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BRANCHED1 Interacts with FLOWERING LOCUS T to Repressthe Floral Transition of the Axillary Meristemsin ArabidopsisC W OA

Masaki Niwa,a Yasufumi Daimon,a,1 Ken-ichi Kurotani,a,2 Asuka Higo,a José L. Pruneda-Paz,b Ghislain Breton,b,3

Nobutaka Mitsuda,c Steve A. Kay,b,4 Masaru Ohme-Takagi,c,d Motomu Endo,a and Takashi Arakia,5

a Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japanb Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, California92093cBioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba 305-8562, Japand Institute for Environmental Science and Technology, Saitama University, Saitama 338-8770, Japan

Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floraltransition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such asBRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWINSISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into thesubtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM).The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interfereswith florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristemdelays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT andTSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.

INTRODUCTION

There is a large degree of developmental plasticity in plant ar-chitecture, which is determined in part by the timing of floraltransition and the branching pattern. The floral transition is in-duced by a systemic flowering signal (florigen), and plant ar-chitecture is often remodeled upon the floral transition in theshoot apical meristem (SAM). In the rosette-forming plant Arab-idopsis thaliana, floral transition is followed by rapid internodeelongation and activation of axillary bud development in in-ductive long-day (LD) conditions. Since axillary bud developmentproceeds differently than that of the main shoot, the systemic

flowering signal may also be perceived differently in the twolocations.Appropriate regulation of axillary bud development is ach-

ieved through integrating internal and environmental signals ateach of the three developmental stages: axillary meristem (AM)initiation, bud differentiation, and shoot elongation. Amongthem, the regulation of AM initiation and shoot elongation hasbeen studied intensively. Molecular genetic approaches havehelped identify genes required for initiation of AMs. In Arabi-dopsis, REVOLUTA and related HD-ZIP genes are required tospecify adaxiality of lateral organs and act to form AMs (Talbertet al., 1995; Emery et al., 2003). AM initiation requires the GRASfamily gene LATERAL SUPPRESOR (Schumacher et al., 1999;Greb et al., 2003) and the Myb-like transcription factor (TF)REGULATOR OF AXILLARY MERISTEM1 (Keller et al., 2006;Müller et al., 2006). On the other hand, axillary shoot elongationis regulated by multiple plant hormones. Auxin produced in themain shoot suppresses axillary shoot elongation (Dun et al.,2006). Strigolactones, carotenoid-derived hormones producedin roots, also act as negative regulatory factors (Gomez-Roldanet al., 2008; Umehara et al., 2008; Crawford et al., 2010). Cy-tokinin is thought to be a secondary messenger and acts morelocally in the buds to promote outgrowth.Compared with initiation and elongation of the axillary bud,

regulation of differentiation including floral transition is far lessunderstood. One gene expressed in the axillary bud known toregulate differentiation is BRANCHED1/TEOSINTE BRANCHED1-LIKE1 (BRC1/TBL1; hereafter referred as BRC1). BRC1 encodesa member of the TEOSINTE BRANCHED1, CYCLOIDEA, and PCF

1Current address: Graduate School of Agricultural and Life Sciences,University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.2 Current address: Bioscience and Biotechnology Center, NagoyaUniversity, Chikusa-ku, Nagoya 464-8601, Japan.3 Current address: Department of Integrative Biology and Pharmacology,University of Texas Health Science Center at Houston, Houston, TX77030.4 Current address: Dana and David Dornsife College of Letters, Arts, andSciences, University of Southern California, Los Angeles, CA 90089.5 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Takashi Araki ([email protected]).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.112.109090

The Plant Cell, Vol. 25: 1228–1242, April 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

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(TCP) TF family and was identified as an ortholog of maize (Zeamays) Teosinte branched1 (Tb1) (Doebley et al., 1997; Aguilar-Martínez et al., 2007; Finlayson, 2007). It was suggested that Tb1-like genes act downstream of strigolactone signaling to suppressdevelopment of axillary shoots in Arabidopsis and pea (Pisumsativum) (Aguilar-Martínez et al., 2007; Finlayson, 2007; Breweret al., 2009). Since axillary buds of the brc1 mutant show ad-vanced development compared with the wild type, it was sug-gested that BRC1 acts as a negative regulator of differentiation inaxillary buds (Aguilar-Martínez et al., 2007). The brc1 mutant oc-casionally develops ectopic AMs at cotyledonary axils, suggest-ing that BRC1 also has an inhibitory role in initiation of AMs(Aguilar-Martínez et al., 2007). It was also suggested that BRC1suppresses the elongation of axillary shoots (Aguilar-Martínezet al., 2007; Finlayson, 2007). These pleiotropic effects reflectmultiple roles of BRC1 in axillary bud development. The expres-sion of BRC1 is regulated by environmental signals, such asplanting density (Aguilar-Martínez et al., 2007; Finlayson et al.,2010). Based on these observations, BRC1 was proposed to bean integrator of axillary bud development (Aguilar-Martínez et al.,2007). A BRC1 paralog, BRC2, is thought to have a partially re-dundant but distinct role in axillary bud development (Aguilar-Martínez et al., 2007; Finlayson, 2007; Finlayson et al., 2010).

In Arabidopsis, initiation of axillary bud development is closelylinked to floral transition in the SAM under LD conditions(Hempel and Feldman, 1994). The molecular mechanism con-trolling floral transition is well understood in Arabidopsis andother species. Among the key regulators of floral transition, FTacts as a floral pathway integrator. Both endogenous and en-vironmental signaling pathways are integrated in the transcrip-tional regulation of FT, which encodes a 20-kD protein of thePEBP/RKIP (for phosphatidylethanolamine binding protein/Rafkinase inhibitor protein) family that acts as a long-distanceflowering signal (Kardailsky et al., 1999; Kobayashi et al., 1999;Lifschitz et al., 2006; Corbesier et al., 2007; Jaeger and Wigge,2007; Lin et al., 2007; Mathieu et al., 2007; Tamaki et al., 2007;Notaguchi et al., 2008). FT expression is induced in phloemcompanion cells in cotyledons and leaves under inductive LDconditions (Takada and Goto, 2003). FT protein then moves intothe SAM, where it interacts with the bZIP TF FD, to induce theexpression of a floral meristem identity gene, APETALA1 (AP1)(Abe et al., 2005; Wigge et al., 2005). A recent study in rice(Oryza sativa) showed that the interaction between Hd3a (an FTortholog) and FD1 (an FD ortholog) is not direct but is mediatedby 14-3-3 proteins (Taoka et al., 2011). Os-FD1 interacts witha 14-3-3 protein through a conventional 14-3-3 recognitionmotif R/K-X-X-S-X-P, whereas Os-Hd3a has a noncanonicalbinding site for 14-3-3 proteins (Yaffe et al., 1997; Pnueli et al.,2001; Taoka et al., 2011). Other PEBP/RKIP proteins are alsoinvolved in the regulation of the floral transition in Arabidopsis.TWIN SISTER OF FT (TSF), the paralog of FT, also acts asa floral promoter (Michaels et al., 2005; Yamaguchi et al., 2005).Although TSF has a minor role under LD conditions in which FTis robustly expressed, TSF makes a prominent contribution tothe floral transition under short-day (SD) conditions (Yamaguchiet al., 2005). By contrast, TERMINAL FLOWER1 (TFL1) delaysthe floral transition through interaction with FD (Bradley et al.,1997; Hanano and Goto, 2011). TFL1 also plays a key role in

