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Nucleocytoplasmic Shuttling of BZR1 Mediatedby Phosphorylation Is Essential in ArabidopsisBrassinosteroid Signaling W OA

Hojin Ryu, a,1 Kangmin Kim, a,1 Hyunwoo Cho, a Joonghyuk Park, a Sunghwa Choe, b and Ildoo Hwang a,2

a Department of Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Koreab Department of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Korea

Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating thephosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1(BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and aGSK3 kinase,BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown howthe perception of BR at theplasma membraneconnects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmicshuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for14-3-3binding,another phosphorylation domainin BZR1 is required forthe BIN2-inducednuclear export of BZR1. Mutationsofputative phosphorylation sites in twodistinct domains enhance the nuclear retention of BZR1 and BR responses in transgenicplants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

INTRODUCTION

Brassinosteroids (BRs) are a group of plant steroid hormonesthat function in diverse plant developmental and growth pro-cesses through a signaling cascade from the BR receptor to theexpression of BR target genes (Mandava, 1988; Clouse et al.,1996; Vert et al., 2005). Plants that are defective in BR signalingor biosynthesis display characteristic dwarsm in the light andphotomorphogenesis in the dark (Clouse et al., 1996; Li and

Chory, 1997). BRs bind to the BRASSINOSTEROID INSENSI-TIVE1 (BRI1)/BKI1 receptor complex at the plasma membrane,causing the release of BKI1(He et al., 2000; Wang et al., 2001;Kinoshita et al., 2005; Wang et al., 2006). The subsequentdimerization of BRI1 and BRI1 ASSOCIATED RECEPTORKINASE1 (BAK1) activates a downstream signal transductionpathway that leads to BRI1 EMS SUPPRESSOR1 (BES1) andBRASSINAZOLE RESISTANT1 (BZR1), the key transcriptionalfactors in BR signal transduction (Li et al., 2002; Nam and Li,2002; Wang et al., 2002; Yin et al., 2002). The phosphorylationstatus of BES1 and BZR1 appears to control their signalingactivity (He et al., 2002; Yin et al., 2002) and is in turn determinedby BIN2, a glycogen synthase kinase-3 (GSK3)–like kinase(Choeet al., 2002; Li and Nam, 2002; Perez-Perez et al., 2002), andBRI1 SUPPRESSOR1 (BSU1), a plant-specic phosphatase(Mora-Garcia et al., 2004). BR signals rapidly induce the de-phosphorylation of BES1 and BZR1; however, how this modu-

lates their transcriptional activity is not fully understood.BR-induced dephosphorylation of BES1 and BZR1 and theirconcomitant accumulation in the nucleus aresuggested to resultfrom either nuclear translocation or protein stabilization, corre-lating with a shift from hyperphosphorylation to dephosphory-lation (He et al., 2002; Wang et al., 2002; Yin et al., 2002).

In these characteristics and the high similarity of BIN2 tometazoan GSK3 b , the BR signaling pathway has been proposedto resemble thecanonical Wntsignaling pathway (Heet al., 2002;Yin et al., 2002; Peng and Li, 2003). Recently, however, Vert andChory (2006) reported that the downstream events mediated byBES1 and BIN2 occur constitutively in the nucleus and sug-gested that the phosphorylation status primarily regulates theDNA binding and trans -activation of BES1 rather than its stabilityor subcellular localization. Nevertheless, the rapid shift in BES1and BZR1 phosphorylation status, the nucleocytoplasmic local-ization of BIN2, and the slightly increased nuclear accumulationof BES1 and BZR1 following BR treatment indicate that the dy-namic distributionof BES1 andBZR1 couldprovide another layerof regulation in BR signaling. Moreover, the sequential signalingevents from membrane-associated BR reception to transactiva-tion of BR regulons in the nucleus must be mediated through acytosolic signaling cascade; however, the location and mecha-nism oftheregulatoryeventsin BRsignalingare still largely unknown.

We show here that BZR1 itself functions as a nucleocytoplas-mic signaling mediator by shuttling between the cytoplasm andthe nucleus. The localization of BZR1 is tightly correlated with itsphosphorylation status. We demonstrate that BIN2-mediatedphosphorylation directly induces the nuclear export and subse-quent cytoplasmic retention of BZR1. We also found that the 14-3-3 proteins play a role in the cytoplasmic translocation of BZR1.Mutations of putative phosphorylation and 14-3-3 recognitionsites identiedin BZR1 lead to constitutive nuclear localization of BZR1 and result in a strong BR response, showing bzr1-1D –like

1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thisndings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Ildoo Hwang([email protected]).W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.107.053728

The Plant Cell, Vol. 19: 2749–2762, September 2007, www.plantcell.org ª 2007 American Society of Plant Biologists

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phenotypes. Based on these observations, we propose that thephosphorylation status of BZR1 is important for the subcellulardistributionof BZR1 to modulate thesensitivityand magnitudeof BR signals between the nucleus and the cytosol.

RESULTS

BZR1 Is a Nucleocytoplasmic Protein, and Its SubcellularDistribution Is Interrelated with BIN2 Activity

To understand how BIN2-mediated phosphorylation negativelyregulates BZR1 activity in BR signaling, we rst analyzed the

expressionpatterns of BIN2 using ProBIN2:GUS transgenic lines(Figure 1A) that carry the b -glucuronidase ( GUS ) gene under thecontrol of the BIN2 promoter. Histochemical analysis of trans-genic roots revealed that the expression of BIN2 is regulateddifferently in different developmental zones. BIN2 was ex-

pressed largely in the epidermis of the maturation zone. Bycontrast, GUS activity was specically observed in the vasculartissues but not in the epidermis of the elongation zone. Suchdistinct expression patterns of GUS imply that BIN2 expressionand its regulatory function in the phosphorylation of BZR1 arelikely regulated by upstream signals having tissue and/or devel-opmental specicity.

Figure 1. Nucleocytoplasmic Distribution of BZR1 Negatively Follows BIN2 Activity.

(A) Nuclear localization of BZR1-CFP is decreased in tissues in which BIN2 is actively expressed. The expression of BIN2 was monitored by the BIN2promoter–driven GUS activity at the maturation (top panel) and elongation (bottom panel) zones of 2-week-old roots. Subcellular localization of BZR1-

CFP in the epidermis and vasculature of the corresponding tissues was examined by optical sectioning using confocal laser scanning microscopy. TheCFP signal at the epidermis of the root tip is also presented. Note that autouorescence in wild-type plants (ecotype Columbia [Col-0]) was nearlyundetected. Top, the top of the confocal layers; Middle, an internal layer with the vasculature. The scanning depth from the tissue surface wasdesignated Z.(B) BZR1 is a multiphosphorylated and nucleocytoplasmic protein. The cytoplasmic and nuclear fractions from ProBZR1:BZR1-HA plants wereseparated by centrifugation. W, whole leaf extract; P, protoplast extract; N, nuclear fraction; C, cytosolic fraction. Histone H2B and RHA1 (Sohn et al.,2003) were used as nuclear and cytoplasmic markers, respectively.(C) BZR1-HA is localized in the nucleus and cytoplasm in protoplasts. The cytoplasmic and nuclear fractions from protoplasts transfected with theindicated genes were separated by centrifugation. The proteins were detected with an anti-HA antibody. N, nuclear fraction; C, cytosolic fraction. ARR2-HA and At MPK3-HA were used as nuclear and cytosolic markers, respectively.(D) The hyperphosphorylated BZR1 by BIN2 accumulates mainly in the cytoplasm. Protoplasts were transfected with BRI1-Myc/BZR1-HA or BIN2-Myc/BZR1-HA . The nuclear and cytosolic fractions were probed with an anti-HA antibody. ARR2-HA was cotransfected as a nuclear marker (Hwangand Sheen, 2001).

