TERMINAL FLOWER1 Functions as a MobileTranscriptional Cofactor in the ShootApical Meristem1[OPEN]

Daniela Goretti,a,2 Marina Silvestre,b,2 Silvio Collani,a,2 Tobias Langenecker,c Carla Méndez,b

Francisco Madueño,b,3 and Markus Schmida,d,3,4

aUmeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umea, SwedenbInstituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas,Universidad Politécnica de Valencia (CSIC-UPV), 46022 Valencia, SpaincMax Planck Institute for Developmental Biology, Department of Molecular Biology, 72076 Tuebingen,GermanydBeijing Advanced Innovation Centre for Tree Breeding by Molecular Design, Beijing Forestry University,Beijing 100083, People’s Republic of China

ORCID IDs: 0000-0003-3996-0204 (D.G.); 0000-0002-2485-1761 (M.Si.); 0000-0002-9603-0882 (S.C.); 0000-0002-9905-7183 (T.L.);0000-0003-4257-3627 (C.M.); 0000-0001-7598-3003 (F.M.); 0000-0002-0068-2967 (M.Sc.).

The floral transition is a critical step in the life cycle of flowering plants, and several mechanisms control this finely orchestratedprocess. TERMINAL FLOWER1 (TFL1) is a floral repressor and close relative of the florigen, FLOWERING LOCUS T (FT). Duringthe floral transition, TFL1 expression is up-regulated in the inflorescence apex to maintain the indeterminate growth of the shootapical meristem (SAM). Both TFL1 and FT are mobile proteins, but they move in different ways. FT moves from the leaves to theSAM, while TFL1 appears to move within the SAM. The importance of TFL1 movement for its function in the regulation offlowering time and shoot indeterminacy and its molecular function are still largely unclear. Our results using Arabidopsis(Arabidopsis thaliana) indicate that TFL1 moves from its place of expression in the center of the SAM to the meristem layer L1and that the movement in the SAM is required for the regulation of the floral transition. Chromatin immunoprecipitationsequencing and RNA sequencing demonstrated that TFL1 functions as a cotranscription factor that associates with andregulates the expression of hundreds of genes. These newly identified direct TFL1 targets provide the possibility to discovernew roles for TFL1 in the regulation of floral transition and inflorescence development.

Plants have to tightly control the switch from vege-tative to reproductive growth to ensure reproductivesuccess. Several factors, such as sugars, hormones,temperature, quality of the light, and the length of theday (photoperiod), can influence flowering time. InArabidopsis (Arabidopsis thaliana), photoperiod ismainly perceived in the leaves, where the exposure toinductive long-day (LD) conditions promotes the ex-pression of the main florigen, FLOWERING LOCUS T(FT), in the phloem companion cells (An et al., 2004;Corbesier et al., 2007). FT protein is then loaded in thesieve elements and transported to the shoot apicalmeristem (SAM), where it regulates the switch to re-productive development (Corbesier et al., 2007; JaegerandWigge, 2007; Mathieu et al., 2007). At the SAM, FThas been proposed to act as a transcription cofactor ina floral activation complex (FAC), that includes 14-3-3proteins and the basic leucine zipper (bZIP) tran-scription factor FD (Pnueli et al., 2001; Abe et al., 2005;Wigge et al., 2005; Hanano and Goto, 2011; Taokaet al., 2011).FT is a member of the phosphatidylethanolamine-

binding protein (PEBP) family, which in Arabidopsisis composed of six members that fall into three major

clades: MOTHER OF FT (MFT)-like, FT-like, and TER-MINAL FLOWER1 (TFL1)-like (Karlgren et al., 2011).MFT, which forms the oldest branch of the PEBP family,is expressed in gametophytes and acts during seedgermination (Xi et al., 2010; Wang et al., 2015). TWINSISTER OF FT is the gene most closely related to FT,and the two proteins have been shown to act in partredundantly to promote flowering in response to LD(Yamaguchi et al., 2005; Fornara et al., 2010;Wang et al.,2015). The main representative of the third PEBP pro-tein subgroup is TFL1, and it inhibits flowering as dothe two other members of the clade, ARABIDOPSISTHALIANA CENTRORADIALIS homolog (ATC) andBROTHER OF FT (BFT; Mimida et al., 2001; Yoo et al.,2010). Both ATC and BFT have been shown to interactwith FD and to repress APETALA1 (AP1) expression(Huang et al., 2012; Ryu et al., 2014). Furthermore,similar to FT, ATC can move over a long distance fromleaves to the SAM (Huang et al., 2012).TFL1 is the best characterized member of its clade

and a key regulator of inflorescence development andflowering time. The primary sequences of TFL1 and FTare highly similar, but the two proteins have beensuggested to have opposite molecular functions, acting

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as a repressor and activator of flowering, respectively(Ahn et al., 2006; Hanano and Goto, 2011). Mutants atthe TFL1 locus flower earlier compared with the wildtype, both in terms of days to flowering and numberof leaves (Shannon and Meeks-Wagner, 1991). Its roleas a flowering time regulator is further confirmed bythe late-flowering phenotype of 35Spro::TFL1 plants(Ratcliffe et al., 1998).