maintenance of inflorescence meristems. In the tfl1 mutant un-der LD conditions, the main inflorescence is terminated witha fused floral structure and the axillary shoots are replaced bysingle flowers (Shannon and Meeks-Wagner, 1991; Alvarezet al., 1992).In Arabidopsis, when floral transition is induced in the SAM,

preexisting primordia develop into cauline leaves and newlyformed primordia become floral buds. AMs are initiated anddevelop basipetally after floral transition of the SAM in LDconditions (Hempel and Feldman, 1994). It was suggested thatAMs are at first insensitive to flowering signals since they formvegetative nodes before making flowers even though a sufficientamount of the systemic flowering signal is produced to inducefloral transition in the SAM (Hempel and Feldman, 1994; Grbi�cand Bleecker, 1996). Therefore, florigen activity and/or percep-tion are likely to be differently modulated in AMs than in theSAM.In this study, we identified BRC1 as an interactor of FT without

mediation by a 14-3-3 protein and demonstrated that BRC1interacts with TSF but not TFL1. Our results indicate that BRC1modulates the florigen activity in the AMs and confers a specificmode of regulation to the floral transition in AMs.

RESULTS

BRC1 Interacts with FT

The fact that FT acts with the bZIP protein FD in a transcriptionalcomplex prompted us to seek other TFs as possible interactingproteins. To identify TFs that can interact with FT, we performedyeast two-hybrid screening using two different TF libraries. Ro-botized screening of the first library (Steve Kay Lab) revealed 64candidates when the threshold was set to twofold higher than theaverage (see Supplemental Table 1 online). By manual screeningof a second TF library (Mitsuda et al., 2010), 10 TFs were found toshow reproducible interaction with FT (see Supplemental Figure 1online). Interestingly, five candidates in the first screening and fourin the second were TFs belonging to the TCP family. Becausesome TCP family members were not represented in the two TFlibraries, we performed yeast two-hybrid assays with the in-dividual TCP transcriptional factors that have been reported to beinvolved in various developmental processes. Among them,BRC1 reproducibly interacted with FT in yeast two-hybrid assays(Figure 1A). We focused on BRC1 as a good candidate to be a linkbetween the floral transition and concomitant architectural re-modeling through branch development.We further investigated interaction between BRC1 and two FT

homologs, TSF (in the FT clade) and TFL1 (in the TFL1 clade),by yeast two-hybrid assay. TSF also showed interaction withBRC1, while TFL1 did not (Figure 1A). No interactions betweenthe TCP protein BRC2, which is closely related to BRC1 (Aguilar-Martínez et al., 2007), and these PEBP/RKIP proteins were de-tected in our yeast two-hybrid experiments (Figure 1A).Protein interaction between FT and BRC1 was further con-

firmed in plant cells by bimolecular fluorescence complemen-tation (BiFC) assays in Nicotiana benthamiana and in vitro bypull-down assays. A clear BiFC signal was detected in the

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nucleus, indicating that FT interacts with BRC1 in plant cells andthat the FT-BRC1 complex is localized to the nucleus (Figure1B). BRC1 was able to pull down FT in the in vitro assay, in-dicating that their interaction is likely direct (Figure 1C).

Analysis using a series of deletion constructs showed that theC-terminal region of BRC1 including the conserved R domain isdispensable for interaction with FT (Figure 1D). However, re-moval of the entire C terminus after the TCP domain disruptedthe interaction (see Supplemental Figure 2 online). In addition,a relatively small deletion of the N-terminal 74 amino acid resi-dues abolished the interaction (Figure 1D). These results sug-gest the N-terminal region of BRC1 is necessary but not sufficientfor interaction with FT.

14-3-3 Proteins Do Not Mediate the Interaction betweenBRC1 and FT

In rice, 14-3-3 proteins mediate the interaction between Hd3a(an FT ortholog) and FD1 (Taoka et al., 2011). Since Arabidopsis

FT can interact with 14-3-3 proteins, it is likely that they mediateFT–FD interaction in Arabidopsis as well (Pnueli et al., 2001).This raises the question whether 14-3-3 proteins also mediatethe interaction between BRC1 and FT. To explore whether 14-3-3 proteins mediate the interaction between BRC1 and FT, twosets of yeast two-hybrid assay were performed. First, we ex-amined whether point mutations in FT that abolish interactionwith 14-3-3 proteins also affect interaction with BRC1. All threemutant FT proteins tested still showed interaction with BRC1,suggesting that 14-3-3 proteins do not mediate the interaction(Figure 2A). BRC1 has a sequence conforming to a recognitionmotif of 14-3-3 proteins (R/K-X-X-S-X-P) in the C-terminal re-gion (Yaffe et al., 1997; Taoka et al., 2011; Figure 2B). Mutationin this sequence (S390A) did not abolish interaction with FT,further supporting that 14-3-3 proteins are not required for theinteraction (Figure 2A). Second, Ala scanning was performed toidentify residues in FT required for interaction with BRC1.Among 46 mutant FT proteins tested, seven did not show in-teraction with BRC1 but could interact with a 14-3-3 protein

Figure 1. Protein Interaction between BRC1 and FT.

(A) Yeast two-hybrid analysis of protein interaction between BRC1, BRC2, or GRF3 and FT, TSF, or TFL1. Clones containing each combination of baitand prey vectors were grown on nonselective medium (+His) or on selective medium (2His). For the clones containing BRC1, 3-amino-1,2,4-triazole (3-AT) was added to the medium (left two rows). pCUY and pGAD424 empty vectors were used as a negative control (BD [binding domain] and AD[activation domain], respectively).(B) BiFC analysis of the interaction between BRC1 and FT in N. benthamiana leaf epidermal cells. The N-terminal fragment of EYFP was fused to BRC1(YN-BRC1) and the C-terminal fragment of EYFP to FT (FT-YC). The fusion proteins were transiently expressed under the cauliflower mosaic virus 35Spromoter through agroinfiltration. EYFP fluorescence (top panels) and EYFP fluorescence merged with a bright-field (BF) image (bottom panels) areshown. Combinations in which one of the fusion constructs was replaced with a fragment of EYFP (YN or YC) were used as negative controls (centraland right columns). Arrowheads indicate complemented EYFP fluorescence in the nucleus. Bar = 200 mm.(C) In vitro pull-down assay demonstrating the interaction between BRC1 and FT. Closed arrowhead, open arrowhead, and arrow indicate T7-FT, GST-BRC1, and GST, respectively.(D) Yeast two-hybrid assay of protein interaction between FT and truncated versions of BRC1. TCP, TCP domain; R, R domain; a.a., amino acids.

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(GENERAL REGULATORY FACTOR3 [GRF3; 14-3-3 psi]) (Figure2C; see Supplemental Figure 3 online). When these residueswere plotted on the FT three-dimensional structure model (Ahnet al., 2006; Protein Data Bank ID 1WKP), the region required forthe interaction with BRC1 was distinct from the interaction in-terface between FT and 14-3-3 proteins (Taoka et al., 2011;Figure 2D). These results, taken together, indicate that the in-teraction between FT and BRC1 is not mediated by 14-3-3. Thus,BRC1 interacts with FT in a different manner than FD does.