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To test whether the nuclear localization of BZR1 has tissuespecicity and is correlated with the spatial distribution of BIN2,we monitored the subcellular localization of BZR1-CFP (cyanuorescent protein) (Wang et al., 2002) where BIN2 was highlyexpressed. We examined BZR1-CFP signals not only in the

epidermis but in the vascular tissues of different developmentalzones of roots by optical sectioning (Figure 1A; see Supplemen-tal Figures 1 and 2 online). As reported previously, BZR1 waslocalized mainly in the nucleus around root tips (Zhao et al.,2002). Interestingly, however, we found that BZR1-CFP signalswere observed in the cytosol as well as the nucleus of otherdevelopmental zones. The relative intensities of nuclear BZR1-CFP were variable among developmental zones (Figure 1A). Inthematuration zone, thenuclear BZR1 was rarelydetectedin theepidermis, whereas strong nuclear signal was observed in theregions around vascular tissues (top panel, Z ¼ 27 mm), whereBIN2 expression appeared to be minimized. In the elongationzone, BZR1-CFP was localized mainly in the nucleus of theepidermis (bottom panel). Notably, the level of nuclear BZR1-

CFP waslargely decreasedin thevascular tissues (bottom panel,Z ¼ 36 mm), which have increased BIN2 expression. On theotherhand, the BZR1-CFP signal in the nucleus was not detected inthe epidermis of the elongation zone of Arabidopsis thalianaroots grown in a medium containing brassinazole (BRZ), abrassinosteroid biosynthesis inhibitor (see Supplemental Figure3 online), indicating that the nuclear localization of BZR1 ispositively regulated by the BR signal. Taken together, theseobservations suggest that BZR1 is localized in both the nucleusand the cytosol.

Subcellular localization of BZR1 interrelated with BIN2 ex-pression implicates BIN2-catalyzed phosphorylation of BZR1 incontrolling the subcellular localization of BZR1. To test thispossibility, we examined the spatial distribution and the phos-phorylation status of BZR1-HA (hemagglutinin) under the controlof its own promoter in transgenic plants. Nuclear and cytoplasmicfractions were prepared from protoplasts of the transgenic plants,and BZR1 proteins were detected with an anti-HA antibody.BZR1 proteins were present as multiple phosphorylated bands(Figure 1B). Interestingly, hyperphosphorylated and hypophos-phorylated forms exhibited distinct spatial distribution in eitherthe cytoplasmic or the nuclear fraction (Figure 1B). The hypo-phosphorylated forms (the lower bands) were predominantlypresent in the nuclear fraction, whereas the hyperphosphory-lated form (the highest band) was found exclusively in thecytoplasmic fraction, suggesting that the phosphorylation statusis closely correlated with the localization. BZR1 proteins tran-siently expressed in protoplasts also showed a similar subcel-lular distribution pattern (Figure 1C). We then used a protoplasttransient expression system to test whether the subcellularlocalization of BZR1 is modulated by BIN2-mediated phosphor-ylation (Figure 1D). Protoplasts were transformed with HA-tagged BZR1 and myc-tagged BIN2 or BRI1 . Coexpression of BIN2, a GSK3-like kinase, produced slower migrating BZR1bands and resulted in the cytoplasmic localization of BZR1(Figure 1D). In the presence of BR and coexpressed BRI1, whichfavors the dephosphorylation of BZR1, BZR1 proteins werelocalized exclusively in the nucleus, again suggesting that thelocalization of BZR1 is determined by its phosphorylation status.

BZR1 Is a Nucleocytoplasmic Shuttling Protein, and ItsNuclear Export Is Largely Regulated by BIN2

To investigate the spatial dynamics of BZR1, we fused BZR1 togreen uorescent protein (GFP) and monitored the subcellulardistribution of BZR1 in the presence of BIN2 and its mutantshaving differentcatalyticefcienciesin phosphorylation events ina protoplast system. We rst examined the transcription of CPD ,a BR-responsive gene (Mathur et al., 1998; Noguchi et al., 1999),in protoplasts after BR treatment to determine whether BR sig-naling is properly transmitted in Arabidopsis mesophyll proto-plasts. BR treatment reduced the expression level of CPD inprotoplasts as much as in seedlings(see SupplementalFigure 4A online). With a proper change in the phosphorylation status of BZR1 by BR treatment, this result suggests that the protoplastsystem is suitable for the elucidation of BR signaling mecha-nisms. Then, HA-tagged BIN2, bin2-1 , or BIN2K69R was cotrans-fected into protoplasts with GFP-tagged BZR1 (Figure 2A).BZR1-GFP proteins were localized mainly in the nucleus but

were also observed in the cytosol, as determined by the relativeintensities of GFP and red uorescent protein (RFP) uorescentsignals (Figure 2A; see Supplemental Figure 4B online). BothBIN2 and the gain-of-function bin2-1 mutant (Li and Nam, 2002)shifted the localization of BZR1 from the nucleus to the cyto-plasm, whereas no shift was observed with BIN2 K69R , a kinase-dead loss-of-function mutant (Zhao et al., 2002), demonstratingthat the kinase activity of BIN2 correlates with both the cyto-plasmic retention of BZR1 and its phosphorylation status. Theseresults suggest that BIN2-catalyzed phosphorylation of BZR1causes the active translocation of BZR1 proteins from thenucleus to the cytoplasm. At GSK1 and ASKd z, BIN2 homologsin Arabidopsis group II GSK3 (Jonak and Hirt, 2002; Vert andChory,2006) affectedthe localization of BZR1 in a mannersimilar

to BIN2 and also interacted physically with BZR1 (see Supple-mental Figure 5 online), indicating that the group II GSK3 kinasessharethe functional redundancy for the subcellular distribution of BZR1 and the negative regulation of BR signaling.

The cytoplasmic localization of BZR1 might be explained byBIN2-dependent phosphorylation of de novosynthesizedproteinsin the cytoplasm and then subsequent sequestration in the cyto-plasm. Another possibility is that nucleus-localizedBIN2 mediatesthe exportof BZR1 fromthe nucleus by phosphorylating BZR1. Totest these alternative hypotheses, we examined the localization of BZR1-GFP in protoplastsunder tightlycontrolledBIN2expressionusing a glucocorticoid receptor–inducible gene expression system(Figures2Band2C) (Yanagisawaetal., 2003).BIN2expressionwasinduced by dexamethasone (DEX) in a concentration- and time-dependent manner (Figure 2B). Without BIN2 induction by DEX,BZR1-GFP was localized mainly in the nucleus (Figure 2C). Follow-ing the induction of BIN2 expression, localization of BZR1 shiftedfrom the nucleus to the cytoplasm, suggesting that preexistingnuclear BZR1 was exported to the cytoplasm after phosphoryla-tion. To test this hypothesis further, we examined the effect of leptomycin B, a potent inhibitor of exportin-dependent nuclearexport(Haasen etal., 1999),on thelocalizationofBZR1 (Figure 2D).Treatment with leptomycin B followed by BIN2 induction withDEX caused the retention of BZR1 in the nucleus, proving thatphosphorylation by BIN2 induces the nuclear export of BZR1.