TFL1 is also involved in the maintenance of the SAM,allowing indeterminate growth of the inflorescence, sothat in tfl1 mutants the inflorescence SAM is convertedinto a terminal floral meristem (Shannon and Meeks-Wagner, 1991). During the vegetative phase, when newrosette leaves are produced, TFL1 is only expressed atlow levels in the center of the SAM (Bradley et al., 1997;Ratcliffe et al., 1999; Conti and Bradley, 2007). At thetime of the switch from the vegetative to the repro-ductive phase, the SAM is converted into an inflores-cence meristem that starts to produce cauline leaves. Atthis stage, TFL1 expression is strongly up-regulated,first in axillary meristems and soon after in the SAM(Bradley et al., 1997; Conti and Bradley, 2007). Laterduring the development of the inflorescence, TFL1 andfloral meristem identity genes are expressed in distinctdomains in the main shoot apex. TFL1 mRNA isdetected predominantly in the inner part of the centralzone of the SAM, while LEAFY (LFY), AP1, and CAU-LIFLOWER (CAL) mRNAs accumulate at the peripheryof the apex where new lateral floral meristems originate(Ratcliffe et al., 1998, 1999; Baumann et al., 2015). Thisspatial separation between TFL1 and the floral meri-stem identity genes is essential to the maintenanceof the inflorescence. For example, in tfl1 mutants, theformation of the terminal floral meristem is enhanced

by the ectopic expression of LFY in the meristem(Baumann et al., 2015). On the other hand, strong lfyand ap1 mutants have floral meristems converted intoinflorescence meristems (Schultz and Haughn, 1991;Weigel et al., 1992; Bowman et al., 1993). It is worthnoting that TFL1 and LFY (or AP1)mutually inhibit eachother (Ratcliffe et al., 1999). As a consequence, TFL1 andthe floral meristem identity genes are expressed in spe-cific domains of the SAM, facilitating flower develop-ment while at the same time ensuring indeterminategrowth of the plant during the reproductive phase.

The molecular function of TFL1 is still elusive. Theprotein has been reported to be mobile within the SAM,suggesting that it might regulate meristem develop-ment in a non-cell-autonomous manner (Conti andBradley, 2007). Specifically, it has been shown that theTFL1 protein can move from the center toward theperiphery of the SAM, where it prevents the expressionof floral meristem genes (Conti and Bradley, 2007).However, whether this protein movement is critical forTFL1 function has not been addressed. At the subcel-lular level, TFL1 has been detected in the nucleus andcytoplasm (Conti and Bradley, 2007; Hanano and Goto,2011). In the cytoplasm, TFL1 has been suggested toparticipate in endomembrane trafficking of proteins tostorage vesicles (Sohn et al., 2007). In the nucleus, TFL1has been shown to interact with the bZIP transcriptionfactor FD, giving rise to the hypothesis that TFL1 andFT might compete for the formation of the FAC (Abeet al., 2005; Wigge et al., 2005; Hanano and Goto, 2011;Taoka et al., 2011; Kaneko-Suzuki et al., 2018). Ac-cordingly, a TFL1-containing FAC that could either betranscriptionally inactive or actively repress the for-mation of flowers would be formed in the center of theSAM, whereas the formation of an FT-containing FACwould actively promote floral initiation at the periph-ery of the inflorescence meristem. In agreement withthis hypothesis, it has been shown that TFL1 can beconverted into a transcriptional activator when fusedwith the strong VP16 activation domain (Hanano andGoto, 2011). However, direct evidence that TFL1 func-tions as a transcription cofactor and the genome-wideset of its targets are still missing.

Here, we expressed TFL1-Venus fluorescent proteinfusion proteins under the control of TFL1 regulatoryelements (gTFL1), already described by Serrano-Mislataet al. (2016), to investigate the role of protein movementin the SAM during the floral transition. We observedthat the movement of TFL1 from the central meristemtoward the periphery of the SAM is necessary for TFL1function and contributes to the regulation of the switchfrom vegetative to reproductive growth. Furthermore,confocal imaging demonstrated that TFL1movement inthe SAM can occur in either direction, inward or out-ward. To assess whether TFL1 associates with DNAand can function in a transcriptional complex, we per-formed chromatin immunoprecipitation sequencing(ChIP-seq) using a gTFL1-1xVenus reporter line to iden-tify loci bound by TFL1 at the genome-wide scale. Incombination with results obtained by RNA sequencing

1This work was supported by the Spanish Ministerio de CienciaInnovación y Universidades (grant no. BIO2015-64307-R to F.M.) andFEDER (grant no. PGC2018-099232-B-I00 to F.M.) and by the KnutandAliceWallenberg Foundation (grant no. KAW 2016.0025 to M.Sc.).Additional support was provided by the Umeå Plant Science Centre(UPSC) through VINNOVA. M.Si. was supported by an Formaciónde Personal Universitario (FPU) contract from the Spanish Minis-terio de Educación, Cultura y Deporte, and C.M. was supportedby a Santiago Grisolía fellowship from the Generalitat Valenciana.