Whether candidates from the yeast two-hybrid screen usingthe second TF library interact with FT through 14-3-3 proteins

was also investigated. A bZIP TF, bZIP30, interacted with 14-3-3proteins and failed to interact with the mutant FT that cannotinteract with 14-3-3 proteins. This suggests that 14-3-3 isprobably required to mediate the FT-bZIP30 interaction (seeSupplemental Figure 1 online). Other candidates interacted withat least one of the mutant FT proteins that cannot interact with14-3-3 proteins, indicating they probably do not require 14-3-3proteins for interaction with FT (see Supplemental Figure 1online).

FT Protein Can Move to an Axillary Bud from theSubtending Leaf

BRC1 is expressed in AMs and the brc1mutant shows defectivephenotypes specifically in the axillary buds (Aguilar-Martínezet al., 2007; Finlayson, 2007). On the other hand, FT is expressedin the leaf vasculature (Takada and Goto, 2003; Yamaguchi et al.,2005; Notaguchi et al., 2008), and FT protein moves into the SAMto promote floral transition (Corbesier et al., 2007; Jaeger andWigge, 2007; Mathieu et al., 2007; Notaguchi et al., 2008). Theobservation that BRC1 and FT protein can interact in plant cellsand in vitro strongly suggests that FT also has a role in the axillarybuds.To explore the possible expression of FT in the axillary

buds, we examined the expression pattern of FT using gFT:GUS (for b-glucuronidase) plants (Notaguchi et al., 2008).Because of its large molecular mass (88.9 kD), FT-GUS fusionprotein is a good indicator of expression pattern. It does notseem to move out of the cells where originally expressed(Notaguchi et al., 2008) and did not promote flowering whenexpressed in the leaf (see Supplemental Figure 4 online). Aspreviously reported, GUS staining was observed in vascularbundles toward the periphery of the rosette leaf blade in gFT:GUS plants (Takada and Goto, 2003; Yamaguchi et al., 2005;Notaguchi et al., 2008; Figure 3A). As in the rosette leaves, FTexpression was detected in the vasculature in the distal part ofcauline leaves but not in the axillary bud (Figure 3B). Furtherobservations of sections of axillary buds did not reveal stainingin the buds at the cauline or rosette leaf axils (Figures 3C and3D; see Supplemental Figure 4 online). We next examinedwhether FT protein expressed in a leaf blade moves into theaxillary bud subtended by the leaf using the ProHSP18.2:FT-EGFP-inducible reporter system (Notaguchi et al., 2008). In-duction of FT-EGFP (for enhanced green fluorescent protein)protein in a single leaf blade was confirmed after local tran-sient heat treatment (see Supplemental Figure 5 online). Oneleaf blade was heated for 2 h to induce FT-EGFP fusion pro-tein, and 24 h after the end of the heat treatment, FT-EGFPfluorescence in the axillary bud subtended by the heat-treatedleaf was observed (Figure 3E). When the heat-treated leafblade was removed by cutting the base of the leaf blade im-mediately after the heat treatment, fluorescence was not de-tected in the axillary bud (Figure 3F). These results showedthat FT-EGFP fusion protein expressed in the leaf blade canmove into the axillary bud. Based on expression in leaves andthe ability to move into the axillary bud from the subtendingleaf, we concluded that FT protein is likely to be present inaxillary buds.

Figure 2. 14-3-3 Proteins Do Not Mediate the Interaction between FTand BRC1.

(A) Yeast two-hybrid analysis of interaction between BRC1 and mutantFT proteins defective in interaction with 14-3-3 proteins. T66I, P94L,G102D, and P75L are mutant FT proteins, the former three of which donot interact with 14-3-3 proteins. S390A is a mutated BRC1 in whicha Ser residue in a putative 14-3-3 recognition site (S390) was substitutedby Ala. GRF3 was chosen as a representative 14-3-3 protein. Yeastswere grown on nonselective medium (+His) or on selective medium(2His and 2His+3-AT). WT, the wild type.(B) Schematic diagram of BRC1 protein depicting the putative 14-3-3recognition site. TCP, TCP domain; R, R domain; a.a., amino acids.(C) Yeast two-hybrid assay of interaction between mutant FT proteinsand BRC1 or GRF3. Representative results for seven FT mutants de-fective in interaction with the BRC1 protein and a mutant defective ininteraction with GRF3 were shown. These mutants were obtained byscreening of Ala-scanned FT mutants (see Supplemental Figure 3 online).Yeasts were grown on nonselective medium (+His) or on selective me-dium (2His and 2His+3-AT).(D) Amino acid residues essential for interaction with BRC1 ([C], in-dicated by magenta) or 14-3-3 proteins ([A], indicated by yellow; Taokaet al., 2011) on a ribbon model of the FT protein.


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brc1-2 Mutants Show Accelerated Floral Transitionin the AMs

In order to gain insight into the physiological role of the in-teraction between FT and BRC1, we looked for relevant phe-notypes in the brc1-2 (tbl1-1) mutant (Alonso et al., 2003;Aguilar-Martínez et al., 2007; Finlayson, 2007). As previouslyreported, more axillary shoots grew out in the brc1-2 mutantthan in the wild type (Aguilar-Martínez et al., 2007; Finlayson,2007; Figure 4A). We also confirmed that the development ofAMs is more advanced in brc1-2 than in the wild type (Aguilar-Martínez et al., 2007; Figure 4B). Further observation indicatedthat almost all the existing axillary buds proceeded to re-productive phase in brc1-2 mutant shortly after bolting of themain shoot. By contrast, lower axillary buds remained vegetativein the wild type (Figure 4B). Furthermore, the axillary shoots ofthe uppermost nodes formed no leaves in many brc1-2 plants,whereas two leaves were usually formed in wild-type plants(Figure 4A). Quantification of leaf number showed that brc1-2has fewer leaves on the axillary shoots at every position com-pared with the wild type (Figure 4C). A similar phenotype wasobserved for the lowest cauline and uppermost rosette nodes ina previous report (Finlayson et al., 2010).Because leaf number is generally considered to reflect the

timing of the floral transition, we assumed that the reduction ofleaf number in brc1-2 indicates earlier floral transition in theAMs. To confirm this possibility in the context of gene expres-sion, we examined AP1 expression in the AMs using a ProAP1:GUS reporter (Hempel et al., 1997). In the SAM, the timing of theonset of AP1 expression did not differ between brc1-2 and wild-type backgrounds, in accordance with the fact that the leafnumber of the main shoot did not significantly differ between thetwo genotypes. By contrast, the floral transition of the AMsproceeded differently between the brc1-2 mutant and wild-typeplants. Eighteen days after germination, almost all the brc1-2plants (92%; 22/24) had at least one AP1-expressing axillarybud, whereas only about a half of the wild-type plants (53%; 16/30) did. The quantification of AP1-expressing buds indicatedthat the brc1-2 mutant formed more reproductive axillary budsthan did the wild type (Figures 4D and 4E). Introduction ofa genomic fragment of BRC1 into brc1-2 complemented the leafnumber phenotype (see Supplemental Figure 6 online). There-fore, we concluded that floral transition of the AM is acceleratedin the brc1-2 mutant.

The ft-2 Mutation Is Epistatic to the brc1-2 Mutation

The observations that the number of leaves on the axillaryshoots differed depending on the node positions in the wild typeand that floral transition of AMs in the brc1-2 mutant was ac-celerated imply that floral transition of AMs is a regulated pro-cess (Figure 4). Furthermore, the observation that FT proteincould move into the axillary buds suggests that FT could beinvolved in this regulation. To address this, we examined floraltransition of AMs in ft-2 single and brc1-2 ft-2 double mutants.Compared with the wild type, the ft-2 mutant formed moreleaves on its axillary shoots (Figure 5A), suggesting that floraltransition in AMs was delayed in the ft-2 mutant. The brc1-2 ft-2

Figure 3. FT Expression Pattern and Movement of FT Protein into Ax-illary Buds.