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Figure 2. BIN2 Expression Leads to BZR1 Transport from the Nucleus to the Cytosol.

(A) BIN2 increases the cytoplasmic localization of BZR1. BZR1-GFP was cotransfected into protoplasts with BIN2-HA , bin2-1-HA , or BIN2K69R -HA and

examined by confocal microscopy. RFP-ARR2 was used as a nuclear marker. The uorescent signal intensities of BZR1-GFP and RFP-ARR2 weredetermined along a line drawn on the confocal images using LSM Image Browser Rel. 4.0 software.(B) BIN2-HA expression is specically induced by DEX in a time- and concentration-dependent manner. Rubisco large subunit (RbcL) was used as aloading control.(C) BIN2 induces the nuclear export of BZR1. Protoplasts were cotransfected with 8OP:BIN2-HA , Pro35S:VP16-GR , and BZR1-GFP . After incubationfor 4 h to express GFP-tagged BZR1 , BIN2 expression was induced by DEX treatment. 0 hr, time of DEX induction.(D) Leptomycin B (LMB) blocks the BIN2-mediated nuclear export of BZR1. Protoplasts were transfected with the plasmids and incubated for 4 h asdescribed for (C) . The transformed protoplasts were treated with leptomycin B for 1 h and then further treated with DEX.

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BR-Activated PP2A Phosphatases Rapidly Induce theNuclear Translocation of BZR1

BZR1 proteins must be located in the nucleus to modulate thetranscription of BR-responsive genes, meaning that BR signalsmust attenuate the BIN2-mediated cytosolic localization of BZR1. To test this, we coexpressed BZR1-GFP with BRI1 andBIN2 in Arabidopsis protoplasts. Without BR treatment, BZR1-GFP was located mainly in the cytoplasm (Figure 3A). However,BR increased the nuclear accumulation of BZR1 and BRI1facilitated BR-induced nuclear localization, conrming that theBRI1-mediated BR signal inhibits the cytosolic translocation of BZR1 by BIN2. The effect of BR was due to neither the alteredexpression nor the stability changes of BIN2 by epi -brassinolide(BL) treatment (Figure 3B). We also monitored the phosphory-lation status of HA-tagged BZR1 to examine the correlationbetween phosphorylation and subcellular distribution (Figure3C). BR converted phosphorylated BZR1 proteins to the de-phosphorylated state in cells coexpressing BRI1 and BIN2,

suggesting that BR-induced BRI1 activation potently attenuatesBIN2 kinase activity.The BIN2 action was suggested to be counteracted by

BSU1, having features of both PP1- and PP2A-type phospha-tase, which have several homologs in Arabidopsis (Mora-Garciaet al., 2004). To test whether the dephosphorylation and subse-quent nuclear localization of BZR1 were mediated by PP2A-typephosphatases, we monitored the BR-induced nuclear localiza-tion ofBZR1in the presence ofthe PP1 and PP2A phosphatase–specic inhibitor, okadaic acid. When protoplasts expressingBZR1-GFP and BIN2-HA were treated with BR, BZR1 waslocalized to the nucleus (Figure 3D). However, 10 nM okadaicacid, a concentration that inhibits PP2A phosphatase, inhibitedthe translocation of BZR1 from the cytosol to the nucleus even in

the presence of BL, suggesting that cytosolic PP2A-type phos-phatases may mediate the BR-induced nuclear translocation of BZR1. To investigate whether BSU1 is involved in the nuclearlocalization of BZR1, we transiently coexpressed BZR1 with HA-tagged BIN2and BSU1in mesophyllprotoplasts. BothBSU1-HA and BSU1-YFP facilitated the BR-induced dephosphorylation of BZR1, indicating that the tagged BSU1 proteins are functional inprotoplasts (Figure 3E). BSU1 rapidly enhanced the nuclearaccumulation of BZR1 upon BR treatment even in the presenceof BIN2 (Figure 3F). Thus, BZR1 could be dephosphorylated inboth the cytoplasm and the nucleus by BR-activated PP2A phosphatases. BSU1 would counteract the BIN2-mediated cy-toplasmic translocation of BZR1 in the nucleus. Therefore, wepropose that BZR1 is a nucleocytoplasmic shuttling proteinwhose localization is tightly regulated by its phosphorylationstatus, which is determined by the opposing actions of BIN2 andphosphatases, including BSU1.

Two Putative Phosphorylation Domains Are Important forthe Nuclear Export of BZR1

We next analyzed the primary structure of BZR1 for putativephosphorylation sitesand usedsite-directed mutagenesis to testthe roles of the Ser/Thr residues in regulating subcellular distri-bution. We substituted each of 25 Ser/Thr residues, which were

previously proposed as possible phosphorylation sites (Wanget al., 2002), with Ala and screened for mutants that have alteredsubcellular distribution by BIN2. We identied 10 residues inBZR1 that affect the BIN2-induced nuclear export of BZR1 inprotoplasts and grouped them into two distinct domains by

their phosphorylation status (Figure 4A; see Supplemental Fig-ure 6 online). We followed two Ser mutants, BZR1 S130A andBZR1 S134A , as representatives of the rst domain. BZR1 S130A

and BZR1 S134A mutants appeared to be insensitive to BIN2,remaining localized in the nucleus (Figure 4B). The dephosphor-ylated proteins were signicantly enriched in these mutants(Figure 4C). We then examined the effect of BIN2 on the phos-phorylation status of the selected mutants. GFP-tagged wild-type BZR1 proteins exhibited both hyperphosphorylated andhypophosphorylated forms, but the cotransfection of BIN2 largelyinduced the hyperphosphorylation of wild-type BZR1 proteins(Figure 4C). However, substantial amounts of BZR1 S130A andBZR1 S134A still remained in the dephosphorylated state even inthe presence of BIN2 (Figure 4C). The double BZR1 S130/134A

mutation further enhanced the dephosphorylation of BZR1 andthe insensitivity to BIN2. These data suggest that the putativephosphorylation residues of the rst domain, including Ser-130and Ser-134, could be direct targets of BIN2 required for thenuclear export of BZR1.

Mutations at Ser-173 and Thr-177 in BZR1 also disrupted thecytoplasmic translocation of BZR1 induced by BIN2 (Figure 4B).However, BZR1 S173A and BZR1 T177A mutants were still hyper-phosphorylated by BIN2 at a similar level to wild-type BZR1(Figure 4C). These data indicate that Ser-173 and Thr-177residues may not be direct targets of BIN2 but are still importantas regulatory sites for theBIN2-mediated nuclear exportof BZR1by an unknown mechanism. Alternatively, it is also possible thatthese residues are phosphorylated by BIN2, although phosphor-ylation was not detected in our gel-shift assays.

