least three individual experiments were performed for each combination. Tobacco mesophyll protoplasts were isolated and incubated in liquid medium containing 10 lΜ BL for 2 h. Specific BiFC protein–protein interactions were examined using epifluorescence microscopy. Images were taken with an Olympus DP71 camera, using Cell^A Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Quantification of fluorescent protein signal All images were analyzed with ImageJ (http://imagejnih.gov/ij/). To measure the ratio between nuclear and cytoplasmic signals of HSP90.1–GFP, HSP90.3–GFP, and BIN2–GFP lines for each cell, a small area of fixed size was defined, and measurements of integrated densities were taken from representative areas within the nucleus, cytoplasm, and background (central vacuole) of each cell. Three repeated measurements were performed for each cell, and the average of background values was then subtracted from the average values for the nuclear and cytoplasmic signals. The nuclear/cytoplasmic signal ratio was then calculated for each cell. The average nuclear/cytoplasmic signal ratio and standard error for independent T2 transgenic plants were calculated from measurements of at least seven cells from each plant. Line scan measurements spanning both the nucleus and cytoplasm were also carried out, and representative plot profiles of sample measurements are presented. Statistical analysis of fluorescence density data was performed using the IBM SPSS Statistics v 20.0 (IBM Corp., Armonk, NY, USA). Phenotypic analyses of hypocotyl sections Hypocotyls of 60-d-old wild-type, hsp90-1, hsp90-3 and bri1-4 mutant Arabidopsis plants were fixed in 50% ethanol, 10% formaldehyde, and 5% acetic acid. Hand-cut sections were performed using common razor blades and were observed under UV light. Bioinformatics Predictions of potential nuclear localization signals (NLS) and nuclear export sequences (NES) were carried out using NL Stradamus (http://www.moseslab.csb.utoronto.ca/NLStradamus) and NetNES (http://www.cbs.dtu.dk/services/NetNES). Subcellular localization predictions were performed using PSORT New Phytologist (2014) 203: 743–757 www.newphytologist.com

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(http://psort.org), the Support Vector Machine (SVM)-based system (http://array.bioengr.uic.edu/subnuclear.htm) and the PredictProtein server (https://www.predictprotein.org/).

Results Nuclear HSP90.1 and HSP90.3 localization depends on active HSP90 The Arabidopsis HSP90 gene family consists of seven members, four of which are referred as cytosolic and three as organelle isoforms (Milioni & Hatzopoulos, 1997; Krishna & Gloor, 2001). Even though the cytosolic members have extensive homologies, the four genes can be distinguished into two groups: AtHSP90.1 consists of four exons and is a bona fide heat-induced gene, while AtHSP90.2–.4 consist of three exons and are considered almost constitutively expressed and highly homologous (Milioni & Hatzopoulos, 1997). Computational analysis of the four cytosolic HSP90 proteins predicted that all members contain nuclear localization signals (NLSs) and nuclear export sequences (NESs; Fig. 1). A clear difference between HSP90.1 and the other cytosolic members is the presence of an additional NLS motif and a higher nuclear prediction score (Fig. 1). To test these predictions experimentally, we generated green fluorescent protein (GFP)-tagged fusions with HSP90.1 and HSP90.3 driven by their respective native promoters (Fig. S1). The products of the HSP90.1::HSP90.1–GFP and HSP90.3:: HSP90.3–GFP constructs showed prominent nuclear localization in root and hypocotyl cells (Figs 2, S2). Nevertheless, cytoplasmic signal, although less intense, was also apparent. Visual differences were quantified and depicted as nuclear/cytoplasmic fluorescence density ratios. The relative nuclear/cytoplasmic fluorescence density ratios of HSP90.1–GFP and HSP90.3–GFP were 5.57 and 5.7, respectively (Fig. 2). HSP90 nuclear localization has also been demonstrated in mammals and Drosophila (Gasc et al., 1990; Sawarkar et al., 2012). The effect of heat or cold stress on HSP90.1–GFP and HSP90.3–GFP subcellular localization was investigated. Neither heat (37°C, 2 h) nor cold stress (4°C, 2 h) had any effect on the subcellular localization of HSP90.1–GFP and HSP90.3–GFP fusion proteins. HSP90.3–GFP had a distinct spot/speckle-like nuclear appearance (Fig. S2d,g), while HSP90.1–GFP exhibited a rather compact nuclear appearance (Fig. S2a,c,f). Taken together, both HSP90.1 and HSP90.3 cytosolic members had a prominent nuclear localization. To investigate whether active HSP90 is a prerequisite for nuclear localization, the drug GDA, which binds to HSP90 with high affinity and specifically inhibits its chaperone activity, was used (Roe et al., 1999). The biological effect of GDA at 2 lΜ concentration was monitored 5 d after treatment (Queitsch et al., 2002; Lachowiec et al., 2013). Both HSP90.1–GFP and HSP90.3–GFP had a fuzzy nuclear appearance and green fluorescence was noticeably diminished in the nucleus within the first 24 h of drug treatment while the cytosolic signal became more intense. The relative nuclear/cytoplasmic fluorescence density ratio of both HSP90–GFP fusion proteins decreased to c. 1.78 at 24 h after the addition of GDA (Fig. 2a, Table S2). This 3.2-fold New Phytologist (2014) 203: 743–757 www.newphytologist.com