2These authors contributed equally to the article.3Senior authors.4Author for contact: markus.schmid@umu.se.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Markus Schmid (markus.schmid@umu.se).

D.G., S.C., F.M., andM.Sc. developed and designed the experimentsand interpreted the data; M.Si. characterized the GR lines, performedthe RNA-seq experiment, and validated data by ChIP-PCR and RT-qPCR; D.G. established some reporter lines, analyzed flowering time,and performed confocal experiments and ChIP-seq; S.C. carried out theChIP-seq experiments and analyzed RNA-seq and ChIP-seq data; T.L.and C.M. established some transgenic lines; D.G. and M.Sc. wrote thearticle with input from all the authors.

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(RNA-seq) using a 35Spro::TFL1-GR line, in which TFL1has been fused to the glucocorticoid receptor (GR) do-main, we identified 115 direct targets of TFL1, which,among others, includes the major floral meristem iden-tity gene LFY. In addition, we found that G-box motifswere highly enriched among the TFL1 target sites andthat many of the bound regions were shared with re-gions previously reported to be bound by FD (Collaniet al., 2019). Nevertheless, many loci specifically boundand regulated byTFL1were not bound by FD, indicatingthat TFL1 participates in different transcriptional com-plexes at the apex that might regulate diverse aspects ofthe floral transition and inflorescence development.

RESULTS

Movement in the SAM Is Required for TFL1 Function

TFL1 in Arabidopsis has been shown to move fromits place of expression in the center toward the pe-riphery of the SAM (Conti and Bradley, 2007). To test ifthe protein movement within the SAM is actually re-quired for its function, we first established a genomicTFL1 rescue construct (hereafter named gTFL1), con-sisting of the 592-bp upstream promoter sequence, allexons and introns, as well as 3,396 bp of the down-stream sequence (Fig. 1A). The gTFL1 fragment, whichis very similar to one used by Serrano-Mislata et al.(2016), contains all regulatory elements essential forcorrect temporal and spatial expression of TFL1.However, gTFL1 lacks the cis-regulatory region V (lo-cated at13.3–3.6 kb from the stop codon) that has beenreported to harbor an enhancer element that boosts TFL1expression (Serrano-Mislata et al., 2016). The gTFL1 con-struct restored flowering time to almost that of the wildtype (Supplemental Fig. S1), indicating that the clonedgTFL1 sequence contains the essential cis-regulatory ele-ments for the correct expression of the transgene.Reasoning that the ability of TFL1 to move from cell

to cell would be influenced by the size of the protein, wenext generated fusion proteins in which gTFL1 wastagged in frame with either a single or three copies of

Figure 1. C-terminal Venus-tagged TFL1 protein is functional. A,Schematic representation of the gTFL1 sequence used in this study. Darkblue boxes indicate 59 and 39 untranslated regions, light blue boxes areexons, and junctions are introns. Dashed boxes indicate regulatoryregions as described by Serrano-Mislata et al. (2016). B, Flowering time

distribution of T1 plants carrying the gTFL1 transgene fused in frame toeither 1xVenus or 3xVenus compared with Col-0 and the tfl1-1mutant.C, Flowering time distribution of homozygous T4 plants constitutivelyexpressing the TFL1 coding sequence (CDS) under the control of the 35Spromoter. Data for four lines originating from two independent T1plants are shown. D, Flowering time distribution of homozygous T4plants constitutively expressing the TFL1 CDS fused to a single or tripleC-terminal Venus tag under the control of the 35S promoter. Data forthree different lines originating from independent T1 plants are shown.Plants were grown under LD conditions. Numbers (n) on the x axisindicate the number of plants. Boxes span the first (Q1) to third (Q3)quartiles, and thick black lines indicate the median value (Q2) for eachgroup; the bottom whisker corresponds to Q1 2 1.5 3 IQR (inter-quartile range), and the top whisker corresponds to Q3 1 1.5 3 IQR.Groups with different letters are significantly different from each otherafter Tukey-Kramer correction (P , 0.05).