Ser-173 and Thr-177 Are Critical for Interaction with the14-3-3 Proteins in the Nuclear Export of BZR1

We previously generated BZR1 mutants disrupting a predictednuclear export signal, but no alteration in subcellular localizationwas observed, implying that the nuclear export of BZR1 mightrequire a guidance protein (data not shown). 14-3-3 proteins areknown to play diverse regulatory roles as molecular chaperonesin signaling networks and have been shown to mediate thenuclear export of Repression of Shoot Growth (RSG) protein in Arabidopsis GA signaling (Igarashi et al., 2001). Interestingly, thesecond putative phosphorylation domain of BZR1 encompass-ing Ser-173 contains a potential mode II type 14-3-3 binding sitefrom the 169th to the 175th residues, RISNSCP (Figure 5A). Wethus examined the physical interaction between BZR1 and Arabidopsis 14-3-3 proteins in yeast two-hybrid assays (Figure5B). We found that 14-3-3 k and 14-3-3 e proteins interactedspecically with BZR1. 14-3-3 proteins are known to interactspecically with the conserved motif designated mode II(RXXXpSXP), and phosphorylation of a conserved Ser residueis required for this interaction (Muslin et al., 1996; Aitken, 2006).Ser-173 in the putative 14-3-3 binding motif of BZR1 is likely tobe phosphorylated for 14-3-3 binding (Figure 5A). We also


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Figure 3. Nuclear Translocation of BZR1 Is Induced by BR-Activated PP2A Phosphatase.

(A) BR induces the nuclear translocation of BZR1. BZR1-GFP was cotransfected with BRI1-HA and BIN2-HA into protoplasts and expressed for 5 h.The protoplasts were then treated with epi -BL for 2 h. 0 hr, time of BR treatment. Note that BRI1 expression facilitates the BR-induced nuclearlocalization of BZR1.(B) The expression levels of BRI1-HA and BIN2-HA were similar in transformed cells.(C) BRI1 facilitates the BR-mediated dephosphorylation of BZR1 in protoplasts. HA-tagged BES1 and BZR1 were cotransfected into protoplasts withthe indicated amounts of BRI1-HA and 4 mg of BIN2-HA . After 4 h of transfection, the protoplasts were incubated with cycloheximide for 30 min beforetreatment with BR for 1 h.(D) Okadaic acid, a PP2A phosphatase inhibitor, blocks BR-induced nuclear translocation of BZR1. Protoplasts were transfected with BIN2-HA andBZR1-GFP and incubated for 5 h to express the effectors. The transfected protoplasts were treated with 1 mM epi -BL alone and 10 or 30 nM okadaicacid (OA) together for 3 h.(E) BSU1acceleratesthe BR-induceddephosphorylationof BZR1. Afterincubationfor 4 h, protoplaststransfected with BSU-HA or BSU1-YFP and BZR1-HA weretreated withor without epi -BLfor1 h inthe presence ofcycloheximide. BSU-HAand BZR1-HA proteinsweredetected usingan anti-HA antibody.(F) BSU1 enhances nuclear localization. Four hours after transfection, protoplasts expressing BSU1-HA , BIN2-HA , and BZR1-GFP were incubatedwithout or with epi -BL for 1 h in the presence of cycloheximide. Subcellular localization of BZR1-GFP was followed by uorescence microscopy.

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predicted that the adjacent Thr-177 residue might have a similarrole. However, mutations of these residues did not affect theinteraction of BZR1 with 14-3-3 proteins in the yeast two-hybridassay (data not shown). We thus examined the in vivo interactionof BZR1 and 14-3-3 proteins using coimmunoprecipitation stud-

ies. HA-tagged BZR1 , BZR1S173A

, or BZR1T177A

was cotrans-fected with myc-tagged 14-3-3 k or 14-3-3 e into Arabidopsisprotoplasts. When the whole protoplast lysate was immunopre-cipitated with an anti-myc antibody, HA-tagged BZR1 but notBZR1 S173A or BZR1 T177A was pulled down with myc-tagged14-3-3 proteins (Figure 5C). We also found that BZR1 S130/134A

coimmunoprecipitated with 14-3-3 proteins (data not shown).These data indicate that 14-3-3 proteins bind to BZR1 throughthe second phosphorylation domain containing Ser-173 andThr-177 and modulate the subcellular distribution of BZR1. Wehere suggest that at least two putative phosphorylation domainsof BZR1 regulate the nucleocytoplasmic shuttling of BZR1 viadistinct regulatory mechanisms.

Nuclear Retention of BZR1 Mutants Leads to ConstitutiveBR Responses in Arabidopsis , Independently of thePhosphorylation Status

To investigate the physiological role of BZR1 subcellular local-ization, we generated transgenic plants that express BZR1 , bzr1-1D , BZR1 S130/134A , BZR1 S173A , or BZR1 T177A under thecontrol of the 35S promoter. All of the dark-grown transgenicseedlings showed similar hypocotyl elongation to the wild type(Figure 6A). However, the bzr1-1D , BZR1 S130/134A , BZR1 S173A ,and BZR1 T177A transgeniclines were relatively resistant to the BRbiosynthetic inhibitor BRZ. These lines had longer hypocotylsthan wild-type and transgenic lines that overexpress BZR1,similar to the bzr1-1D mutant. Hypocotyl and root elongation of these transgenic lines grown in long-day conditions was moresensitive to exogenous BL (Figure 6B; see Supplemental Figure7A online). Notably, the BZR1 T177A line under the control of the35S promoter respondedto BL similarly to the ProBRI:BRI1-GFPtransgenic lines (Nam and Li, 2002), which showed typical BRhypersensitivity, whereas the bri1-5 mutant was not affected byBL. Three-week-old BZR1 S130/134A , BZR1 S173A , and BZR1 T177A

transgenic lines also showed curled leaves and dwarsm, whichwas typically observed in the bzr1-1D transgenic plants (Figure6C). The expression pattern of the transgene was veried byRT-PCR and immunoblotting with an anti-HA antibody (see Sup-plemental Figures 7B and 7C online). As shown by transient ex-pression in protoplasts (Figure 4B), BZR1 S130/134A was mainlydephosphorylated, whereas BZR1 S173A and BZR1 T177A werehighly phosphorylated in the transgenic plants. The protein levelof BZR1 T177A was similar to that of BZR1.

We then examined the localization of BZR1 T177A in the trans-genic lines by subcellular fractionation (Figure 6D). Consistentwith the nucleocytoplasmic localization of BZR1 under its ownpromoter shown in Figure 1B, overexpressed wild-type BZR1proteins also localized to the nucleus and cytosol (Figure 6D),with an apparent distribution of the phosphorylated forms in thecytosolic fraction. By contrast, the phosphorylated form of BZR1 T177A was localized mainly in the nucleus, further support-ing the role of Thr-177 in the nuclear export of BZR1 in planta.

Figure 4. Identication of Putative Phosphorylation Residues Requiredfor Nuclear Export of BZR1 by BIN2.

(A) Ten putative phosphorylation residues in two distinct domains,marked by asterisks, are involved in BIN2-medated cytoplasmic trans-location of BZR1. The Ser-130/Ser-134 and Ser-173/Thr-177 residues in

red were selected in this study as representatives for the rst and seconddomains, respectively.(B) Mutations of putative phosphorylation sites blocked the nuclearexport of BZR1. BIN2 was cotransfected into protoplasts with BZR1-GFP or mutated BZR1-GFP as indicated.(C) Mutations of putative phosphorylation sites in BZR1 partly abolishedBIN2-mediated phosphorylation. GFP-tagged BZR1 or its mutants wereexpressed in protoplasts with or without BIN2 proteins. Protein expres-sion patterns of BZR1 and its mutants were analyzed with an anti-GFPantibody. BIN2 expression was probed with an anti-HA antibody.