reduction in nuclear/cytoplasmic fluorescence ratio when compared with untreated seedlings indicated a rather rapid exclusion of HSP90.1 and HSP90.3 from the nucleus. Further reduction (c. 4.0- and 6.7-fold) was estimated when transgenic lines were treated for 3 and 5 d with GDA, respectively (Fig. 2a, Table S2). Nuclear localization of HSP90.1 or HSP90.3 was not affected in seedlings treated with DMSO (Fig. S3). The negative exponential distribution curves depicting the nucleocytoplasmic shuttling were very similar for both HSP90–GFP proteins when HSP90s were blocked by the specific inhibitor GDA (Fig. 2b). Therefore, active HSP90 is a prerequisite for the nuclear accumulation of HSP90.1 and HSP90.3. HSP90.1 and HSP90.3 interact with BIN2 The well-established role of HSP90 proteins in glucocorticoid receptors’ nuclear translocation and the dependence of GSK3b on HSP90 to form functional intermediates (Lochhead et al., 2006; Cooper et al., 2011) in Wnt signaling prompted us to investigate whether HSP90 interacts with BIN2 physically. HSP90.1 and HSP90.3 interacted strongly with BIN2 in yeast two-hybrid assays (Fig. 3a). The interactions between BIN2 and HSP90.1 or HSP90.3 were also confirmed in planta by BiFC assays in N. benthamiana leaves (Fig. 3b). Nuclear fluorescence caused by the reconstruction of YFP was observed predominately in nuclei of epidermal cells of leaves that coexpressed either HSP90.1–YFPc or HSP90.3–YFPc and BIN2–YFPn, whereas fluorescence in nuclei of control leaves that coexpressed either HSP90.1–YFPc or HSP90.3–YFPc and YFPn or YFPc and BIN2–YFPn was below detection limits (Fig. S4). This interaction was also substantiated in coimmunoprecipitation experiments. Proteins were extracted from tobacco leaves expressing HSP90.3–HA–YFPc and BIN2–cMyc–YFPn transiently. HSP90.3–HA was immunopurified using the anti-HA antibody and the myc-tagged BIN2 was copurified with HSP90.3–HA. The results showed that HSP90.3 interacted with BIN2. This interaction was profound in the nuclear extracts (Fig. 3c). Inhibited HSP90s redirect BIN2 nuclear localization HSP90.1 and HSP90.3 interacted strongly with BIN2. BIN2– GFP had a similar spot-like pattern to HSP90.3–GFP in the nucleus (Fig. S2e,d); nevertheless fluorescence was also detected in the cytoplasm. To investigate whether active HSP90 is a critical component of the canonical GSK3-like BIN2 nuclear localization, we examined the impact of the GDA inhibitor. The relative nuclear/cytoplasmic fluorescence density ratio was calculated to be 5.16 under control conditions (Fig. 4, Table S3). GDA-treated BIN2–GFP seedlings showed a gradual accumulation of BIN2 in the cytosol in a time-dependent mode. The relative nuclear/cytoplasmic fluorescence density ratio moderately decreased to 4.76 after treatment with GDA for 24 h. When transgenes were treated for 3 and 5 d with GDA, the relative ratios further decreased to 1.89 and 0.85, respectively (Fig. 4, Table S3). The rate of decrease of the BIN2 relative fluorescence ratio was lower than those of HSP90.1 and HSP90.3 after 24 h Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 1 Nuclear signature motifs in heat shock proteins 90 (HSP90s). (a) Multiple amino acid sequence alignment of the Arabidopsis cytoplasmic HSP90.1 (At5g52640), HSP90.2 (At5g56010), HSP90.3 (At5g56030) and HSP90.4 (At5g56000) proteins. Red and blue letters highlight nuclear localization signals (NLSs) and nuclear export signals (NESs), respectively. The HSP90.1 contains two NLSs. Gaps indicated by dashes have been introduced to maximize alignment. Asterisks (*) denote identical amino acid residues. Two dots (:) and one dot (.) denote conservative and semiconservative substitutions, respectively. Numbers indicate the amino acid residue position. (b) In silico analysis of the predicted signature motifs and nuclear localization using different software programs.