(A) to (D) FT expression patterns monitored by GUS staining of gFT:GUSplant. Whole-mount preparations of a rosette leaf (A) and the main stemof a plant with the axillary bud subtended by a cauline leaf (B). Resinsections of the axillary buds subtended by a cauline leaf (C) and bya rosette leaf (D). Arrowhead in (B) indicates staining in the peripheralregion of the cauline leaf.(E) and (F) Movement of FT-EGFP into an axillary bud from the sub-tending leaf. EGFP fluorescence merged with a bright-field image of theaxillary bud is shown. FT-EGFP expression was induced from theProHSP18.2:FT-EGFP transgene by heat treatment of the leaf blade ofa rosette leaf (see Supplemental Figure 5 online). EGFP fluorescence wasobserved 24 h after the end of the heat treatment. The treated leaf bladewas either left intact (E) or cut off immediately after the heat treatment toprevent FT-EGFP export (F). The cut site is not seen in (F).ap, apical meristem; ax, axillary meristem; cl, cauline leaf; fl, floral bud; pt,petiole of the heat-treated rosette leaf; rl, rosette leaf; st, stem. Bar in(A) = 1 mm and bars in other panels = 100 mm.

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Figure 4. Early Floral Transition of the Axillary Shoots in brc1 Mutants.

(A) Axillary shoot phenotype of the wild type (Col) and brc1-2 mutant. Successive axillary shoots with the subtending leaf of 44-d-old plants are shown.Arrowheads and arrows indicate vegetative nodes and the first flowers in the most apical branches. Plus numbers represent cauline-leaf axils in theacropetal direction, and minus numbers represent rosette-leaf axils in the basipetal direction as shown in the right diagram. In the diagram, open circles,arrows, and bars represent flowers, shoots, and leaves, respectively.(B) Developmental stages of the buds in the axils of cotyledons (c1 and c2) and rosette leaves (L1 to L15) of 10 plants of wild-type Col (left) and brc1-2(right). Developmental stages were defined according to Aguilar-Martínez et al. (2007). Gray indicates absence of the leaf node such that the position isoccupied by a flower. Observations were made on the days when the first floral buds became visible (before bolting), the main shoot became 1-cm long(1 cm bolting), and the main shoot became 5-cm long (5 cm bolting).(C) Number of leaves formed on axillary shoots at the five axil positions of the wild type (Col) and brc1-2mutant grown in LD. The axil position is definedin (A). Leaves in the basal part of axillary shoots without internodes are classified as axillary rosette leaves (ARL) and those with elongated internodes ascauline leaves (CL). Error bars indicate the SD (n = 9). The differences significant between Col and brc1-2 in two-tailed multiple t test with Bonferronicorrection (P < 0.05) are indicated with asterisks.(D) and (E) Expression of AP1 in the axillary buds monitored by ProAP1:GUS. Number of GUS-positive axillary buds in ProAP1:GUS plants in wild-type(Col) and brc1-2 background grown under LD (D). Error bars indicate the SD (n $ 21). The differences significant in two-tailed multiple t test withBonferroni correction (P < 0.05) with respect to the wild type (Col) are indicated with asterisks. Representative 22-d-old ProAP1:GUS plants in wild-type(Col) and brc1-2 backgrounds (E). Arrowheads indicate GUS-positive axillary buds at rosette-leaf axils.


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double mutant formed almost the same number of leaves as theft-2 single mutant on axillary shoots of cauline leaves (Figure5A). Thus, the acceleration of AM floral transition observed in thebrc1-2 mutant requires functional FT in cauline axils. In axillaryshoots of rosette leaves, the brc1-2 ft-2 mutant formed fewerleaves than the ft-2 mutant (Figure 5A). The elongation of axillaryshoots at the rosette axils was also enhanced in brc1-2 ft-2compared with ft-2 (Figure 5B).

In the ft mutant background, TSF promotes the floral transi-tion during prolonged vegetative phase (Yamaguchi et al., 2005;Hiraoka et al., 2013). Because our yeast two-hybrid assayshowed that TSF is also able to interact with BRC1 (Figure 1A),TSF is likely to be responsible for the acceleration of floraltransition in the AMs at rosette axils in the brc1-2 ft-2mutant. Totest this hypothesis, the phenotype of the brc1-2 ft-2 tsf-1 triplemutant was examined. As observed in the brc1-2 ft-2 and ft-2

Figure 5. Delayed Floral Transition of Axillary Shoots in ft and ft tsf Mutants and Genetic Interaction with brc1.

(A) Number of leaves formed on the axillary shoots at the five axil positions of the wild type (Col) and brc1-2, ft-2, and brc1-2 ft-2 mutants grown in LD.Error bars indicate the SD (n = 12 for Col and brc1-2; n = 24 for ft-2 and brc1-2 ft-2). The differences significant between ft-2 and brc1-2 ft-2 in two-tailedmultiple t test with Bonferroni correction (P < 0.05) are indicated with asterisks. ARL, axillary rosette leaves; CL, cauline leaves.(B) Representative plants of ft-2 and brc1-2 ft-2 mutants grown in LD for 60 d Arrows indicate elongated axillary shoots at rosette axils.(C) Number of leaves formed on the axillary shoots at the three axil positions of the wild type (Col) and brc1-2, ft-2 tsf-1, and brc1-2 ft-2 tsf-1 (triple)mutants grown in LD. Error bars indicate the SD (n = 12). The differences significant between ft-2 tsf-1 and brc1-2 ft-2 tsf-1 in two-tailed multiple t testwith Bonferroni correction (P < 0.05) are indicated with asterisks.(D) Axillary shoot phenotype of ft-2 tsf-1 and brc1-2 ft-2 tsf-1 mutants. Top panel shows the rosette part of the plants, and bottom panels show theuppermost rosette leaf with the axillary bud or shoot. Note that most of the primary rosette leaves on the main axis were decaying or senescent.Arrowheads in the bottom panels indicate the senescent rosette leaves. Bars = 5 mm.Designation of the axil positions and classification of leaves in (A) and (C) are as in Figure 4.[See online article for color version of this figure.]

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mutants, brc1-2 ft-2 tsf-1 formed almost the same number ofleaves as the ft-2 tsf-1 double mutant in axillary shoots ofcauline leaves (Figure 5C). The ft-2 tsf-1 double mutant did notform elongated axillary shoots on its rosette axils under ourexperimental conditions (Hiraoka et al., 2013; Figure 5D). In thebrc1-2 ft-2 tsf-1 triple mutant, rosette leaves did form axillarybuds and developed many leaves, but over 100 d after bolting ofthe main shoot, the buds failed to make flowers and did notelongate (Figure 5D). These results suggest that prompt floraltransition and subsequent elongation of the axillary shoots in thebrc1-2 mutant require either FT or TSF functions.

In contrast with the ft-2 single and ft-2 tsf-1 double mutants,both of which were epistatic to the brc1-2 mutant, the tfl1-17mutant showed an additive effect on reduction in leaf number ofaxillary shoots in the double mutant with brc1-2 (see SupplementalFigure 7 online). Taking this together with the results of yeasttwo-hybrid assay (Figure 1A), BRC1 and TFL1 are likely to actindependently to suppress floral transition in AMs.