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These results also indicate that the nuclear retention of BZR1 T177A may increase the binding capacity of BZR1 to itstarget genes and confer strong BR responses on the transgeniclines. To test this idea, we performed the chromatin immuno-precipitation (ChIP) assay on the CPD promoter (Figure 6E). The

HA-tagged BZR1 and BZR1T177A

proteins in transgenic lineswere immunoprecipitated with an anti-HA monoclonal antibody.The CPD target sequences thatwerecoimmunoprecipitated withBZR1-HA or BZR1 T177A -HA were amplied by standard PCR.The amount of the CPD promoter precipitated from theBZR1 T177A lines was slightly higher than that precipitated fromthe BZR1 lines, suggesting that BZR1 T177A represses CPDexpression by direct binding to regulatory cis elements. Consis-tent with strong BR response phenotypes, the expression of theBZR1 target genes CPD and DWF4 was strongly reduced in thetransgenic lines that ectopically express the mutated BZR1transgenes (Figure 6F). It is notable that DWF4 transcriptionwas greatly suppressed in the Pro35S:BZR1 T177A lines, similar tothe level inthe bzr1-1D lines (Figure 6F)(Mora-Garcia et al., 2004;

He et al., 2005). These results together indicate that increasednuclear retention of BZR1 by either its dephosphorylation or itsinhibition of 14-3-3 interaction leads to BZR1 activation andstrong BR responses in Arabidopsis .

DISCUSSION

BZR1 is a highly phosphorylatable protein and is rapidly de-phosphorylated by brassinosteroid treatment. Various in vitro/ in vivo studies have suggested that the phosphorylation statusof BZR1 wasregulated by BIN2 andBL. Theroleof thephosphor-ylation of BZR1 or BZR2/BES1 has been proposed to regulate avariety of molecular events: protein stability, cytoplasmic reten-tion,multimerization,and inhibition of the transactivation/repressionof target genesvia directinteraction with cis elements (Vert et al.,2005; Vert and Chory, 2006). Our study showed that BIN2-mediated phosphorylation also caused the nuclear export of BZR1 (Figure 2), whereas BR induced the nuclear translocationof the protein. This result was correlated with previous observa-tions that the nuclear localization of BZR1 and BZR2/BES1 wasenhanced by exogenous BR treatment (Wang et al., 2002; Yinet al., 2002). However, other studies suggested that BZR1 andBZR2/BES1 are constitutively localized in the nucleus (Zhaoet al., 2002; Vert and Chory, 2006). The discrepancy in BZR1localization might be explained by differences in the analyzedtissues, developmental stages of the plants, and experimentalconditions. The nuclear export of BZR1 appears to be minimaldue to a strong input of BR signals in expanding tissues likehypocotyls and root tips of young seedlings, resulting in eitherconstitutive or dominant nuclear retention of BZR1. However,dynamic redistribution of the nuclear BZR1 could have physio-logical importance in many cells under regulatory conditions inwhich the BR signal is needed to be desensitized or BIN2 activityis increased. Under such conditions, BIN2 could mediate thecytoplasmic translocation of BZR1 from the nucleus and thusminimize BR signaling activity in cells. Phosphorylation-mediatednucleocytoplasmic shuttling of BZR1 allows plants to respondswiftly to environmental or developmental stimuli without the denovo synthesis of BZR1. Multiple regulatory mechanisms for

Figure 5. 14-3-3 Proteins Interact with BZR1 and Prevent BZR1 fromBeing Constitutively Active by Mediating Nuclear Export.

(A) Putative 14-3-3 binding site of BZR1. Two 14-3-3 binding sitesequences (mode I and mode II) are aligned against the BZR1 sequence.X, any given amino acid; Y, Tyr; R, Arg; I, Ile; pS, phospho-Ser; N, Asn; C,Cys; P, Pro; –, absence of an amino acid residue.

(B) BZR1 interacts with the 14-3-3 proteins in yeast two-hybrid assays.BZR1 and 14-3-3 k /14-3-3 e were cloned into pGADT7 and pGBKT7vectors, respectively. BIN2 was used for a positive interaction control.Transformed yeast cells were examined for interaction on syntheticmedium lacking Leu, Trp, and His with 3 mM 3-aminotriazole.(C) Mutations at Ser-173 and Thr-177 of the 14-3-3 binding site of BZR1abolish in vivo interaction with 14-3-3 k and 14-3-3 e proteins. HA-taggedBZR1 , BZR1 S173A , or BZR1 T177A was cotransfected into protoplasts withmyc-tagged 14-3-3 k or 14-3-3 e . 14-3-3 proteins were immunoprecipi-tated with anti-myc monoclonal antibody, and BZR1 proteins weredetected with an anti-HA antibody. Theasterisk and arrowheads indicatenonspecic bands and 14-3-3-myc, respectively.

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Figure 6. Mutations of Putative Phosphorylation Residues Enhance BR Responses in Transgenic Plants.

(A) BZR1 S130/134A , BZR1 S173A , and BZR1 T177A mutations suppress the BR-deciency phenotype. Transgenic seedlings harboring HA-tagged bzr1-1D ,


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BZR1 and BZR2/BES1 likely contribute to tight regulation of themagnitude and sensitivity of BR signals in plant growth and de-velopment.

One important nding in our study is that, in a BR-stimulatedgrowth state, cytosolicBZR1 itself may be an efcient transmitter

of BR signals from receptor kinases at the plasma membrane toinuence the expression of BR-responsive genes in the nucleus.This process must be delicately regulated by a BR-activatedtoggleswitch composedof a kinaseBIN2 andphosphatases likeBSU1. Dissociation of BRI1 from BKI1, a negative regulator of BRI1 signaling, upon BR perception leads to the activation of anas yet unidentied cytosolic phosphatase or modication of BSU1, which might shift theequilibrium between BIN2 andBSU1toward dephosphorylation and translocation of BZR1 to thenucleus. Simultaneously, BIN2 might be subjected to negativeregulation, such as protein degradation, to facilitate the nucleartranslocation of BZR1. It is also possible that the temporal andspatial regulation of BIN2 or BSU1 expression modulates BZR1function to act as a cytoplasmic signal transmitter. This is

supported by the interrelation between the subcellular distribu-tion of BZR1 and the tissue and developmental specicity of BIN2 expression (Figure 1A). Furthermore, BIN2 expression wasnot detected in the elongating region of dark-grown hypocotyls(data not shown); by contrast, BSU1 was reported to be highlyexpressed in expanding hypocotyls but completely silenced infully expanded stems andleaves (Mora-Garciaet al., 2004).Suchopposite expression patterns between BIN2 and BSU1 couldexplain the constitutive localization of BZR1 in the nucleus of rapidly elongating tissues. In other tissues, different degrees of subcellular distribution of BZR1 and possibly BZR2/BES1 mightbe determined by the differential activity of BIN2 and BSU1,which could contribute to the optimization of BR signaling duringplant development.

In this study, we identied at least two domains carryingputative phosphorylation sites that modulated the subcellularlocalization of BZR1 (Figure 4). Positioned in the rst domain,Ser-130 and Ser-134 residues are critical for the nuclear export

of BZR1. Mutations of these residues enriched the dephosphor-ylation form of BZR1 and abolished BIN2-mediated phosphor-ylation, which is well correlated with increased nuclear retentionof BZR1. As the putative phosphorylation residues, includingSer-130 and Ser-134, in the rst domain are positioned in the

conserved S/TxxxS/T GSK3 phosphorylation motifs, they arelikely to be direct targets for BIN2 kinase. It is possible that thephosphorylation degree in this domain may directly determinethe DNA binding afnity of BZR1 on cis elements of the BR-responsive genes. The second domain containing Ser-173 andThr-177 is also important for the nuclear localization of BZR1.However, as opposed to the mutants in the rst domain, S173A and T177A mutants were still sensitive to BIN2-mediated phos-phorylation. The hyperphosphorylated state of these mutants isprobably due to phosphorylation in the rst domain. This resultindicated that phosphorylation of the rst domain alone may notbe sufcient to mediate the nuclear export and that phosphory-lation of the second domain may also be required, implying thatthe second domain is essential for the nuclear export of BZR1.