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the fluorescent proteins Venus (gTFL1-1xVenus orgTFL1-3xVenus) and GFP (gTFL1-1xGFP or gTFL1-3xGFP) at its C terminus. The constructs were trans-formed into the tfl1-1 mutant, and flowering time of T1plants was scored. Only gTFL1-1xVenus plants (T1)showed significant rescue of the early-flowering phe-notype of the tfl1-1mutant, whereas most of the gTFL1-3xVenus plants were still early flowering (Fig. 1B).gTFL1-1xGFP suppressed the formation of a terminalflower and restored indeterminate growth of the apicalinflorescence in more than 50% of the T1 lines (Table 1;Supplemental Fig. S2). In contrast, indeterminate growthwas observed in less than 10% of the T1 gTFL1-3xGFPlines (Table 1; Supplemental Fig. S2). Together, thesefindings suggest that either TFL1 movement was im-paired by the large 3xVenus and 3xGFP tags, therebypreventing it from rescuing the tfl1-1 mutant, or alter-natively that the 3x-tagged proteins had lost their activ-ity. To distinguish between these two possibilities, weexpressed the unmodified and the tagged versions ofTFL1 from the constitutive 35S promoter in Columbia-0(Col-0). As expected, independent stably transformed T4homozygous lines carrying 35Spro::TFL1 flowered verylate when compared with Col-0 (Fig. 1C; SupplementalFig. S3, A–C), confirming previous results (Ratcliffe et al.,1998;Hanano andGoto, 2011). Importantly, latefloweringwas also observed in the 35Spro::TFL1-1xVenus and35Spro::TFL1-3xVenus lines (Fig. 1D; Supplemental Fig. S3,D–F), indicating that the presence of C-terminal 1xVenusor 3xVenus tags did not affect the ability of TFL1 to repressflowering and that the TFL1 protein retained activityirrespective of the size of the tag. The most parsimoniousexplanation for these findings is that the 3xVenus (and3xGFP) tag impairs TFL1 protein movement in the SAMand that this movement is important for normal TFL1function in the control of the floral transition.

TFL1-1xVenus Is Detected in the EpidermalMeristem Layer

To visualize the localization of TFL1 in vivo moreprecisely, we established homozygous single and triple

Venus-tagged T4 lines. Two selected gTFL1-1xVenuslines largely complemented the early-flowering phe-notype of the tfl1-1mutant, both in terms of total leavesand days to flowering, and displayed indeterminategrowth in 100% of the plants (Fig. 2A; SupplementalFig. S4). Next, we investigated the distribution of TFL1-1xVenus protein in the SAM by confocal microscopy.For this, plants were grown under inductive LD con-ditions and images were taken before and after thefloral transition. In 13-d-old plants, before the transitionto flowering, TFL1-1xVenus was detectable only in theaxillary meristems (Supplemental Fig. S5A). However,2 d later (15 d after sowing [DAS]), when floral transi-tion was occurring, TFL1-1xVenus appeared also in theSAM, where it was detected in the central zone and inmore peripheral regions, including the epidermal layer(L1; Fig. 2B; Supplemental Figs. S5, A and B, and S6A).Transverse sections of apices collected 15 DAS showedenrichment of TFL1-1xVenus especially in the adaxialregions of the axillary meristems (Supplemental Fig.S5B). In contrast to gTFL1-1xVenus, gTFL1-3xVenusfailed to rescue the tfl1-1 mutant phenotype, and theplants transitioned to flowering relatively early at ap-proximately 13 DAS (Fig. 2A; Supplemental Fig. S4). Inaddition, growth of all established T4 gTFL1-3xVenuslineswas determinate. At the time of floral transition, 13DAS, TFL1-3xVenus was detectable only in the innerzone but absent from the peripheral region of the mer-istem (Fig. 2B; Supplemental Fig. S6A). The distribution ofTFL1-3xVenus resembles that of theGUS reporter line 0.6-G-3.3 described by Serrano-Mislata et al. (2016), which isinteresting as the large GUS protein is thought to actlargely cell autonomously (Kim et al., 2003).

To verify that the observed differences in the distri-bution of 1xVenus- and 3xVenus-tagged TFL1 proteinwere not due to differences in gene expression, weperformed reverse transcription quantitative PCR (RT-qPCR) on the transgenes in dissected apices collectedfrom gTFL1-1xVenus and gTFL1-3xVenus plants. Theresults clearly showed that gTFL1-1xVenus and gTFL1-3xVenuswere expressed at comparable levels in all lines(Supplemental Fig. S6B). Taken together, the floweringtime and protein localization results demonstrated that

Table 1. Inflorescence phenotypes of Col-0, tfl1-1, and transgenic T1 lines

The numbers of individuals with determinate and indeterminate inflorescence were determined in LD-grown Col-0, tfl1-1, and independent T1 lines in the tfl1-1 background transformed with various TFL1constructs. See also Supplemental Figure S2.