At5g56010 -----MADAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSDALDKIRFESLTDKS 55 At5g56000 -----MADAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSDALDKIRFESLTDKS 55 At5g52640 MADVQMADAETFAFQAEINQLLSLIINTFYSNKEIFLRELISNSSDALDKIRFESLTDKS 60 ******************************************************* At5g56030 KLDGQPELFIHIIPDKTNNTLTIIDSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADV 115 At5g56010 KLDGQPELFIHIIPDKTNNTLTIIDSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADV 115 At5g56000 KLDGQPELFIHIIPDKTNNTLTIIDSGIGMTKADLVNNLGTIARSGTKEFMEALAAGADV 115 At5g52640 KLDGQPELFIRLVPDKSNKTLSIIDSGIGMTKADLVNNLGTIARSGTKEFMEALQAGADV 120 **********:::***:*:**:******************************** ***** At5g56030 SMIGQFGVGFYSAYLVADKVVVTTKHNDDEQYVWESQAGGSFTVTRDTSGETLGRGTKMV 175 At5g56010 SMIGQFGVGFYSAYLVADKVVVTTKHNDDEQYVWESQAGGSFTVTRDTSGEALGRGTKMV 175 At5g56000 SMIGQFGVGFYSAYLVADKVVVTTKHNDDEQYVWESQAGGSFTVTRDTSGEALGRGTKMI 175 At5g52640 SMIGQFGVGFYSAYLVAEKVVVTTKHNDDEQYVWESQAGGSFTVTRDVDGEPLGRGTKIT 180 *****************:*****************************..**.******: At5g56030 LYLKEDQLEYLEERRLKDLVKKHSEFISYPISLWIEKTIEKEISDDEEEEE-KKDEEGKV 234 At5g56010 LYLKEDQMEYIEERRLKDLVKKHSEFISYPISLWIEKTIEKEISDDEEEEE-KKDEEGKV 234 At5g56000 LYLKEDQMEYIEERRLKDLVKKHSEFISYPISLWIEKTIEKEISDDEEEEE-KKDEEGKV 234 At5g52640 LFLKDDQLEYLEER--RDLVKKHSEFISYPIYLWTEKTTEKEISDDEDEDEPKKENEGEV 238 *:**:**:**:*** :************** ** *** ********:*:* **::**:* At5g56030 EEVDEEKE KEEKKKKKIKEVSHEWDLVNKQKPIWMRKPEEINKEEYAAFYKSLSNDWEEH 294 At5g56010 EEVDEEKE KEEKKKKKIKEVSHEWDLVNKQKPIWMRKPEEINKEEYAAFYKSLSNDWEEH 294 At5g56000 EEIDEEKE KEEKKKKKIKEVTHEWDLVNKQKPIWMRKPEEINKEEYAAFYKSLSNDWEEH 294 At5g52640 EEVDEEKE KDGKKKKKIKEVSHEWELINKQKPIWLRKPEEITKEEYAAFYKSLTNDWEDH 298 **:***** *: *********:***:*:*******:******.***********:****:* At5g56030 LAVKHFSVEGQLEFKAILFVPKRAPFDLFDTKKKPNNIKLYVRRVFIMDNCEDIIPEYLG 354 At5g56010 LAVKHFSVEGQLEFKAILFVPKRAPFDLFDTKKKPNNIKLYVRRVFIMDNCEDIIPEYLG 354 At5g56000 LAVKHFSVEGQLEFKAILFVPKRAPFDLFDTKKKPNNIKLYVRRVFIMDNCEDIIPDYLG 354 At5g52640 LAVKHFSVEGQLEFKAILFVPKRAPFDLFDTRKKLNNIKLYVRRVFIMDNCEELIPEYLS 358 *******************************:** *****************::**:**. At5g56030 FVKGIVDSEDLPLNISRETLQQNKILKVIRKNLVKKCLELFFEIAENKEDYNKFYEAFSK 414 At5g56010 FVKGIVDSEDLPLNISRETLQQNKILKVIRKNLVKKCLELFFEIAENKEDYNKFYEAFSK 414 At5g56000 FVKGIVDSEDLPLNISRETLQQNKILKVIRKNLVKKCLELFFEIAENKEDYNKFYEAFSK 414 At5g52640 FVKGVVDSDDLPLNISRETLQQNKILKVIRKNLVKKCIEMFNEIAENKEDYTKFYEAFSK 418 ****:***:****************************:*:* *********.******** At5g56030 NLKLGIHEDSQNRTKIAELLRYHSTKSGDELTSLKDYVTRMKEGQNDIFYITGESKKAVE 474 At5g56010 NLKLGIHEDSQNRTKIAELLRYHSTKSGDELTSLKDYVTRMKEGQNDIFYITGESKKAVE 474 At5g56000 NLKLGIHEDSQNRTKIAELLRYHSTKSGDELTSLKDYVTRMKEGQNEIFYITGESKKAVE 474 At5g52640 NLKLGIHEDSQNRGKIADLLRYHSTKSGDEMTSFKDYVTRMKEGQKDIFYITGESKKAVE 478 ************* ***:************:**:***********::************* At5g56030 NSPFLEKLKKKGIEVLYMVDAIDEYAIGQLKEFEGKKLVSATKEGLKLD-ETEDEKKKKE 533 At5g56010 NSPFLEKLKKKGIEVLYMVDAIDEYAIGQLKEFEGKKLVSATKEGLKLD-ETEDEKKKKE 533 At5g56000 NSPFLEKLKKKGYEVLYMVDAIDEYAIGQLKEFEGKKLVSATKEGLKLE-ETDDEKKKKE 533 At5g52640 NSPFLERLKKRGYEVLYMVDAIDEYAVGQLKEYDGKKLVSATKEGLKLEDETEEEKKKRE 538 ******:***:* *************:*****::**************: **::****:* At5g56030 ELKEKFEGLCKVIKDVLGDKVEKVIVSDRVVDSPCCLVTGEYGWTANMERIMKAQALRDS 593 At5g56010 ELKEKFEGLCKVIKDVLGDKVEKVIVSDRVVDSPCCLVTGEYGWTANMERIMKAQALRDS 593 At5g56000 ELKEKFEGLCKVIKDVLGDKVEKVIVSDRVVDSPCCLVTGEYGWTANMERIMKAQALKDS 593 At5g52640 EKKKSFENLCKTIKEILGDKVEKVVVSDRIVDSPCCLVTGEYGWTANMERIMKAQALRDS 598 * *:.**.***.**::********:****:***************************:** At5g56030 SMAGYMSSKKTMEINPENSIMDELRKRADADKNDKSVKDLVLLLFETALLTSGFSLDEPN 653 At5g56010 SMGGYMSSKKTMEINPENSIMDELRKRADADKNDKSVKDLVLLLFETALLTSGFSLDEPN 653 At5g56000 NTGGYMSSKKTMEINPENSIMDELRKRAEADKNDKSVKDLVLLLFETALLTSGFSLDEPN 653 At5g52640 SMSGYMSSKKTMEINPDNGIMEELRKRAEADKNDKSVKDLVMLLYETALLTSGFSLDEPN 658 . .*************:*.**:******:************:**:*************** At5g56030 TFGSRIHRM LKLGLSIDDDDAVEADAEMPPLEDDADAEGSKMEEVD 699 At5g56010 TFGSRIHRM LKLGLSIDDDDVVEADADMPPLEDDADAEGSKMEEVD 699 At5g56000 TFGSRIHRM LKLGLSIEEDDAVEADAEMPPLEDDADAEGSKMEEVD 699 At5g52640 TFAARIHRM LKLGLSIDEDENVEEDGDMPELEEDAAEE-SKMEEVD 703 **.:************ ::*: ** *.:** **:** * *******

(b) pSORT Subnuclear compartment prediction NLStradamus NetNES

Hsp90.1 Nucleus 0.96 Nuclear lamina

aa:243-256, 0.9 aa:534-542, 0.75 aa:668-674, 0.841 aa: amino acid position

incubation with GDA. However, the BIN2 relative decrease rate was similar to those of HSP90.1 and HSP90.3 at 3 and 5 d incubation of the transgenes with GDA (Tables S2,S3). Similarly, the BIN2 relative nuclear/cytosolic signal density followed a negative exponential distribution curve similar to that of the HSP90.1 or Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Hsp90.2 Nucleus 0.82 Nuclear lamina

Hsp90.3 Nucleus 0.82 Nuclear lamina

Hsp90.4 Nucleus 0.82 Nuclear lamina

aa:243-252, 0.85

aa:243-252, 0.85

aa:243-252, 0.85

aa:663-669, 1.181

aa:662-669, 1.191

aa:663-669, 1.191

HSP90.3 distribution when the HSP90 was blocked by GDA (Fig. 4b, Tables S2,S3). The nuclear localization of BIN2–GFP was not affected in seedlings treated with DMSO (Fig. S3). To validate the specificity of GDA treatment, we tested whether the inhibitor affects the nuclear localization of the New Phytologist (2014) 203: 743–757 www.newphytologist.com

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Fig. 2 Geldanamycin (GDA) induces changes in the nucleocytoplasmic partitioning of the heat shock protein 90 (HSP90). (a) Effect of GDA on the subcellular localization of HSP90.1–GFP (green fluorescent protein) and HSP90.3–GFP. Transgenic Arabidopsis thaliana seedlings expressing HSP90.1–GFP and HSP90.3–GFP were treated with 2 lΜ GDA for 0 (control), 1, 3 or 5 d. GFP images were obtained by using epifluorescene microscopy. The numbers in each image show the average ratios between nuclear and cytoplasmic fluorescence signal densities and standard errors, calculated from at least seven cells for each treatment. White lines inside the images show the areas used for line scan measurements that yielded plot profiles shown in the lower panels. n, nuclear signal; c, cytoplasmic signal. Bars, 20 lm. (b) Kinetics of the GDA-induced nuclear exodus of HSP90.1–GFP (red) and HSP90.3–GFP (blue). Data were obtained using the average ratios between nuclear and cytoplasmic signal densities.