The second putative phosphorylation domain of BZR1 has ashort stretch of amino acid sequence that is similar to the con-served 14-3-3 recognition motif, designated mode II RXYXpSXPresidues (Aitken, 2006) (Figure 5). It is well known that phosphor-ylation of the fth Ser residue is required for proper interaction of the 14-3-3 proteins with interacting partners. Interestingly, BZR1appears to interact with 14-3-3 k and 14-3-3 e proteins, and Ser-173 and Thr-177 residues in the putative 14-3-3 binding domainof BZR1 areessential for this interaction (Figure 5).We also foundthat two additional isoforms, 14-3-3 v and 14-3-3 f , also interactwith BZR1 in the yeast two-hybrid system (data not shown).Besides mutation at Ser-173 of the putative binding domain,mutation at Thr-177 also abolished interaction with the 14-3-3proteins and resulted in the constitutive nuclear localization of BZR1. These results strongly suggest that Ser-173 or Thr-177 of BZR1 is directly targeted by phosphorylation for binding to the14-3-3 proteins. However, it remains unknown whether Ser-173is directly phosphorylated by BIN2 or a yet unidentied kinase in

Figure 6. (continued).

BZR1 S130/134A , BZR1 S173A , or BZR1 T177A driven by the35S promoter display a BRZ-resistant phenotype similar to that of the bzr1-1D mutant. Seedlings

were grown without or with BRZ (top panel) for 5 d in the dark, and the hypocotyl length of each seedling was measured. Error bars indicate SD ( n ¼ 10).(B) The BZR1 mutations result in increased BR sensitivity in hypocotyl elongation. Transgenic lines that overexpress BZR1 S130/134A -HA, BZR1 S173A -HA,or BZR1 T177A -HA show BR hypersensitivity similar to the Pro35S:bzr1-1D and ProBRI1:BRI1-GFP plants. The bri1-5 mutant is insensitive to BR.

Seedlings were grown on Murashige and Skoog medium containing various concentration of epi -BL for 6 d, and the hypocotyl length of each seedlingwas measured. Error bars indicate SD ( n ¼ 10).(C) Phenotypes of mutated BZR1 transgenic leaves (top panel) and plants (bottom panel) grown for 3 weeks under long-day conditions in soil. Themutants show BR-response phenotypes similar to that of the bzr1-1D mutant.(D) The BZR1 T177A mutation abolishes the cytoplasmic localization of hyperphosphorylated BZR1. Subcellular fractionation was performed withprotoplasts isolated from Pro35S:BZR1-HA and Pro35S:BZR1 T177A -HA transgenic lines. Note that the ectopic expression of BZR1 does not affect itssubcellular distribution in the transgenic line. W, whole leaf extract; P, protoplast extract; N, nuclear fraction; C, cytosolic fraction. Histone H2B andRHA1 were used as the nuclear and cytoplasmic markers, respectively.(E) The BZR1 T177A mutation in the 14-3-3 binding site does not affect the DNA binding capacity of BZR1. ChIP assays of the CPD promoter wereperformed with BZR1-HA or BZR1 T177A -HA transgenic plants using anti-HA monoclonal antibody. The specic primer set (He et al., 2005) was used toanalyze the CPD promoter bound to the proteins.(F) Expression of BR biosynthesis genes is suppressed in BZR1 S130/134A -HA, BZR1 S173A -HA, and BZR1 T177A -HA transgenic lines. The transcript levelsof CPD and DWF4 were quantied using quantitative real-time PCR. UBQ10 was used for normalization of the data. Error bars indicate SD ( n ¼ 3).

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BR signaling. Although the BR-induced nuclear retention of BZR1 and the constitutive nuclear localization of BZR1 S173A

and BZR1 T177A mutants in protoplasts and transgenic plants arewell correlated with a critical role of 14-3-3 proteins in thesubcellular distribution of BZR1, it was difcult to test whether

14-3-3 interaction directly mediates the export of BZR1 from thenucleus, in part due to functional redundancy and high levels of 14-3-3 proteins. However, several studies have suggested that14-3-3 proteins mediate the subcellular localizationof interactingprotein partners (Igarashi et al., 2001). Furthermore, BZR1 ap-peared to be exported from the nucleus to thecytosol via CRM1/ exportin1 receptors (Figure 2G) (Fornerod et al., 1997).

We generated a BZR1 mutant disrupting a predicted nuclearexport motif, MEDLELTL, but no alteration in subcellular local-ization was observed (data not shown). This indicates that thenuclear export of BZR1 is likely accompanied by exportingmachinery complex formation, in part with binding to 14-3-3proteins as molecular chaperones (Igarashi et al., 2001). How-ever, it is also possible that 14-3-3 proteins mediate the cyto-

plasmic retention of BZR1 proteins. All of the interacting 14-3-3proteins localize to both the nucleus and the cytosol. 14-3-3proteins were also shown to interact with BAK1, its homologSERK1, and the barley ( Hordeum vulgare ) homolog Hv BAK1(Rienties et al., 2005; Karlova et al., 2006; Schoonheim et al.,2007),further supporting important roles of 14-3-3proteinsin BRsignaling. 14-3-3 binding has recently emerged as a key regu-latory mechanismin plant signalingas well as in yeast andanimalsystems. For example, 14-3-3 proteins negatively regulate nu-clear localization of the bZIP transcriptional factor RSG tomodulate gibberellin signaling in Arabidopsis (Igarashi et al.,2001; Ishida et al., 2004). A similar role of 14-3-3 proteins wasalso reported in DBP1 (tobacco protein phosphatase1) (Carrascoet al., 2006). Therefore, themodulation of signaling output viathesubcellular redistribution of critical components by interactionwith 14-3-3 proteins could be a conserved mode of regulation inplant developmental signaling.

BZR1 S130/134A , BZR1 S173A , and BZR1 T177A mutations con-ferred increasedBR responses in transgenic plants. In particular,BZR1 S173A and BZR1 T177A , but not wild-type BZR1, caused thestrong bzr1-1D –like phenotypes, although its phosphorylationstatus was similar to that of the wild type. We also found thatBZR1 T177A proteins were dephosphorylated by BL treatment(data not shown) and bound to a target CPD promoter in plants(Figure 6D). This result indicates that phosphorylation in the rstdomain is not sufcient to inhibit BZR1 activity, but phosphory-lation in the second domain is required for full inhibition of BZR1in vivo, even though BIN2-mediated phosphorylation inhibitsDNA binding and transactivation of a BZR1 homolog, BZR2/BES1, invitro (Vert and Chory, 2006). It is likely that enhanced nuclearlocalization of BZR1 increases the pool size of BSU1 substratesin the nucleus. This could enhance DNA binding capacity to itstarget promoters and thus induce strong BR responses in theBR-stimulated state, which would lead to BR hypersensitivityand bzr1-1D –like phenotypes in the transgenic plants. It is alsopossible that an interaction between 14-3-3 and the phosphor-ylated second domain could block the DNA binding motif byeither modulating the multimerization of BZR1 or regulating in-teraction with other proteins or exporting machinery. BZR1 T177A

wouldrelieve the inhibition induced by interaction between BZR1and 14-3-3s and thus maintain the binding activity of the DNA binding motif. In any case, the phosphorylation status of the twodomains of BZR1 appears to have distinct and diverse roles inBR signaling. This further suggests that multiple regulatory

switches are required to ne-tune the signaling output of BZR1in BR signaling. The subcellular redistribution of BZR1 followingphosphorylation would be essential for normal BR responses inmany cells and would provide another regulatory layer to tightlycontrol the output strength of BR signaling in various tissues anddevelopmental stages of plants.