GenotypeInflorescence Phenotype

Determinate Indeterminate

Col-0 0 19tfl1-1 20 0gTFL1-1xGFP/tfl1-1 17 21gTFL1-3xGFP/tfl1-1 22 2gTFL1-3xGFP-NLS/tfl1-1 46 0ML1pro::TFL1/tfl1-1 50 1ML1pro::TFL1-NLS/tfl1-1 49 2ML1pro::TFL1-1xGFP/tfl1-1 49 2ML1pro::TFL1-1xGFP-NLS/tfl1-1 52 5

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the presence of TFL1 only in the central zone of themeristem is apparently not sufficient to rescue theearly-flowering phenotype of the tfl1-1 mutant.

Furthermore, these findings provided indirect evidencefor the importance of the movement of TFL1 toward theproximal cell layers of the SAM.

Expression of TFL1 in the Epidermis Partially Rescues thetfl1-1 Early-Flowering Phenotype

To test whether the presence of TFL1 in the epidermallayer of the SAM is important for its function, weexpressed TFL1 under the control of the MERISTEMLAYER1 (ML1) promoter. T1 plants carryingML1pro::TFL1showed a partial rescue of the early-flowering phenotype ofthe tfl1-1mutant (Fig. 3A; Supplemental Fig. S7).However,the terminal flower phenotype was suppressed only inone out of 51 T1 plants examined (Table 1), indicating thatTFL1 expression in the L1 is insufficient to fully comple-ment the mutant.To visualize the protein and its localization in the

SAM, we tagged TFL1 at its C terminus with GFP andexpressed the fusion protein under the control of theML1 promoter. Similar to the lines expressing the un-tagged TFL1 protein, the ML1pro::TFL1-1xGFP plantsdisplayed a partial rescue of the early flowering of tfl1-1, but most individuals were still determinate (Fig. 3A;Table 1; Supplemental Fig. S7). Interestingly, TFL1-1xGFP could be detected several cell layers inwardfrom the L1, reaching the L2 and L3 layers (Fig. 3B).This indicates that movement of TFL1 is most likely notdirectional and the protein moves away from the L1cells in which the transgene is expressed. However, thefinding that TFL1-1xGFP moves toward the center ofthe SAMmakes it impossible to conclude if localizationin the L1 is required for TFL1 function.To prevent the movement of TFL1 from the L1, we

next added a strong nuclear localization signal (NLS) toTFL1 and expressed the resulting TFL1-NLS constructfrom the ML1 promoter. The nucleus-localized TFL1-NLS protein rescued the early-flowering phenotype ofthe tfl1-1mutant to a similar extent to the untagged andGFP-tagged TFL1 proteins (Fig. 3A; Supplemental Fig.S7) but had little effect on the formation of the terminalflower (Table 1). To verify that the NLS was functional,we generated an ML1pro::TFL1-1xGFP-NLS construct.As expected, the GFP fluorescent signal was largelyconfined to the L1 layer, localized to the nucleus, andrescued the early-flowering phenotype of the tfl1-1 mutant similarly to the untagged TFL1 protein(Fig. 3), suggesting that TFL1 works in the nucleus andregulates flowering time at least in part in the L1.

Genome-Wide Targets of TFL1

TFL1 has been shown to interact with transcriptionfactors and, when fused to the strong transcription acti-vation domain VP16, is capable of inducing the expres-sion of floral genes (Hanano and Goto, 2011), suggestingthat TFL1might be involved in transcriptional regulation.However, as there is no indication that it can directly bindDNA, TFL1 likely functions as a transcriptional cofactor.To identify potential target genes of TFL1 and to gain

Figure 2. TFL1 movement in the SAM is required for the regulation offlowering. A, Flowering time distribution of Col-0, tfl1-1, and homo-zygous T4 plants expressing fusion proteins under the control of thenative gTFL1 regulatory elements. Data for two different lines carryingeither 1xVenus- or 3xVenus-tagged proteins are shown. Plants weregrown under LD conditions. Numbers (n) on the x axis indicate thenumber of plants. Boxes span the first (Q1) to third (Q3) quartiles, andthick black lines indicate the median value (Q2) for each group; thebottom whisker corresponds to Q1 2 1.5 3 IQR (interquartile range),and the top whisker corresponds to Q3 1 1.5 3 IQR. Groups withdifferent letters are significantly different from each other after Tukey-Kramer correction (P , 0.05). B, Confocal microscopy images of linesexpressing the mobile (1xVenus tagged; top) or the immobile (3xVenustagged; bottom) form of TFL1. Images of longitudinal sections of themain shoot apex were taken at the time of floral transition, 15 and 13 dafter sowing, respectively. Bars 5 20 mm. BF, Bright field.