Relationship between BRC1 and Flowering-Time Genes

FT acts together with FD to promote floral transition in the SAM.Because FD is also expressed in the AMs (Abe et al., 2005;Wigge et al., 2005; Figure 6A), it is likely that FD also has a role infloral transition of the AMs. Similar to the brc1-2 ft-2 mutant, thebrc1-2 fd-1 mutant showed substantial suppression of the brc1-2 phenotype in terms of the leaf number of axillary shoots(Figure 6B). This indicates that FD also acts as a promoter of thefloral transition in AMs.

It was proposed that AP1, FRUITFULL (FUL), and SU-PRESSOR OF OVEREXPRESSION OF CO1 (SOC1) are down-stream genes of the FT-FD floral transition pathway (Abe et al.,2005; Michaels et al., 2005; Teper-Bamnolker and Samach,2005; Wigge et al., 2005; Yoo et al., 2005; Searle et al., 2006). Toexamine the effect of BRC1 on the expression of these genes,wild-type and brc1-2 mutant plants were grown under non-inductive SD conditions and then transferred to LD. After boltingof the main shoot, a plant section containing the axils of therosette leaves was harvested by removing the main shoot, ex-panded leaves, hypocotyl, and root at three time points (13, 16,and 19 d after the LD shift) and subjected to expression analysis.Quantitative RT-PCR showed that AP1 and FUL were alreadyupregulated 13 d after the LD shift in brc1-2 compared with thewild type (Figure 6C). These two genes were further upregulatedin brc1-2 during further growth in LD but remained at a low level inthe wild type. Although SOC1 expression was not upregulated inbrc1-2 compared with the wild type at 13 d after the LD shift,a gradual increase was observed during the subsequent growthin LD (Figure 6C). The floral meristem identity gene LEAFY (LFY)is expressed in an FT-independent manner. Although LFY ex-pression level was elevated in brc1-2 13 d after the LD shift,unlike AP1 and FUL, subsequent upregulation did not occur atlater time points (see Supplemental Figure 8 online). These re-sults indicate that BRC1 represses photoperiod-dependent ex-pression of FT downstream genes in axillary buds.

SOC1 functions in floral transition of AMs together with FULand other SOC1-related MADS box genes (Melzer et al., 2008;Dorca-Fornell et al., 2011). In accordance with this, the soc1-2

mutant formed many more leaves on axillary shoots compared withthe wild type (Figure 6D). Nevertheless, the soc1-2 mutation onlypartially suppressed the phenotype of the brc1-2 mutant (Figure6D). These results suggest that, unlike the cases of FT and TSF, thebrc1-2 phenotype is not fully dependent on SOC1 function.

BRC1 Can Delay Floral Transition When Expressedin the SAM

The observations described so far indicate that BRC1 has a rolein preventing AMs from undergoing early floral transition. Theabilities of FT to be transported into the axillary buds and tointeract with BRC1 suggest that BRC1 is likely to act throughinhibition of FT function. This raises the possibility that BRC1could delay the floral transition of the main shoot when expressedectopically in the SAM. To test this possibility, the flowering-timephenotype of transgenic plants expressing BRC1 in the SAMunder the control of the FD promoter (ProFD:BRC1) was exam-ined. As expected, ProFD:BRC1 plants showed significant delayin floral transition of the SAM under inductive LD (Figure 7; seeSupplemental Figure 9 online). Previously, it was shown thatoverexpression of BRC1 under the control of cauliflower mosaicvirus 35S promoter resulted in pleiotropic developmental defects(Aguilar-Martínez et al., 2007). In this study, ProFD:BRC1 plantsshowed mild retarded growth in early developmental stages but noobvious morphological alterations (Figure 7A). In SD, the effect ofProFD:BRC1was more subtle (see Supplemental Figure 10 online).

DISCUSSION

In this study, we showed that the BRC1 interacts with FT. BRC1interacts also with TSF, whereas it did not interact with TFL1.This establishes BRC1 as an interactor specific to the FT cladeof PEBP/RKIP proteins. Our study indicates that FT proteinexpressed in the leaf is transported into the axillary bud andpromotes floral transition of the AM. BRC1 expressed in theaxillary bud interacts with FT to inhibit its function. Thus, BRC1,previously proposed to be an integrator of branching signalswithin the axillary buds, plays a role in modulating the activity ofa long-distance florigen signal to delay floral transition in AMs.

FT Is Able to Interact with Various TFs

Yeast two-hybrid screening using TF libraries revealed numer-ous candidate FT-interacting proteins. Although the functionalsignificances of these interactions are beyond the scope of thisstudy, it is to be noted that some of the candidates obtainedhere overlapped with recently reported candidates for inter-actors of FT and related proteins. Yeast two-hybrid screeningusing a cDNA library from inflorescence meristems, floral mer-istems, and floral buds identified bZIP52, TCP8, TCP15, andTCP22 as candidates to interact with FT in Arabidopsis (Liuet al., 2012). Yeast two-hybrid screening with Md-FT1, a homo-log of FT in apple (Malus 3 domestica) showed that Md-VOZ1,Md-TCP2, and Md-TCP4 (belonging to the same clade as At-VOZ1, At-TCP2 and 24, and At-TCP3 and 4, respectively) interactwith Md-FT1 (Mimida et al., 2011). Given the repeated identifi-cation in separate screenings, these TCP, bZIP, and VOZ proteinsare promising candidates for factors acting with FT protein.


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BRC1 Interacts with FT and TSF Proteins without Mediationby 14-3-3 Proteins

We identified BRC1 as specifically interacting with FT and TSF,but not with TFL1. By contrast, many of the previously reportedFT interactors showed interaction with TFL1 as well. For example,

FD and FD PARALOG interact with both FT/TSF and TFL1 (Abeet al., 2005; Wigge et al., 2005). Tomato (Solanum lycopersicum)SELF-PRUNING–interacting proteins (SPAK, SIP4, and SPGB)and maize FD homolog DELAYED FLOWERING also interactwith FT clade and TFL1 clade proteins (Pnueli et al., 2001;Danilevskaya et al., 2008, 2010). For some FT interactors, such as

Figure 6. brc1 and Genes Acting Downstream of Florigen.

(A) Expression of FD in the AMs monitored by ProFD:GUS. Longitudinal (left) and transverse (right) sections of ProFD:GUS plants grown for 18 d underLD conditions are shown. Arrowheads indicate AMs at rosette axils. Dotted lines show the outline of the tissues.(B) Number of leaves formed on the axillary shoots at the five axil positions of the wild type (Col) and brc1-2, fd-1, and brc1-2 fd-1 mutants. Error barsindicate the SD (n = 15). The difference significant between fd-1 and brc1-2 fd-1 in two-tailed multiple t test with Bonferroni correction (P < 0.05) isindicated with an asterisk. ARL, axillary rosette leaves; CL, cauline leaves.(C) Relative expression levels of FT-downstream genes AP1, FUL, and SOC1 after the shift from SD to LD. Plants were grown for 24 SDs andtransferred to LD. Rosette-axil samples were analyzed (see Methods). Error bars indicate the SE for three biological replicates. Differences significant intwo-tailed t test (P < 0.05) with respect to the wild type (Col) are indicated with asterisks.(D) Number of leaves formed on axillary shoots at the five axil positions of the wild type (Col) and brc1-2, soc1-2, and brc1-2 soc1-2mutants. Error barsindicate the SD (n = 9). The differences significant between soc1-2 and brc1-2 soc1-2 in two-tailed multiple t test with Bonferroni correction (P < 0.05) areindicated with asterisks.Designation of the axil positions and classification of leaves in (B) and (D) are as in Figure 4.