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Fig. 3 BIN2 interacts with heat shock protein 90 (HSP90). (a) Yeast two-hybrid assays using BRASSINOSTEROID INSENSITIVE 2 (BIN2) as bait and HSP90.1 or HSP90.3 as prey. Left column, cotransformants grown on (synthetic dropout nutrient medium) SD-Leu-Trp (SD2) demonstrate that both bait and prey plasmids are present in yeast. Positive selection of yeast two-hybrid interactions with minimal selection on SD-Leu-Trp-His medium (SD3). The blue color represents positive interactions between BIN2 and HSP90.1 or HSP90.3 in the presence of X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside, right column). (b) HSP90.1– or HSP90.3–BIN2 interaction is localized mostly in the nucleus. In vivo interactions of BIN2 and HSP90.1 or HSP90.3 were confirmed by bimolecular fluorescence complementation (BiFC). The N-terminal and C-terminal domains of yellow fluorescence protein (YFP) were fused to BIN2 and HSP90.1 or HSP90.3, respectively. Bright field (l), Chl autofluorescence (r), yellow fluorescent protein (g) and overlay images (o/l) of r and g. Bars, 20 lm. (c) Coimmunoprecipitation assays of HSP90.3 with BIN2. Total (t), cytosolic (c) and nuclear (n) protein fractions were extracted from tobacco leaves transiently expressing HSP90.3–HA (HSP90.3 fused with hemagglutinin (HA) epitope) and/or BIN2–c-Myc (BIN2 fused with c-myc fused with c-myc epitope), and immunoprecipitated with anti-HA antibody. The coimmunoprecipitated proteins were detected by anti-c-Myc, antiphosphotyrosine (P-Tyr) or anti-HA antibody. For western blot assays, leaf protein extracts (no immunoprecipitation) were detected with anti-c-Myc, anti-HA or anti-acetyl-histone H3 (Ac-H3) antibody. Arrows indicate BIN2-cMyc protein; arrowheads indicate BIN2-cMyc phosphorylated protein forms; and asterisks indicate an unspecific band which was used as a loading control. New Phytologist (2014) 203: 743–757 www.newphytologist.com

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Fig. 4 BIN2 nucleocytoplasmic trafficking depends on heat shock protein 90 (HSP90) activity. (a) Transgenic Arabidopsis seedlings expressing BIN2–GFP (green fluorescent protein) were treated with 2 lΜ geldanamycin (GDA) for 0 (control), 1, 3 or 5 d. GFP images were obtained using epifluorescene microscopy. The numbers in each image show the average ratios between nuclear and cytoplasmic fluorescence signal densities and standard errors calculated from seven cells for each treatment. White lines inside the images show the areas used for line scan measurements that yielded plot profiles shown in the lower panels. n, nuclear signal; c, cytoplasmic signal. Bars, 20 lm. (b) Kinetics of the GDA-induced nuclear exodus of BIN2–GFP (black), HSP90.1–GFP (red) and HSP90.3–GFP (blue). Data were obtained using the average ratios between nuclear and cytoplasmic densities.

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transcription factors, RHD6 (Menand et al., 2007) and RSL4 (Yi et al., 2010). Seven-day-old RHD6::mch-RHD6 and RSL4:: GFP-RSL4 seedlings were treated with 2 lM GDA and the localization of RHD6 and RSL4 was monitored after 1, 3 and 5 d of treatment (Fig. S5). Intriguingly, GDA treatment had no effect on the nuclear localization of the tested proteins, demonstrating that the effect of GDA on BIN2 localization is specifically routed via HSP90. Our observations strongly imply that active HSP90s are recruited for proper nuclear compartmentalized localization of BIN2 kinase. GDA modulates BR-responsive gene expression similarly to BL Inactivated HSP90.1 and HSP90.3 are exported from the nucleus and are incapable of sustaining BIN2 nuclear localization. This conditional BIN2 exodus from the nucleus most probably affects its specific compartmentalized function to inactivate BZR1 and BES1 transcription factors. To verify the latter, the expression of specific genes known to be affected by the presence/ absence of BRs was tested. BL down-regulates the transcriptional activity of biosynthetic genes such as CPD1 and DWF4, and upregulates TCH4, a BR-responsive gene, while the BR biosynthetic inhibitor brassinazole (BRZ2001) acts in opposite way, up-regulating the former two genes and down-regulating the latter (Mathur et al., 1998; Goda et al., 2002; Iliev et al., 2002; Yin et al., 2002; Tanaka et al., 2005; Kim et al., 2006). If BR-regulated gene expression is mediated via HSP90 function and/or its conditional nuclear shuttling, then GDA that affects BIN2 nuclear localization should modulate the gene transcription pattern similarly to BL. In fact, GDA application decreased the expression of both CPD1 and DWF4 genes and induced the expression of the TCH4 gene (Fig. 5). Consequently, inactive HSP90s and/or improper HSP90.1 and HSP90.3 localization affected the expression of these genes in a similar way to BL. Our Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

Fig. 5 Heat shock protein 90 (HSP90) activity navigates brassinosteroid (BR) response. Semiquantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of DWARF4 (DWF4), Constitutive Photomorphogenesis and Dwarfism (CPD) and Touch Induced Protein 4 (TCH4) gene expression in 10-d-old seedlings from control (Col) and wild-type Arabidopsis thaliana plants treated with 3 lM BRZ2001 (BRZ), 1 lM brassinolide (BL) and 2 lM geldanamycin (GDA), respectively, for 12 h. 18S rRNA gene was used as an internal control. RNA was reversetranscribed and amplified until the mid-log phase using specific primers.

results emphasize that the HSP90 operates as an essential component in sustaining BIN2 activity in nuclear brassinosteroid signaling. To test the effect of BRs and GDA on hypocotyl growth, wildtype Arabidopsis and hsp90.1 or hsp90.3 mutant seedlings were grown on media containing 60 nM BL or 2 lM GDA. Both BR and GDA reduced the hypocotyl length of 7-d-old etiolated wild-type and hsp90.3 mutant Arabidopsis seedlings (Fig. S6). Nevertheless, hypocotyl growth of the etiolated hsp90.1 mutant was reduced and had a comparable hypocotyl length when the mutant was grown in the presence of BL. GDA drastically reduced the hypocotyl length of all seedlings (Fig. S6). BR homeostasis affects HSP90.1 and HSP90.3 expression Nuclear brassinosteroid signaling recruits HSP90s to regulate BR-responsive gene expression. To determine whether BR New Phytologist (2014) 203: 743–757 www.newphytologist.com