Further Research

While this article was under revision, Gampala et al. (2007) andBai et al. (2007) reported that the BIN2-catalyzed phosphoryla-tion of BZR1 inhibits the BR response through the cytosolicretention of BZR1 induced by the interaction between BZR1 and14-3-3 proteins in Arabidopsis and rice ( Oryza sativa ).

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana ecotype Col-0 wasused as thewild type andgeneticbackground for all transgenic lines. Seeds were germinated in mediumcontaining half-strength Gamborg B5 medium(Duchefa) and 1% sucroseunder uorescent light (14-h-light/10-h-dark cycle) at 22 8C. Young seed-lings were further grown either in medium or in soil.

Plasmid Constructs

BRI1 , BSU1 , BIN2, At GSK1 , ASKd z, ASK k , 14-3-3 e , 14-3-3 k , and BZR1cDNAs were cloned into plant expression vectors containing HA, myc, or

GFP tags driven by the35

S C4PPDK promoter (Hwang and Sheen, 2001),as indicated in the gure legends. ARR2 cDNA was fused to monomericRFP coding sequences in a plant expression vector controlled by thecassava vein mosaic virus promoter (Verdaguer et al., 1996). All of themutants were generated with the QuickChange site-directed mutagen-esis kit (Stratagene). For DEX-inducible expression of BIN2, BIN2 cDNA was fused to eight copies of the LexA binding sites as described(Yanagisawa et al., 2003), and the produced plasmid was designated8OP:BIN2-HA . The Pro35S:VP16-GR construct was obtained fromS. Yanagisawa. For the yeast two-hybrid interaction assay, cDNAs of BIN2, bin2-1 , BIN2K69R , At GSK1 , ASKd z, ASK k , 14-3-3 e , and 14-3-3 kwere cloned into the vector pGBKT7, which contains the GAL4 DNA binding domain, and BZR1 was cloned into pGADT7, which contains theGAL4 activation domain (Clontech).

Protoplast Transient Expression Assay andFluorescence Microscopy

For transient expression assays, typically, 4 3 10 4 mesophyll protoplastswere isolated from 4-week-old seedlings, transfected with 20 to 40 mg of plasmid DNA puried by CsCl gradient ultracentrifugation, and thenincubated under constant light at 23 8C (Hwang and Sheen, 2001). Fortransient induction of BIN2-HA expression, 10 mg of 8OP:BIN2-HA and10 mg of Pro35S:VP16-GR were cotransfected into wild-type protoplasts. After 3 h of incubation, the protoplasts were treated with 0, 5, or 10 mMDEX for 1 or 3 h. To test the BIN2-mediated cytosolic translocation of BZR1, 8OP:BIN2-HA , Pro35S:VP16-GR , and BZR1-GFP were cotrans-fected into protoplasts. After 5 h of incubation, the protoplasts were


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incubated with or without 10 mM DEX for 1 or 3 h. For the nuclear exportinhibition study, protoplasts transfected with the genes described abovewere pretreated with 30 nM leptomycin B (Sigma-Aldrich) for 1 h beforeDEXtreatment. Protoplasts were further incubated without or with 10 mMDEX for 5 h. For the nuclear import experiment, BZR1-GFP wascotransfected with BIN2-HA , BIN2-HA and BRI1-HA , or BIN2-HA andBSU1-HA . Transfected protoplasts were incubated for 4.5 h beforetreatment with 100 mM cycloheximide for 30 min and then further treatedwith 1 mM epi -BL for 1 to 3 h. For the nuclear import inhibition study, pro-toplasts transfected with the genes described above were pretreatedwith okadaic acid (10 and 30 nM) (Sigma-Aldrich) and 100 mM cyclohex-imide for 30 min before BR treatment. All transient transfection experi-ments were repeated at least four times. GFP and RFP uorescence wasobserved with either a confocal laser scanning microscope (LSM 510Meta system; Carl Zeiss MicroImaging) or a uorescence microscope(Axioplan; Carl Zeiss MicroImaging). The signal intensities of GFP andRFP were quantitatively determined using LSM Image Browser Rel. 4.0software (Carl ZeissMicroImaging). To detectBZR1 localizationin plants,theroot tip, elongation zone, and maturation zone of 2-week-old Pro35S:BZR1-CFP transgenic plants (Wang et al., 2002) grown on half-strengthGamborg B5 medium with or without BRZ (1 mM) were observed with a

confocal laser scanning microscope.

Protein–Protein Interaction Assays

For interaction among GSKs, 14-3-3, and BZR1, the yeast strain AH109was transformedwith pGBKT7 vector expressing BIN2 , At GSK1 , ASKd z , ASK k , 14-3-3 e , or 14-3-3 k and pGADT7 vector expressing BZR1 ,BZR1 S173A , or BZR1 T177A . Transformed cells were grown on syntheticmedium minus Leu, Trp, and His containing 3 mM 3-aminotriazole ormedium lacking Leu and Trp. To carry out coimmunoprecipitation, HA-tagged BZR1 , BZR1 S173A , or BZR1 T177A was cotransfected with myc-tagged 14-3-3 e or 14-3-3 k and then incubated for 6 h to allow proteinexpression. Total protein was extracted from the transfected protoplastsusing immunoprecipitation buffer (50 mM Tris-HCl, pH 7.5, 75 mM NaCl,5 mM EDTA, 1 mM DTT, 1 3 protease inhibitor cocktail [Roche AppliedScience], and 1% Triton X-100). The protein complex was precipitatedwith monoclonal anti-c-myc antibody (Cell Signaling) and protein A/Gplus–agarose beads (Calbiochem). Precipitated proteins were detectedwith peroxidase-conjugated high-afnity anti-HA antibody (Roche Ap-plied Science).

ChIP Assay

ChIP was performed as described (Gendrel et al., 2002) with a minormodication. Two-week-old seedlings (1 g) of transgenic and wild-typeplants (Col-0 as a control) were cross-linked with 1% formaldehydefollowed by quenching with 125 mM Gly. The grounded tissue wasresuspended with 25 mL of extraction bufferI (0.4 M sucrose, 10 mM Tris-HCl, pH 8.0, 5 mM b -mercaptoethanol, 1 mM phenylmethylsulfonyl

uoride [PMSF], and 13

protease inhibitor cocktail) and ltered throughMiracloth (Calbiochem). The ltrate was centrifuged at 4000 rpm and 4 8Cfor20 min. Thepelletwas resuspended in 1 mL of extraction buffer II (0.25M sucrose, 10 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 1% Triton X-100, 5mM b -mercaptoethanol, 1 mM PMSF, and1 3 protease inhibitor cocktail)and centrifuged at 14,000 rpm and 4 8C for 10 min. The pellet wasresuspended in 300 mL ofextraction buffer III (1.7M sucrose,10 mMTris-HCl, pH 8.0, 0.15% TritonX-100, 2 mM MgCl 2 , 5 m M b -mercaptoethanol,1 mM PMSF, and 1 3 protease inhibitor cocktail) and loaded on top of anequal amount of extraction buffer III, then centrifuged at 14,000 rpm for1 h. The nuclear pellet was resuspended in a nuclei lysis buffer (50 mMTris-HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF, and 1 3 proteaseinhibitor cocktail) and sonicated to yield DNA fragments of ; 0.5 to 1 kb.