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insight into the transcriptional networks in which TFL1might be involved, we adopted a ChIP-seq approach.

To identify regions in the genome bound by TFL1-containing protein complexes, we sampled apices (in-cluding the SAM, young organ primordia, and the

closest axillary meristems) of 15-d-old gTFL1-1xVenus#2.9.1 plants, as we had previously established that thisline shows rescue of the early-flowering phenotype ofthe tfl1-1 mutant and the TFL1-1xVenus protein alsowas detectable in the SAM at this time point (Fig. 2).

ChIP-seq identified 1,891, 1,752, and 1,140 regionsenriched for TFL1 in the three biological replicates, ofwhich 971 peaks were shared between all the replicates(Supplemental Data S1). Subsequent analyses wereperformed on this set of 971 high-confidence peaks thatcorrespond to 952 unique genes, since some loci con-tained more than one region enriched for TFL1. Anno-tation of the peak regions showed that the majority(71.8%) mapped in promoter regions (Fig. 4, A and B),lending support to the idea that TFL1 acts as a cofactorin transcriptional complexes. De novo motif analysisusing Multiple EM for Motif Elicitation (MEME)-ChIP(Machanick andBailey, 2011) revealed aG-box (CACGTG)as the most highly overrepresented binding sites in thesequences underlying the peaks (Fig. 4C; SupplementalFig. S8). GeneOntology (GO) analysis showed that TFL1is enriched on genes involved in cellular and metabolicprocesses, response to stimuli, and single-organismprocesses (Supplemental Fig. S9A).

Among the genes that showed strong enrichment ofTFL1were several important regulators of the circadianclock, flowering time, and floral development. For ex-ample, we detected enrichment of TFL1 on the pro-moters of CIRCADIAN CLOCK ASSOCIATED1 (CCA1)and LATE ELONGATED HYPOCOTYL (LHY), whichare components of the core circadian oscillator in Ara-bidopsis (Fig. 4D; Alabadí et al., 2001; Mizoguchi et al.,2002).We also observed enrichment of TFL1 on the corepromoter of CONSTANS (CO; Fig. 4D), a key player inthe photoperiod pathway that is circadian regulatedand induces flowering in response to inductive day-length (Suárez-López et al., 2001). Interestingly, TFL1was also recruited to the promoter of NUCLEARFACTOR-YB2 (NF-YB2; Fig. 4D), which has beenshown to interact with CO and induce flowering bypromoting FT expression in the leaf (Kumimoto et al.,2008, 2010). TFL1 was also strongly enriched on thesecond exon of the floral meristem identity gene LFYand the promoters of TREHALOSE-6-PHOSPHATEPHOSPHATASE H (TPPH) and PHYTOCHROME-INTERACTING FACTOR5 (PIF5; Fig. 4D), involved insugar metabolism and in metabolic signaling to thecircadian clock, respectively (Vandesteene et al., 2012;Shor et al., 2017). Taken together, these results dem-onstrate that TFL1-containing protein complexes binddirectly to and might regulate the expression of im-portant flowering time and plant development genes.

Genes Regulated by TFL1 in the Main Apex at theFloral Transition

To determine which of the bound genes were tran-scriptionally regulated by TFL1, we transformed thetfl1-1 mutant with a construct carrying TFL1 fused to

Figure 3. Expression of TFL1 in the epidermis partially rescues theearly-flowering phenotype of the tfl1-1 mutant. A, Flowering time dis-tribution of T1 plants expressing the TFL1 CDS under the control of theepidermis-specific ML1 promoter, without any tag, tagged with eitherGFP or an NLS, or both. Data from Col-0 and the tfl1-1 mutant plantsserve as controls. Plants were grown under LD conditions. Numbers (n)on the x axis indicate the number of independent T1 plants. Boxes spanthe first (Q1) to third (Q3) quartiles, and thick black lines indicate themedian value (Q2) for each group; the bottom whisker corresponds toQ12 1.53 IQR (interquartile range), and the top whisker correspondsto Q3 1 1.5 3 IQR. Groups with different letters are significantly dif-ferent from each other after Tukey-Kramer correction (P , 0.05). B,Confocal microscopy images taken during the floral transition of T2 linesexpressing TFL1-1xGFP under the control of theML1 promoter, without (top)orwith (bottom) aC-terminalNLS. Imagesof longitudinal sectionsof themainshoot apex were taken 15 d after sowing. Bars5 20 mm. BF, Bright field.