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homeostasis has an impact on HSP90 gene expression, ucu1-1 (BIN2) mutant plants (Perez-Perez et al., 2002) that accumulate high amounts of BRs were tested. Semiquantitative RT-PCR analysis revealed that AtHSP90.3 mRNA was increased in the ucu1-1 mutant compared with that of wild-type seedlings, while AtHSP90.1 mRNA levels were almost undetectable (Fig. 6b). However, transcript abundances of both AtHSP90.1 and AtHSP90.3 genes were higher in heat-stressed ucu1-1 plants than that in stressed wild-type plants. In order to evaluate whether the transcript accumulation was a result of selective altered site-specific expression, transgenes bearing the promoters of AtHSP90.1 and AtHSP90.3 genes (Haralampidis et al., 2002; Prasinos et al., 2005) driving b-glucuronidase (GUS) were crossed to the ucu1-1 mutant. Under normal or heat-stress conditions, GUS activity driven by AtHSP90.3 promoter increased at least 10-fold in ucu1-1 mutant background with respect to wild-type plants. Strong GUS staining was detected in mature male and female floral tissues of stressed and unstressed F2 ucu1-1 mutant plants (Fig. 6a), showing a similar pattern of site-specific expression to wild-type plants bearing the respective construct. Recently, chip-on-chip data analysis showed that AtHSP90.3 belongs to the high confidence BR-regulated BZR1-target (BRBT) group of genes (Sun et al., 2010), corroborating these results. However, almost no AtHSP90.1 promoter activity was detected in mature floral tissues of ucu1-1 mutant plants under normal conditions as assessed by GUS staining. Interestingly, under heatstress conditions, GUS staining was prominent and comparable to that observed in Arabidopsis plants bearing the respective construct (Fig. S7). Taken together, the interplay between the BR homeostasis (BR-insensitive 2 mutant), and HSP90.1 and (a)

HSP90.3 gene expression emphasizes the crosstalk between BRs and HSP90. BRs alter HSP90.1 and HSP90.3 nucleocytoplasmic shuttling In metazoans HSP90 is a crucial component in steroid and Wnt signaling pathways, affecting a number of physiological and developmental processes (Picard, 2006). Mutations on BRI1 and BIN2 genes showed a range of phenotypes from plant architecture to flowering (Yin et al., 2002; Clouse, 2011). hsp90 mutants in Arabidopsis show a plethora of phenotypes. Nevertheless, certain phenotypic types showed reduced fertility, abnormal vascular development (Samakovli et al., 2007) and extreme stunt phenotypes (Fig. 7a), resembling the loss- or gain-of-function mutants in BR signaling pathways (Clouse et al., 1996; Yin et al., 2002). Brassinosteroid-deficient or -insensitive mutants generally display cell growth phenotypes, particularly affecting hypocotyl elongation (Chory et al., 1991; Li et al., 1996; Wang et al., 2001, 2002). It has been reported that BRs are involved in the early events of cambium to phloem or xylem cell differentiation and formation (Iwasaki & Shibaoka, 1991; Yamamoto et al., 2001; Iba~ nes et al., 2009). Observation of transverse hypocotyl sections of hsp90.1, hsp90.3, and bri1-4 mutant plants revealed that lignified materials represented by autofluorescence were completely or partially lost in the presumptive secondary xylem (Fig. 7b), reminiscent of BR-deficient mutants. To test whether the BR signaling pathway has an effect on HSP90s spatial subcellular topology, HSP90–GFP transgenic lines were treated with different concentrations of the BR analog, BL. Interestingly, BL application resulted in a massive exodus of (b)

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Fig. 6 Brassinosteroid (BR) homeostasis affects HSP90 expression. ucu1-1 mutant background induces HSP90.3 gene expression. (a) HSP90.3::GUS (b-glucoronidase) qualitative analysis in Arabidopsis thaliana wild-type (Col; upper panels) or in ucu1-1 mutant background (lower panels) under normal (left panels) or heat stress conditions at 37°C for 2 h (right panels). Bars, 1 mm. (b) Semiquantitative expression profile analysis of HSP90.1 (90.1) and HSP90.3 (90.3) genes in 15-d-old seedlings from control (Col) and ucu1-1 (ultracurvata 1-1) mutant plants under normal (left panel) and heat stress conditions (right panel). The Adenine Phosphoribosyl Transferase 1 (APT1) gene was amplified as an internal control. (c) GUS quantitative analysis of the above lines under normal (white) or heat stress conditions (black). Error bars,  SD. New Phytologist (2014) 203: 743–757 www.newphytologist.com