Thechromatin solution wasdiluted 10-foldwithChIP dilution buffer(1.1%TritonX-100, 1.2mM EDTA, 16.7 mM Tris-HCl,pH 8.0, and167 mM NaCl)and precleared with protein A–agarose beads equilibrated with salmonsperm DNA.

Protein–DNA complexes in 1 mL of the precleared chromatin solutionwere immunoprecipitated with 5 mL of a high-afnity anti-HA antibody(Roche) overnight at 4 8C and extracted by incubating with 40 mL of salmon sperm DNA/protein A–agarose beads for 1 h at 4 8C. After severalwashes with a washing buffer (150 mM NaCl, 0.2% SDS, 0.5% TritonX-100, 2 mM EDTA, and 20 mM Tris-HCl, pH 8.0), the immunocomplexeswere eluted twice from the beads with 250 mL of an elution buffer (1%SDS and 0.1 M NaHCO 3 ) and then reverse cross-linked for 6 h with a nalconcentration of 250 mM NaCl at 65 8C. After removing all proteins withproteinase K, DNA was puried by phenol-chloroform extraction andethanol precipitation. Precipitated DNA was resuspended with 50 mL of 13 TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) and used as atemplate for PCR analysis. The cis elements of the CPD promoter wereamplied using a pair of gene-specic primers (5 9-CCATTGAAGAAGAA-GATGATGATGA-3 9 and 5 9-AAGAAAAGAAGAAGAATGCAAAAGAG-3 9 ). A small aliquot after preclearing was reverse cross-linked for use as aninput control. PCRconditions were as follows: 95 8C for 5 min,35 cyclesof

extension (94 8C for 30 s, 61 8C for 45 s, and 72 8C for 20 s), and 72 8C for3 min. The amplied bands were visualized on a 2% agarose gel.

Fractionation of Subcellular Organelles and Immunodetection

Nuclear and cytoplasmic fractions in protoplasts expressing HA-taggedproteins were separated as described previously (Yanagisawa et al.,2003). Protoplasts were lysed with a buffer (20 mM Tris-HCl, pH 7.0,250 mM sucrose, 25% glycerol, 20 mM KCl, 2 mM EDTA, 2.5 mM MgCl 2 ,30 mM b -mercaptoethanol, 1 3 protease inhibitor cocktail, and 0.7%Triton X-100) and fractionated by centrifugation at 3000 g. The superna-tant was taken as the cytosolic fraction. The pellet was further washedwith a resuspension buffer (20 mM Tris-HCl, pH 7.0, 25% glycerol,2.5 mM MgCl 2 , and 30 mM b -mercaptoethanol) and reconstituted as thenuclear fraction. Each fraction was resolved by 10% SDS-PAGE, fol-

lowed by immunodetection with a peroxidase-conjugated high-afnityanti-HA antibody, anti-RHA1, and anti-histone H2B (Upstate). For theimmunoblot analyses,3 to 20 mg of proteins from protoplasts orseedlingswas analyzed by 10% SDS-PAGE and detected with a horseradishperoxidase–conjugated anti-HA, anti-myc, anti-actin (MP Biomedicals),or anti-GFP (Clontech) antibody.

Physiological Analysis of Transgenic Arabidopsis

To generate transgenicplants overexpressing various forms of BZR1 , thecoding sequences were cloned into pCB302ES containing the 35Spromoter and the HA epitope tag (Hwang and Sheen, 2001) and inte-grated into the Arabidopsis genome by the Agrobacterium tumefaciens –mediated oral dipping method (Clough and Bent, 1998). To driveendogenous levels of protein expression, the promoter and coding

regions (3.4 kb) of BZR1 were cloned into pCB302 containing the HA epitope tag (Xiang et al., 1999; Hwang and Sheen, 2001). The transgeneexpression was veried by immunoblotting with an anti-HA antibody andquantitativereal-time PCRwithsets of gene-specic forward primers andan HA tag–specic reverse primer. For hypocotyl and root elongationassays, T3 homozygous seeds were plated on Gamborg B5 mediumcontaining 1%sucrose and1 mM BRZ(Sekimata etal., 2001)or BL(0.001,0.01, 0.1, and 1 mM) (Sigma-Aldrich) and grown for 5 to 8 d in the dark orunder 14-h-light/10-h-dark conditions. To determine expression levels of the CPD and DWF4 genes, total RNA was isolated from each transgenicline. Quantitative real-time PCR was performed according to the instruc-tions provided for the LightCycler 2.0 (Roche) with the SYBR PremixExTaq system (Takara). To investigate the BIN2 expression pattern in

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planta, the 3.4-kb Arabidopsis BIN2 promoter was cloned into the vectorpCAMBIA 1303. Two-week-old T3 homozygous plants were stained withX-Gluc (Duchefa).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Subcellular Localization of BZR1-CFP in theMaturation Zone of Arabidopsis Roots.

Supplemental Figure 2. Subcellular Localization of BZR1-CFP in theElongation Zone of Arabidopsis Roots.

Supplemental Figure 3. Subcellular Localization of BZR1-CFP in theElongation Zone of Arabidopsis Roots Grown on BRZ-ContainingMedium.

Supplemental Figure 4. Mesophyll Protoplasts and Whole PlantsShow a Similar Response to BR.

Supplemental Figure 5. Group II At GSKs Have Functional Redun-dancy in BR Signal Transduction in Arabidopsis .

Supplemental Figure 6. Identication of Putative PhosphorylationResidues Required for the Nuclear Export of BZR1 by BIN2.

Supplemental Figure 7. Transgene Expression and Physiological Analysis of Nucleus-Accumulated BZR1 Mutants.

ACKNOWLEDGMENTS

We thank Jen Sheen and Joe Kieber for comments on the manuscriptand Zhiyong Wang for the Pro35S:BZR1-CFP seeds. This work wassupported by grants from the Plant Diversity Research Center and theKorea Science and Engineering Foundation (Grant M10601000194-06N0100-19410), the Plant Signaling Network Research Center, andthe Korea Research Foundation (Grants KRF-2005-070-C00129 andR08-2003-000-10819-0). K.K. was supported by the POSTECH CoreResearch Fund.

Received June 17, 2007; revised August 13, 2007; accepted August 28,2007; published September 14, 2007.

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2762 The Plant Cell

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DOI 10.1105/tpc.107.053728; originally published online September 14, 2007;2007;19;2749-2762Plant Cell

Hojin Ryu, Kangmin Kim, Hyunwoo Cho, Joonghyuk Park, Sunghwa Choe and Ildoo HwangBrassinosteroid Signaling

ArabidopsisNucleocytoplasmic Shuttling of BZR1 Mediated by Phosphorylation Is Essential in

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