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FT-INTERACTING PROTEIN1 and other factors from thescreening mentioned above, it remains to be examined whetherthey interact with TFL1 clade proteins (Mimida et al., 2011; Liuet al., 2012). FT clade and TFL1 clade proteins seem to sharecommon interactors through the mediation of 14-3-3 proteins.FT and TFL1 share the ability to interact with a common subset of14-3-3 isoforms (e.g., GRF3) (Figure 1A). In rice, 14-3-3 proteinsmediate the formation of the Hd3a-FD1 (corresponding to FT-FDin Arabidopsis) complex (Taoka et al., 2011). In tomato, the SELF-PRUNING–interacting factors mentioned above also interact with14-3-3 proteins (Pnueli et al., 2001). By contrast, BRC1 does notseem to require 14-3-3 proteins for interaction with FT.

It is likely that FT interacts with a variety of proteins using 14-3-3 proteins as a hub. Here, bZIP30 was found to be a putative 14-3-3–dependent FT interactor, whereas other candidates seemedto be independent of 14-3-3 proteins (see Supplemental Figure 1online). These observations indicate that interaction mediated by14-3-3 is not the only way in which FT interacts with TFs, raisingthe possibility that the protein complex containing FT has morecomponents than known at the moment. Another possibility isthat FT uses different sets of partners in different cells or for differentfunctions.

BRC1 Is a Negative Regulator of Floral Transition in AMs

FT acts as a systemic flowering signal (florigen) and moves fromthe leaves to the SAM (Corbesier et al., 2007; Jaeger and Wigge,

2007; Lin et al., 2007; Mathieu et al., 2007; Tamaki et al., 2007;Notaguchi et al., 2008). FT protein expressed under the HSP18.2promoter in a single leaf also moved into the subtending axillarybud (Figure 3). In addition, ft-2 mutants showed delayed floraltransition in AMs (Figure 5). Taken together, these findingsdemonstrate that FT is a key regulator of floral transition in AMsas well as in the SAM. Recent work showed that FT is involved inthe regulation of axillary shoot elongation (Hiraoka et al., 2013),indicating that FT has an additional role in axillary buds after thetransition.On the other hand, BRC1 was previously proposed to be an

integrating repressor of axillary bud development (Aguilar-Martínezet al., 2007). BRC1 suppresses the initiation, differentiation, andelongation of axillary buds (Aguilar-Martínez et al., 2007; Finlayson,2007). In this study, the brc1-2mutant showed a reduction in thenumber of leaves on axillary shoots, as was, in part, reportedpreviously (Finlayson et al., 2010), and earlier onset of AP1expression in the axillary buds (Figure 4). Furthermore, whenectopically expressed in the SAM, BRC1 delayed floral transi-tion of the main shoot, with an increase in the number of leavesformed on the main axis (Figure 7). These observations, takentogether, strongly suggest that BRC1 acts as a negative regu-lator of the floral transition in AMs.Since BRC1 protein interacts with FT and TSF proteins, and

brc1 and ft (tsf) mutants showed opposite phenotypes in theAMs, BRC1 and FT play antagonistic roles likely through theirprotein–protein interaction. Genetic analyses indicated that theft-2 and tsf-1 mutations are epistatic to brc1-2, suggesting thatBRC1 inhibits FT and TSF functions (Figure 5). This is supportedby the SD-to-LD shift transcript profiling experiment, wheregenes downstream of FT were overinduced in the brc1-2mutant(Figure 6).TFL1 also antagonizes FT function, but through competitive

interaction with the common partner, FD (Bradley et al., 1997;Abe et al., 2005; Wigge et al., 2005; Hanano and Goto, 2011).Under LD conditions, when florigen activity is high, the main(primary) inflorescence of the tfl1 mutant makes only a fewflowers and terminates soon after bolting (Shannon and Meeks-Wagner, 1991; Alvarez et al., 1992). In addition, TFL1 is ex-pressed in the AMs as well as in the SAM, and some axillaryshoots are replaced by solitary flowers in tfl1 mutants (Shannonand Meeks-Wagner, 1991; Alvarez et al., 1992; Bradley et al.,1997), indicating TFL1 is also involved in the suppression ofphase transition in AMs. However, BRC1 showed no interactionwith TFL1, and the tfl1-17 brc1-2 double mutant showed anadditive reduction in the leaf number of axillary shoots under SDconditions (see Supplemental Figure 7 online). These resultssuggest that TFL1 and BRC1 act independently.Although the mechanism by which BRC1 modulates the ac-

tivity of FT/TSF remains to be clarified, the observation that FTinteracts with BRC1 through a different region than the 14-3-3protein interaction surface (Figure 2) supports the possibility thatBRC1 binds to the FT/14-3-3/FD complex. In the deducedstructure of the FT/14-3-3/FD complex based on Taoka et al.(2011), the positioning of the region of FT responsible for in-teraction with BRC1 would allow binding of BRC1 to the com-plex (see Supplemental Figure 11 online). If this is the case,BRC1 could inhibit the transcriptional activator function of the

Figure 7. Ectopic Expression of BRC1 Delays Floral Transition in theSAM.

(A) Thirty-day-old plants of wild type (Col) and two independent lines ofProFD:BRC1 (#1 and #2) grown in LD.(B) Flowering time measured by the number of rosette leaves (RL) andcauline leaves (CL). Error bars indicate the SD (n = 15). The differencessignificant in two-tailed Dunnett’s test (P < 0.01) in the total leaf numberswith respect to Col are indicated with asterisks.[See online article for color version of this figure.]


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FT/14-3-3/FD complex. However, the possibility that BRC1traps FT and prevents it from interacting with 14-3-3 proteinscannot be excluded.

Possible Conservation of the Antagonistic Mechanismbetween BRC1 and FT

The BRC1-like and FT-like genes seem to be conserved amongspecies. BRC1 was first identified as a maize Tb1 ortholog inArabidopsis (Aguilar-Martínez et al., 2007; Finlayson, 2007).Similar to the phenotype of Arabidopsis brc1 mutants, maize tb1mutants show increased numbers of elongated axillary shoots(Doebley et al., 1995). Based on comparative genomics andexpression analysis, it was suggested that Zea mays CEN-TRORADIALIS8 (ZCN8) is a maize counterpart of FT (Danilevskayaet al., 2008; Meng et al., 2011; Lazakis et al., 2011). The FDorthologous gene Zm-DLF1 was also identified. Mutation inZm-DLF1 resulted in a late-flowering phenotype (Muszynskiet al., 2006). Zm-ZCN8 interacts with Zm-DLF1, indicating thatthe FT-FD flowering activator complex is also conserved inmaize. TB1/FINE CULM1 from rice is orthologous to Zm-Tb1,and the fc1 mutant shows enhanced tillering (Takeda et al.,2003). Os-Hd3a (an FT ortholog) and Os-FD1 interact with eachother and promote floral transition in rice (Taoka et al., 2011).The tomato BRC1b and SINGLE FLOWER TRUSS genes, BRC1and FT orthologs, respectively, were also characterized(Lifschitz et al., 2006; Martín-Trillo et al., 2011). These studiessuggest that there is functional conservation of the FT-like andBRC1-like genes as florigens and integrators of the axillary buddevelopment, respectively. Our study proposes that these twopathways interact in the AMs of Arabidopsis. It is worth in-vestigating whether BRC1-like genes have conserved functionin regulation of floral transition in AMs and whether FT-like andBRC1-like proteins interact with each other in these species.