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Fig. 7 Crosstalk between heat shock proteins 90 (HSP90s) and brassinosteroids (BRs). (a) Wild-type Arabidopsis (wt) and hsp90.1 and hsp90.3 mutant plants show stunt phenotypes. (b) Disorganized secondary xylem architecture in hypocotyls of hsp90.1, hsp90.3 and brassinosteroid insensitive 1-4 (bri14) mutants. Transverse sections of hypocotyls from 60-d-old plants (wt), hsp90.1, hsp90.3 and bri1-4 mutants observed under UV light. v, vessels; fib, fibers; x, xylem; bars, 500 lm. (c) Effect of BL on the nuclear localization of HSP90.1–GFP (green fluorescent protein) and HSP90.3–GFP. Transgenic Arabidopsis seedlings expressing HSP90.1–GFP and HSP90.3–GFP were treated with 1 lM brassinolide (BL) for 1, 2, 3 or 5 d and images of the GFP signal were obtained by using epifluorescence microscopy. The numbers in each image show the average ratios between nuclear and cytoplasmic fluorescence signal densities and standard errors were calculated from seven cells for each treatment. White lines inside the images show the areas used for line scan measurements that yielded plot profiles shown in the lower panels. n, nuclear signal; c, cytoplasmic signal. Bars, 20 lm. (d) Kinetics of geldanamycin (GDA)- or BL-induced nuclear exodus of HSP90.1–GFP, HSP90.3–GFP and GDA-induced nuclear exodus of BIN2–GFP. Data were obtained using the average ratios between nuclear and cytoplasmic signal densities. Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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HSP90.1–GFP and HSP90.3–GFP from the nucleus (Fig. 7c). BL application redirected the HSP90.1 and HSP90.3 subcellular localization in a similar manner to that observed when transgenes were treated with GDA. An average 2.2-fold decrease of the relative nuclear/cytoplasmic fluorescence density ratio was seen when transgenes were treated with 10 6 M BL for 24 h (Fig. 7c, Table S4). A marked shift in the localization of HSP90.1 and HSP90.3 out of the nucleus was observed in 2, 3 or 5 d BL-treated lines. The relative nucleus/cytoplasmic fluorescence density ratio decreased c. 4.2-, 4.6- and 6.7-fold after 2, 3 and 5 d of BL treatment, respectively (Fig. 7c, Table S4). As auxin alone did not affect the nuclear localization of HSP90.1 and HSP90.3, the massive exodus from the nucleus is a BL-specific response (Fig. S8). The rate of HSP90.1 or HSP90.3 depletion from the nucleus was affected by BL concentration in a time-dependent mode. It is interesting to note that an average 7.3-fold decrease of the relative nuclear/cytoplasmic fluorescence density ratio was estimated when transgenes were treated with 10 5 M BL for 24 h. In fact, at 24 h the cytoplasmic fluorescence density was higher than that estimated in the nucleus (Fig. S9), showing that both HSP90s were rapidly excluded from the nucleus and mainly accumulated in the cytoplasm. Further incubation for 2, 3, and 5 d had no effect on the relative nuclear/cytoplasmic fluorescence density ratio, showing that within the first 24 h, all the HSP90.1 or HSP90.3 was depleted from the nucleus (Fig. S9). There is a sharper decline in the relative nucleus/cytoplasmic fluorescence density ratio in transgenes treated with 10 5 M BL than in those treated with 10 7 M BL. Nevertheless, the relative nucleus/cytoplasmic fluorescence density ratio decreased c. sevenfold after 5 d of BL application irrespective of the concentration (Fig. S9). To confirm this observation, we followed the HSP90.1–GFP nucleocytoplasmic shuttling during the first 24 h after addition of 1 lΜ BL. BL treatment triggered nuclear depletion of the HSP90.1 protein as early as 15 min and this depletion increased for at least 4 h (Fig. S10). Collectively, the negative exponential distribution curves illustrating the nucleocytoplasmic shuttling of the HSP90.1–GFP and HSP90.3–GFP in the presence of BL were very similar to those derived when GDA was applied to HSP90.1- and HSP90.3–GFP or BIN2–GFP lines (Figs 2, 7, S9). Data analysis resulted in monotonic exponential functions and their first derivatives are expressed as N(t) = N0e et, where N(t) stands for the nuclear/cytoplasmic density fluorescence ratio at time t; N0 is the initial ratio, that is the ratio at time t = 0, and e is the ‘exodus rate’ constant from the nucleus (Notes S1). In the BIN2–GFP transgenic line treated with GDA, the first derivative of the relative nuclear/cytoplasmic fluorescence density ratio was N(t) = 3.8001e 0.36266t (R2 = 0.9786). In HSP90–GFP lines treated with 10 6 Μ BL, the first derivative of the relative nuclear/cytoplasmic fluorescence density ratio was N(t) = 3.8001e 0.3626t (R2 = 0.7885) for HSP90.1 and N(t) = 3.9219e 0.336t (R2 = 0.8368) for HSP90.3. These functions are graphically depicted in Figs 2, 4, and 7. Taken together, the three equations have a very similar e constant, which varies in the range 0.336–0.3626. Intriguingly, the exponential functions estimated by the nucleocytoplasmic New Phytologist (2014) 203: 743–757 www.newphytologist.com

fluorescence ratio rates under different applications clearly depicted a link between active HSP90.1 or HSP90.3, BIN2 nuclear localization and BR signaling (Notes S1). BIN2 interacts with HSP90.1 and HSP90.3 in a site-specific and BR-dependent mode The attenuation of BIN2 nuclear localization following GDA application, the exodus of HSP90.1 and HSP90.3 from the nucleus in BL-treated transgenes, and the physical interaction of the two proteins prompted us to investigate the compartmentalized specific interaction of the two HSP90s with the kinase when BL was applied. Tobacco epidermal cells coexpressing either HSP90.1–YFPc or HSP90.3–YFPc and BIN2–YFPn were used to isolate protoplasts. In the absence of BL, strong YFP fluorescence signal resided predominantly in the nucleus of tobacco protoplast cells. However, in BL-treated protoplasts, YFP fluorescence was mainly apparent in the cytoplasm (Fig. 8). Fluorescence in the nuclei of control protoplasts that coexpressed either HSP90.1–YFPc or HSP90.3–YFPc and YFPn or YFPc and BIN2–YFPn was below detection limits (Fig. S4). Quantification of the relative nucleus/cytoplasmic fluorescence density ratio in BL-treated and control tobacco protoplasts also revealed a rapid nuclear depletion of the HSP90.1– or HSP90.3–BIN2 complex (Fig. 8). Therefore, our data showed that the localization of HSP90.1– or HSP90.3–BIN2 interaction is BR-dependent.

Discussion Most studies have focused on cytosolic HSP90 activity influencing cellular homeostasis via client stabilization. In the nucleus, the chaperone activity has been implicated in a signal-dependent disassembly of transcriptional complexes (Freeman & Yamamoto, 2002), the stabilization of Trx (Tariq et al., 2009), and the optimization of the RNA polymerase II pausing complex (Sawarkar et al., 2012). The results presented here demonstrate the prominent role of HSP90.1 and HSP90.3 in BR nuclear signaling in plants. As BR signaling crosstalks with other plant hormone circuitries, such as auxin and GA signaling pathways (Vert et al., 2008; Clouse, 2011), HSP90 action could ultimately result in a broader control of cellular behavior, as seen in Samakovli et al. (2007). HSP90 interaction with a diverse set of kinases presents a small, though highly important, part of the global picture of its role in the modulation of cellular circuitries (Taipale et al., 2012). Even though a role for HSP90 in BR action has long been suggested, it was only recently experimentally evaluated by the interaction of HSP90 with BES1 (Lachowiec et al., 2013; Shigeta et al., 2014). Our studies linking HSP90 with BR signaling were based on the chaperone’s crucial role in steroid receptor competence and Wnt signaling, the similarities of animal Wnt with the plant BR signaling process, and the resemblance between the HSP90 mutant and BR mutant phenotypes. The employment of biochemical approaches using HSP90 and BR signaling inhibitors or ligands, and the genetic and molecular analyses allowed us to propose a dynamic role for HSP90 recruitment in the nuclear Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 8 Brassinosteroids (BRs) redirect heat shock protein 90 (HSP90)–BIN2 interaction in the cytoplasm. In vivo interactions between BIN2 and HSP90.1 or HSP90.3 were confirmed by bimolecular fluorescence complementation (BiFC). The N-terminal and C-terminal domains of yellow fluorescent protein (YFP) were fused to BIN2 and HSP90s, respectively. Protoplasts isolated from cotransformed tobacco epidermal cells were untreated with brassinolide (–BL) or treated with 10 lM BL (+BL) for 2–4 h. The relative fluorescence density in the nucleus was calculated for each protoplast cell. The average fluorescence density in the nucleus and standard error for independent protoplasts were calculated from measurements of at least 10 cells for each treatment. Bright field (l), Chl autofluorescence (r), YFP (g) and overlay images (o/l) of r and g. Bars, 20 lm. Arrows indicate the position of the nucleus.