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the GR domain under the control of the 35S constitutivepromoter (35Spro::TFL1-GR). In the absence of a ligand,GR fusion proteins are efficiently retained in the cyto-plasm and only imported into the nucleus after applica-tion of a steroid hormone ligand such as dexamethasone(DEX). Constitutively expressed GR fusion proteins have

been widely used to identify targets of transcription fac-tors in plants (Gómez-Mena et al., 2005; Kaufmann et al.,2010; Reymond et al., 2012). As our ChIP-seq results in-dicated that TFL1was associatedwithDNA,we reasonedthat a TFL1-GR fusion protein would be retained in thecytoplasm, thereby preventing it from executing its

Figure 4. Identification of TFL1 targets at the SAM during the floral transition. A, Annotation of the locations of high-confidencepeaks found in three biological replicates in the gTFL1-1xVenus line by ChIP-seq according to genome features. UTR, Un-translated region. B, Distribution of the distance of the center of the 971 peaks shared between the three biological ChIP-seqreplicates to the nearest transcription start site (TSS). C, Motif of the most highly enriched TFL1 complex-binding site identified byde novo prediction. D, Examples of peaks from the gTFL1-1xVenus line for selected loci. Pileup values shown on the y axis werecalculated with MACS2. A red box under each peak indicates the region of significant enrichment as determined by MACS2.Black arrows indicate the positions of the translation start site (ATG) and the direction of gene transcription.

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nuclear function. Expression of TFL1-GR from the 35Spromoter was thus predicted to delay flowering in aDEX-inducible manner, which could be used in a tran-scriptomic approach to identify TFL1-regulated genes.

To test whether the TFL1-GR fusion protein wasfunctional, plants were grown under noninductiveshort-day (SD) conditions for 3 weeks and then shiftedto LD to induce flowering. DEX or mock solution at30 mM was directly applied to the shoot apex every 72 hfrom the time plants had been shifted to LD until theyhad bolted. Col-0 plants started to bolt 15 d after theshift to LD, independently of whether they had beentreated with DEX or mock solution (Supplemental Fig.S10A). In contrast, bolting was substantially delayed intwo 35Spro::TFL1-GR tfl1-1 (T4) lines (#4.7 and #7.9)after application of DEX, while mock-treated plantsflowered very early, as expected for a tfl1-1 mutant(Supplemental Fig. S10A). Line #7.9, in particular, dis-played a homogenous delay in flowering time, with50% of the plants flowering 22 d after the shift to LDversus 15 d of Col-0 and 7 d of the same #7.9 line mocktreated (Supplemental Fig. S10, A and B). In this line,continuous application of DEX prevented flowering inapproximately 20% of the individuals tested at the endof the experiment, 55 d after the shift to LD and thebeginning of the DEX treatments. These initial analysesindicated that the TFL1-GR system was functional andthat plants responded to DEX treatment as expected. Inaddition, these analyses provide further indirect evi-dence that TFL1 needs to enter the nucleus in order toregulate flowering time.

To determine the optimal time of DEX treatments forsubsequent transcriptome analyses of TFL1 induction,we applied 30 mM DEX 4, 5, or 6 d after shifting to LD atZeitgeber time (ZT)12.While application of DEX 4 and5 d after the shift essentially blocked the induction ofLFY and AP1 expression completely, application after6 d allowed for an initial expression of these twogenes thatwas subsequently repressed by TFL1-GR (SupplementalFig. S10, C andD). Further experiments demonstrated thatmock- and DEX-treated plants showed strong differencesin AP1 and LFY expression 24 h after the treatment(Supplemental Fig. S10, E and F). Based on these initialresults, we performed a transcriptome-wide RNA-seqexpression analysis using RNA isolated from apices of35Spro::TFL1-GR (#7.9) plants 24 h after they had beentreated with either 30 mM DEX or mock solution atZT12 6 d after the shift to LD, following the initialcultivation in SD for 21 d.

In total, this RNA-seq experiment identified 1,379genes that were differentially expressed betweenmock- and DEX-treated samples (false discoveryrate, 0.001, fold change. 1.5; Fig. 5A; SupplementalData S2). Of these, 462 were down-regulated and 917were up-regulated in response to DEX treatment(Supplemental Data S2). It is worth noting that amongthe genes significantly down-regulated by TFL1-GR,we could detect the floral identity genes LFY,CAL, andFRUITFULL. Overall, GO analyses showed that cate-gories related to cellular and metabolic processes were

overrepresented among the genes differentially expressedby induction of TFL1-GR (Supplemental Fig. S9B).

Intersection of RNA-Seq and ChIP-Seq Data Sets

To identify which of the 1,379 differentially expressedgenes were also bound by TFL1-containing proteincomplexes, we intersected data from the RNA-seq andChIP-seq experiments. Of the 952 genes that wereenriched for TFL1-1xVenus, 115 (12%) were also differ-entially expressed (Fig. 5B; Supplemental Data S3). Ofthese, 36 genes were down-regulated and 79 were up-regulated at the SAM upon induction of TFL1-GR byDEX (Fig. 5B; Supplemental Data S3).