The Significance of Regulating AM Floral Transition withFlorigen and BRC1

In wild-type Arabidopsis, not all axillary buds elongate, and inProAP1:GUS plants, buds formed at lower axils remain vege-tative for longer after bolting of the main shoot (Figure 4). Thismay contribute to preventing existent buds from elongating si-multaneously and preparing for future alterations in environ-mental and autonomous conditions.

AM-specific modulation of florigen activity by BRC1 might bemore important under adverse growth conditions, when flow-ering is accelerated. For example, an increased number ofneighboring plants leads to a change in light quality to a lowratio of red to far-red radiation (reviewed in Smith, 1982). Whenplants grow under far-red-rich light, FT, as an integrator gene ofthe flowering pathway, is upregulated and induces prematurefloral transition (Devlin et al., 2003; Endo et al., 2005; Sessaet al., 2005; Wollenberg et al., 2008). On the other hand, ex-pression of BRC1 is also increased by a high planting densityand in phyB mutants (Aguilar-Martínez et al., 2007; Finlaysonet al., 2010). Although overproduced FT protein potentially ac-tivates the developmental program in undesirable parts of theplant, such as AMs, increased levels of BRC1 will suppress

florigen activity specifically in AMs and thus enable the plant toconcentrate its limited resources on the main shoot and pro-mote reproductive success. Regulation of FT expression byenvironmental and internal signals is well studied (reviewed inSimpson and Dean, 2002; Boss et al., 2004; Fornara et al.,2010), and expression of BRC1 is also affected by external andhormonal cues (Aguilar-Martínez et al., 2007; Finlayson, 2007).These signals would converge to create a balance between FTand BRC1 and regulate floral transition in AMs. This additionalmode of regulation in AMs enables modulation of the timing ofaxillary bud differentiation and thus determines plant architec-ture depending on external and internal conditions.

METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana Columbia-0 (Col) was used as the wild type. TheProAP1:GUS reporter (POP40) was kindly provided by M.F. Yanofsky(University of California at San Diego). The T-DNA insertion mutant brc1-2(tbl1-1) was obtained from the ABRC and corresponds to the lineSALK_091920 previously characterized (Aguilar-Martínez et al., 2007;Finlayson, 2007). Plant materials below were previously described: gFT:GUS (Notaguchi et al., 2008), ProFD:GUS (Abe et al., 2005), ft-2 (Imuraet al., 2012), fd-1 (Abe et al., 2005), and soc1-2 (Lee et al., 2000).ProHSP18.2:FT-EGFP was constructed by fusing a promoter fragment(2853 to21, relative to ATG) of theArabidopsis HSP18.2 gene (Takahashiand Komeda, 1989) with the FT-EGFP open reading frame (ORF). ProFD:BRC1 was constructed based on the previously described ProFD:GUSconstruct (Abe et al., 2005) by replacing theGUSORFwith theBRC1ORF.Seeds were stratified by keeping them at 4°C for 2 to 4 d and thentransferred to 22°C LD (16 h light/8 h dark) with white fluorescent light(;80 mmol m22 s21) or SD (8 h light/16 h dark) with white fluorescent light(;100 mmol m22 s21) conditions. For analyses of the axillary shootphenotypes and FT protein movement, plants were grown on vermiculite.Developmental stages of the axillary buds were determined according toAguilar-Martínez et al. (2007). Observations were made on the days whenthe individual plant first showed a visible floral bud (before bolting),reached 1 cm in height (1 cm bolting), and reached 5 cm in height (5 cmbolting). The leaf number of the axillary shoot was counted after internodeelongation. Leaves at the base of the axillary shoot without a visibleelongated stem were counted as axillary rosette leaves and leaves on theelongated shoots as cauline leaves. For expression analyses, plants weregrown on half-strengthMurashige and Skoogmedium supplemented with0.5% Suc and 0.8% agar.

Plasmid Constructs

Plasmid constructs used in this study and primers used for cloning aredescribed in Supplemental Tables 2 and 3 online.

Yeast Two-Hybrid Assay

For the first yeast two-hybrid screening, a TF library containing 1729 TFswas used (Pruneda-Paz et al., 2009). The ORF of the full-length FT proteinwas cloned in pENTR/D-TOPO (Life Technologies) and then transferred topDEST32 (Life Technologies) using LR recombination reaction. The lacZactivity was measured by b-galactosidase assay. For the secondscreening and two-hybrid assays, the FTORF was cloned in pCUY vector(Suzuki et al., 2001). A TF library containing 1503 TFs constructed inpDEST_GAD424 was described previously (Mitsuda et al., 2010). Plas-mids isolated from positive colonies were transformed into Escherichia

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coli (DH5a) for sequencing. The interaction was confirmed after re-transformation of yeast. pCUY and pGAD424 (Clontech/TAKARA) wereused for construction of the other yeast two-hybrid constructs. Theappropriate plasmids were transformed into a yeast strain AH109(Clontech/Takara) using the lithium acetate method and selected onsynthetic dropout medium plates lacking Leu and Trp. After 3 or 4 d ofincubation at 30°C, yeast cells were transferred to liquid synthetic dropoutmedium lacking Leu and Trp and incubated for 24 h. After measuringOD600, 1 mL of each culture was centrifuged and resuspended in TEbuffer, pH 7.4, to a final OD600 of 0.33. Five microliters of suspended yeastwas spotted on synthetic dropout medium lacking Leu, Trp, and His forthe His requirement test (2His). 3-Amino-1,2,4-triazole (0.5 mM) wasadded to the medium for prevention of nonspecific growth, wherespecified (2His+3-AT). As positive controls, yeast cells were also spottedon synthetic dropout medium lacking Leu and Trp (+His). These plateswere incubated at 30°C and scored for growth of the yeast cells. Tovisualize the location of the residues important for protein interaction, anFT ribbon model was produced using Pymol (http://www.pymol.org) fromthe Protein Data Bank file (Ahn et al., 2006; Protein Data Bank ID 1WKP).

BiFC Assay in N. benthamiana Leaf Epidermal Cells

A split EYFP fragment encoding the N-terminal fragment of EYFP (YN; 1 to155 amino acids) was cloned into pBI121 vector and then BRC1ORF wasinserted in frame after the YN to make YN-BRC1. Other constructs (FT-YC, YN, and YC in pBI121) were described previously (Abe et al., 2005).Nicotiana benthamiana plants were grown under continuous light con-ditions at 23°C. Agrobacterium tumefaciens strain pMP90 containing anappropriate construct was grown at 28°C in L-broth to the stationaryphase. Bacterial cells were harvested by centrifugation and resuspendedin 10 mM MgCl2 and 10 mg/mL acetosyringone (Wako) and left for 3 h atroom temperature. The bacterial suspensions were infiltrated into theabaxial air spaces of leaves of 2- to 4-week-old plants using 1-mL sy-ringes. Plants were kept for 36 h after the infiltration in continuous lightconditions at 23°C. Complemented EYFP fluorescence was excited at515 nm by an argon laser and detected in a range from 530 to 630 nmwithconfocal laser scanning microscopy (FV1000; Olympus).