BR signaling pathway. The proposed role of HSP90 in BR signaling is highlighted by its subcellular compartmentalization, its spatial interaction with BIN2 kinase in a hormone-promoted manner and, subsequently, its involvement in BR-mediated gene transcriptional control. Inhibition of HSP90s alters BIN2 and HSP90.1 or HSP90.3 nuclear localization Nuclear entry of HSP90s could be facilitated via NLS present within the deduced amino acid sequence or could be assisted by its interactions with nuclear targeted proteins. Nuclear Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

localization of HSP90s has recently been determined in plants (Shigeta et al., 2014). Evaluation of the nuclear/cytoplasmic fluorescence density ratio of both HSP90.1–GFP and HSP90.3– GFP fusion constructs in root cells clearly demonstrated the attenuation of HSP90.1 or HSP90.3 localization in the nucleus. In budding yeast, it has been reported that HSP90 probably shuttles between cytoplasm and nucleus during logarithmic growth (Tapia & Morano, 2010). Cold treatment trapped HSP90.3 nuclear localization at loci reminiscent of transcriptionally active foci (Fig. S2). In Drosophila, HSP90 localizes near promoters of many genes and maintains RNA polymerase II pausing via stabilization of the negative elongation factor complex (Sawarkar et al., New Phytologist (2014) 203: 743–757 www.newphytologist.com

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2012). Challenging the HSP90 ATPase activity with a known inhibitor demonstrated that either HSP90.1 or HSP90.3 nuclear localization depends on active HSP90s. A 3.2-fold decrease in the relative nuclear/cytoplasmic fluorescence density ratio for both HSP90–GFP fusion proteins upon 24 h of GDA incubation revealed a rapid exclusion from the nucleus. Geldanamycin application also attenuated BIN2 subcellular localization, signifying that an active HSP90 system is crucial to sustain BIN2 nuclear retention. Previous data have shown that BIN2–GFP distributes more or less evenly among the plasma membrane, cytosol, and the nucleus (Vert & Chory, 2006), suggesting that differential subcellular localization might be an important mechanism in regulating the BIN2 activity exerted on its two nuclear substrates, the transcription factors BZR1 and BES1 (He et al., 2002; Wang et al., 2002; Yin et al., 2002). Our yeast two-hybrid and split-YFP assays showed that GSK3like BIN2 kinase is a novel HSP90.1 or HSP90.3 client. The coimmunoprecipitation analysis confirmed that HSP90.3 associated with nonphosphorylated and phosphorylated forms of BIN2. This interaction was evidenced in the cytoplasm and profoundly present in the nucleus. As HSP90 association/dissociation to client kinases undergoes repeated loops, inhibition of HSP90’s ATPase activity prevents a new round of chaperone association to clients (Krukenberg et al., 2011). This could redirect the client kinases to alternative fates of degradation, aggregation (Taipale et al., 2012), or ectopic subcellular localization (Fig. 4). Therefore, a parallel GSK3b kinase activation mechanism, involving HSP90 activity, exists in plants and animals. BRs promote HSP90.1 and HSP90.3 nuclear depletion, and cytoplasmic BIN2–HSP90 association Brassinosteroids as steroid compounds are not inhibitors of HSP90s. The BL-induced nuclear depletion of HSP90.1 and HSP90.3 reveals that the BR downstream nuclear signaling modulates their trafficking and their transient localization. As BRs’ mode of action is transient and over a short distance (Symons et al., 2008), we consider that the signaling is canalized via the necessity of the HSP90.1 and HSP90.3 action in the nucleus to maintain the metastable client proteins temporally and spatially competent for proper conformation and/or function. In the presence of BL, HSP90.1– or HSP90.3–BIN2 kinase association is prominent in the cytoplasm of tobacco protoplast cells in splitYFP assays. The negative exponential formulas depicting the relative nuclear/cytoplasmic fluorescence density ratios of HSP90.1 or HSP90.3 and BIN2 in the presence of GDA or BL, and the rapid nuclear depletion of HSP90.1 in BL-treated plants signify the link between active HSP90s, nucleocytoplasmic localized HSP90.1– or HSP90.3–BIN2 associations, and BR signaling. The binding of HSP90.1 or HSP90.3 to GSK3-like BIN2 irrespective of the ligand’s presence signifies that BIN2 kinase exploits HSP90 machinery. This could allow for immediate response to the BR signal. It is tempting to suggest that the subcellular distribution of HSP90.1 and HSP90.3 is a dynamic balance of nuclear import and export processes. In animal cells, the dynamic HSP90-mediated cycling of the ligand-bound steroid New Phytologist (2014) 203: 743–757 www.newphytologist.com

New Phytologist receptor is an important factor in receptor trafficking to the nucleus for proper function (Picard, 2002, 2006; Pratt et al., 2004). A decrease in the amount of nuclear HSP90.1 or HSP90.3 by the use of GDA could shift the equilibrium from HSP90.1- or HSP90.3–BIN2 complexes towards free BIN2. In this case, the transient absence of HSP90.1 or HSP90.3 from the nucleus could facilitate the subsequent BR-dependent nuclear cascade. In the light of our experimental data, HSP90 acts as an integral component of the BIN2 activation mechanism. HSP90 acts as an essential component in the regulation of BR-responsive genes Disrupted or inappropriate localized HSP90–kinase interaction makes BIN2 incapable of acting as a negative regulator in the nuclear BR signaling. GDA or BL application had a similar effect on the transcriptional activation/repression of BR-regulated genes (Fig. 5). Noticeably, HSP90 inhibition derepresses BR-responsive genes and represses BR-biosynthetic genes mainly through the BZR1/BES1-mediated transcriptional network. The effect of GDA was apparent at the physiological level. GDA drastically reduced the hypocotyl elongation of etiolated wild-type, hsp90.1 and hsp90.3 mutant seedlings. BL had a similar reduction effect on hypocotyl growth of wild-type and mutant seedlings. Nevertheless, the hypocotyl growth of the hsp90.1 mutant without BL indicated that the BR signaling pathway is open, as in the case of BIN2 redirection from the nucleus in the presence of GDA. In the light of the current results, which are consistent with previous observations (Sawarkar et al., 2012; Taipale et al., 2012), the HSP90 system could work as a master homeostasis regulator that coordinates diverse developmental signaling circuitries. It is well documented that the function of HSP90 is to assist metastable steroid receptors and kinases by conformational stabilization, therefore triggering their ability to be properly activated in a transient and spatial manner (Lochhead et al., 2006; Picard, 2006; Taipale et al., 2012). Given the similarities between Wnt and BR signaling, our data provide insights into how the downstream nuclear BR signaling deactivation, transmitted via the negative regulator BIN2, is perceived via the HSP90 and BIN2 nucleocytoplasmic trafficking. In the absence of BR, HSP90.1 and HSP90.3 in the nucleus assist proper localization and sustainability of BIN2. In the presence of BL, HSP90.1– or HSP90.3–BIN2 complexes drift transiently in the cytoplasm, allowing the signal to proceed via dephosphorylation of the nuclear-localized transcription factors BES1 and BZR1, probably by PP2A (Tang et al., 2011). A model describing the HSP90 action in BR nuclear signaling is depicted in Fig. 9. In metazoans, GSK3b, a BIN2 homolog, requires HSP90 for autophosphorylation and maintenance of its stability and function. Previous studies have highlighted the potential interaction between HSP90 and GSK3b, showing that directed inhibition of HSP90 decreased the GSK3b steady-state protein content, which in turn reduced b-catenin phosphorylation components of the Wnt signaling pathway (Lochhead et al., 2006). The fact that BR signaling is mediated through a cascade of phosphorylation/dephosphorylation events along with the established Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. 9 A model for the role of heat shock protein 90 (HSP90) in brassinosteroid (BR) nuclear signaling. (a) In the absence of BR, BRASSINOSTEROID INSENSITIVE 1 (BRI1) and its coreceptor BRI1 ASSOCIATED RECEPTOR KINASE1 (BAK1) are inactive. The HSP90s are present in the nucleus and the cytoplasm facilitating the transport and retention of BIN2 into the nucleus. They effectively stimulate, by conformational competence, BIN2 kinase activity to phosphorylate and efficiently inactivate BRI1-EMS-SUPPRESSOR 1 (BES1) and BRASSINAZOLE-RESISTANT 1 (BZR1). (b) In the presence of BR, BRI1 facilitates the signaling by the depletion of HSP90s and significant reduction of HSP90–BIN2 complexes in the nucleus. This ultimately inactivates BIN2 by spatial (cytoplasmic) localization and/or conformational incompetence, thus leaving them incapable of phosphorylating the nuclear-localized BES1 and BZR1, allowing BRI1 SUPPRESSORS 1 (BSU1) to dephosphorylate them. BES1 and BZR1 represent a class of plant-specific transcription factors that bind to and activate the promoters of BR-responsive genes or repress BR biosynthetic genes. Arrows and bars represent actions of promotion and inhibition, respectively.

role of HSP90 in GSK3b autophosphorylation (Lochhead et al., 2006) highlights the vital role of HSP90.1 and HSP90.3 in BIN2-dependent nuclear events of the BR signaling pathway. It is well documented that HSP90 associates with a number of kinases (Taipale et al., 2012). As HSP90 crosstalks with diverse developmental circuitries in plants (Samakovli et al., 2007; Shirasu, 2009; Kadota et al., 2010), a signal that induces its depletion from a subcellular organelle, such as the nucleus, could mark a major turning point, as in the case of the BR signaling pathway. Given that diverse gene regulatory circuitries could recruit similar network hubs, it is important to determine how signaling specificity is maintained when different pathways share the same components, such as HSP90s. Interestingly, the phenotypic classes of hsp90 Arabidopsis mutant plants revealed that the members of this system could have overlapping as well as distinctive roles (Samakovli et al., 2007). Herein, the differential expression of the HSP90.1 and HSP90.3 genes in the ucu1-1 mutant background could highlight a comprehensive recruitment of certain HSP90 members or reset a need of a specific member in a cellular circuitry. The functional assignment of the different HSP90 members will shed light on the activation and fine-tuning of signaling pathways and their crosstalk during complex developmental processes.

Acknowledgements We dedicate this article to the memory of Dr Fotis Gazis; time has not been generous to discuss these findings. This work was funded by PENED 01ED148, GSRT/USA-024 and ARISTEIA/ Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

GSRT 1200 grants to P.H. We thank Dr J. Chory and G. Vert for providing the BIN2:BIN2–GFP line; Dr Perez-Perez for providing ucu1-1 mutant; Dr L. Dolan for providing the RSL4:: GFP-RSL4 and RHD6::mCherry-RHD6 lines; Dr T. Asami for providing BRZ2001; Dr G. Banilas for his critical comments and E. Hatzistavrou, A. Thanou, A. Rambou and T. Koutroubis for technical assistance.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Schematic representation of the HSP90.1::HSP90. 1–GFP or HSP90.3::HSP90.3–GFP fusion constructs used for transformation and subcellular localization of the HSP90.1 and HSP90.3 proteins. Fig. S2 Effect of cold and heat stress on HSP90.1 or HSP90.3 and BIN2 localization.

Ó 2014 The Authors New Phytologist Ó 2014 New Phytologist Trust

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Fig. S3 HSP90.1 or HSP90.3 and BIN2 localization in DMSOtreated seedlings. Fig. S4 Controls for HSP90.1 or HSP90.3 and BIN2 interactions by BiFC. Fig. S5 Specific action of GDA on BIN2 nuclear localization. Fig. S6 BL and GDA effect on wild-type, hsp90.1 and hsp90.3 mutant hypocotyl length. Fig. S7 HSP90.1 expression in the ucu1-1 mutant background. Fig. S8 HSP90.1 or HSP90.3 and BIN2 localization in NAAtreated seedlings. Fig. S9 Effect of BL concentration and time on HSP90.1 or HSP90.3 nuclear exodus. Fig. S10 Time-lapse analysis of HSP90.1–GFP localization in Arabidopsis root cells treated with 1 lΜ BL. Table S1 Set of nucleotides used in gene expression analysis and in different fusion translation constructs Table S2 Measurements of fluorescence densities of fixed size areas from HSP90.1–GFP and HSP90.3–GFP lines in the presence or absence of GDA Table S3 Measurements of fluorescence densities of fixed size areas from BIN2–GFP lines in the presence or absence of GDA Table S4 Measurements of fluorescence densities of fixed size areas from HSP90.1–GFP and HSP90.3–GFP lines in the presence of different BL concentrations Notes S1 Computational approach of the HSP90.1–GFP, HSP90.3–GFP and BIN2–GFP fluorescence exodus from the nucleus. Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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