In Vitro Pull-Down Assay

The BRC1 full-length ORFwas cloned into pET41a (Novagen), and FT full-length ORF was cloned into pET24d (Novagen). The plasmids weretransformed into E. coli BL21 (DE3) to express glutathione S-transferase(GST)-BRC1 and T7-FT, respectively. pET41a empty vector was used toexpress GST. For GST pull-down assays, GST-BRC1 or GST bacteriallysatewas incubatedwithGlutathioneSepharose 4Bbeads (GEHealthcare),then 20 mL of the slurry (50% [w/v]) was mixed with 10 mL of T7-FTbacterial lysate and 220 mL of NETN buffer (50 mM Tris-HCl, pH 7.5, 100mM NaCl, 1 mM EDTA, and 0.5% Nonidet P-40) and was gently stirred at4°C for 2 h. The beads were washed thoroughly with the NETN buffer fivetimes and subjected to SDS-PAGE and immunoblot analyses. T7-FTprotein was detected with anti-T7 antibody (Novagen) and visualizedusing ECL plus (GE Healthcare) with Image Quant LAS 4000mini (GEHealthcare).

GUS Staining

Plants were fixed for 15 min in 90% acetone on ice and washed in PBSbuffer twice. Fixed samples were incubated at 37°C for ;16 h withstaining solution (0.5 mg/mL X-Gluc, 50mM sodium phosphate buffer, pH7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and0.2% TritonX-100). For resin sectioning, samples were dehydratedthrough an ethanol series and embedded in Technovit 7100 (HeraeusKulzer) and sectioned at a thickness of 5 mm with a microtome (RM2155;

Leica). The ProAP1:GUS plant was observed with microscopy, and thenumber of stained buds was counted.

Single-Leaf Heat Treatment and Fluorescence Microscopy

ProHSP18.2:FT-EGFP plants grown under LD conditions for 22 d weresubjected to single-leaf heat treatment essentially as described previously(Notaguchi et al., 2008). The leaf blade of the sixth leaf was exposed to37°C for 2 h by placing it in the water-filled space between a heatedcopper plate and a slide glass to ensure good contact. Twenty-four hoursafter heat treatment, plants were embedded in 4% agar gel with PBS andsectioned at a thickness of 80 mm with a vibratome (VIB-1500; Intracel).GFP fluorescence was excited at 488 nm by an argon laser and detectedin a range from 500 to 600 nm with confocal laser scanning microscopy(FV1000; Olympus).

Quantitative RT-PCR Analysis

Plants grown under SD for 24 d were transferred to LD and harvested after13, 16, and 19 d. Rosette-axil samples were obtained by removing themain shoot, leaves, hypocotyl, and root. Total RNA was extracted usingTRIzol reagent (Life Technologies) and treated with DNase I AmplificationGrade (Life Technologies) according to the manufacturer’s instructions.Total RNA (0.5 mg) was reverse transcribed in a 20-mL reaction mixtureusing Transcriptor (Roche Applied Science). The resultant cDNA solutionwas diluted with 30 volumes of water, and 3-mL aliquots were analyzed.Quantitative RT-PCR analyses were performed with the CFX96 system(Bio-Rad Laboratories) using SYBR green I. The primers used are de-scribed in Supplemental Table 4 online. Quantitative RT-PCR resultswere normalized to the geometric means of ASCORBATE PEROXI-DASE3 (APX3; Hazen et al., 2005), REGULATORY PARTICLE TRIPLE-A1A (RPT1a; Michael et al., 2008), and THIOREDOXIN H-TYPE3 (TRX3;Michael et al., 2008) and shown as the average of nine different reactions(biological 3 technical triplicate). Internal control genes used in this studywere selected from nine genes according to Vandesompele et al. (2002).

Accession Numbers

Arabidopsis Genome Initiative locus identifiers for genesmentioned in thisarticle are as follows: At3g18550 (BRC1), At1g65480 (FT ), At1g68800(BRC2), At4g20370 (TSF ), At5g03840 (TFL1), At5g38480 (GRF3),At2g21230 (bZIP30), At4g35900 (FD), At1g69120 (AP1), At5g60910(FUL), At2g45660 (SOC1), At5g61850 (LFY), At4g35000 (APX3), At1g53750(RPT1a), At5g42980 (TRX3), At5g59720 (HSP18.2), At1g06850 (bZIP52),At4g18390 (TCP2), At1g53230 (TCP3), At3g15030 (TCP4), At1g58100(TCP8), At1g69690 (TCP15), At1g72010 (TCP22), and At4g18390 (TCP24).

Supplemental Data

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

Supplemental Figure 1. Yeast Two-Hybrid Assay between FT andPutative FT-Interacting Proteins.

Supplemental Figure 2. Yeast Two-Hybrid Assay between FT andBRC1 Fragments.

Supplemental Figure 3. Yeast Two-Hybrid Assay between Ala-Scanned Mutant FT Proteins and BRC1 or GRF3.

Supplemental Figure 4. Expression Analysis Using FT-GUS FusionProtein.

Supplemental Figure 5.Heat Treatment of Leaf Blades of ProHSP18.2:FT-EGFP Plants.

Supplemental Figure 6. Complementation Test of the brc1-2 MutantUsing a Genomic Fragment of BRC1.


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Supplemental Figure 7. BRC1 and TFL1 Likely Act Independently inthe Regulation of Floral Transition of Axillary Shoots.

Supplemental Figure 8. Relative Expression of LFY after a Shift fromSD to LD.

Supplemental Figure 9. Relative Expression of BRC1 in ProFD:BRC1Plants.

Supplemental Figure 10. Flowering-Time Phenotype of ProFD:BRC1Grown under SD.

Supplemental Figure 11. Amino Acid Residues Essential for theInteraction with BRC1 Shown on a Ribbon Model of FT in the Complexwith 14-3-3 Protein and FD.

Supplemental Table 1. List of Candidate FT-Interacting ProteinsObtained from Yeast Two-Hybrid Screening of the Kay Lab Transcrip-tion Factor Library.

Supplemental Table 2. Constructs.

Supplemental Table 3. Primers Used in Plasmid Construction.

Supplemental Table 4. Primers Used in qRT-PCR.

ACKNOWLEDGMENTS

We thank M.F. Yanofsky (University of California at San Diego) for POP40seeds and the ABRC for brc1-2 seeds. We also thank Ayako Yamaguchi(Kyoto University) for discussion and Yuki Tomita (Kyoto University) andFumie Tobe (National Institute of Advanced Industrial Science andTechnology) for excellent technical assistance. This work was supportedby Grants-in-Aid for Scientific Research on Priority Areas (19060012 and19060016 to T.A.) from the Ministry of Education, Culture, Sports, Science,and Technology of Japan, Grants-in-Aid for the Japan Society for thePromotion of Science Fellows (22-6127 to M.N.), a Grant-in-Aid for YoungScientists (B) (21770064 to N.M.) from the Japan Society for the Promotionof Science, Mitsubishi Foundation (to T.A.), and National Institutes ofHealth Grants GM56006, GM67837, and GM092412 (to S.A.K.).

AUTHOR CONTRIBUTIONS

M.N., Y.D., S.A.K., M.O.-T., M.E., and T.A. designed the research. M.N.,K.K., Y.D., A.H., J.L.P.-P., G.B., and N.M. performed research. M.N., J.L.P.-P,G.B, M.E., and T.A. analyzed data. M.N. and T.A. wrote the article.

Received December 28, 2012; revised March 16, 2013; accepted April 8,2013; published April 23, 2013.

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