These 115 genes comprise the core set of high-confidence TFL1 targets. However, as the RNA-seqand ChIP-seq experiments were performed using dif-ferent experimental systems and plant lines, we con-firmed our results in an independent experiment. Tothis end, we performed ChIP on the DEX-inducible35Spro::TFL1-GR line using an anti-GR antibody andtested enrichment on three target genes by qPCR. Thesegenes were selected based on their known role in reg-ulating the floral transition (LFY) or for their involve-ment in light (PIF5) or trehalose (TPPH) signaling.Consistent with our previous ChIP-seq results, ChIP-PCR detected enrichment of TFL1-GR on the genestested in DEX-treated compared with mock-treatedplants (Fig. 6A; Supplemental Fig. S11). We also exam-ined the expression of these genes in the 35Spro::TFL1-GR line 2 and 26 h after mock and DEX treatments(Fig. 6C). Interestingly, already 2 h after treatment, wecould detect significant changes in the expression level of

Figure 5. Identification of TFL1-regulated genes by RNA-seq. A, Scatter-plot of significantly differentially expressed genes (red) in 35Spro::TFL1-GRplants in response to DEX treatment. B, Three-set Venn diagram depictingthe overlap of TFL1 targets identified by ChIP-seq (TFL1 bound) anddifferentially expressed genes identified by RNA-seq (down-regulatedand up-regulated).

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TPPH and PIF5. Importantly, and in agreement with ourRNA-seq analyses, the expression of LFY and TPPHwasdown-regulated whereas that of PIF5 was significantlyup-regulated in DEX-treated samples in our RT-qPCRanalysis (Fig. 6, B and C).

TFL1 Is an Important Interactor of FD at the SAM

It has previously been shown that TFL1 physicallyinteracts with the bZIP transcription factor FD, an im-portant regulator of flowering time at the SAM (Wiggeet al., 2005; Hanano and Goto, 2011). More recently, itwas demonstrated in vitro that this protein complexcan bind G-box sequences (Collani et al., 2019). Thesame study has shown by ChIP-seq that genome-wideG-boxes are the most strongly enriched binding site ofFD at the SAM. As a G-box is also the most highlyoverrepresented binding site in our TFL1 ChIP-seqanalyses, we compared the list of TFL1 and FD tar-gets. In total, we obtained 358 peak regions that wereshared between the 971 targets of TFL1 identified in thisstudy and the 595 high-confidence FD targets (Collaniet al., 2019; Supplemental Data S4; Supplemental Fig.S12A). The large proportion of shared peaks betweengTFL1-1xVenus and pFD::GFP-FD strongly suggeststhat FD is an important interactor of TFL1 at the SAM.Among the genes bound by both TFL1 and FD areimportant regulators involved in LD photoperiodismand flowering (CCA1, LHY, and NF-YB2), in develop-ment of the reproductive shoot system (HOMEODO-MAIN GLABROUS5, MONOPTEROS, SEUSS-LIKE1,and CYCLING DOF FACTOR5), and in the jasmonatepathway (JASMONATE-ZIM-DOMAINPROTEIN3 [JAZ3],

JAZ6, MYC2, ETHYLENE-RESPONSIVE ELEMENT-BINDINGFACTOR4,UBIQUITIN-SPECIFICPROTEASE13,and JASMONATE RESISTANT1).However, we also found 613 unique peaks for TFL1

and 237 unique peaks for FD (Supplemental Data S5and S6). Since we detected a considerable number ofpeaks specific for TFL1, we can assume that TFL1 bindsDNA also through interactions with other DNA-bindingproteins (Supplemental Data S5; Supplemental Fig.S12B). For example, TFL1, but not FD, binds to genesthat are involved in Fru-1,6-bisphosphate metabolism(CHLOROPLASTIC FRUCTOSE-1,6-BISPHOSPHATASE1and CYTOSOLIC FRUCTOSE-1,6-BISPHOSPHATASE)and in the trehalose pathway (TPPA, TPPE, TPPJ,TPPH, and TREHALOSE-6-PHOSPHATE SYNTHASE6[TPS6]). Which transcription factors, besides FD, inter-act with TFL1 is currently not known. However, giventhe strong overrepresentation of G-box motifs amongthe genes bound specifically by TFL1 (not shared withFD), these yet unknown interaction partnersmost likelyinclude DNA-binding proteins with a preference for aG-box, such as other bZIP or basic helix-loop-helix(bHLH) proteins.

DISCUSSION

Movement of TFL1 within the Shoot Meristem

All multicellular living beings rely on mobile, long-distance and cell-to-cell signals to coordinate growthand development of different parts of the organism.The production of signaling molecules is temporallyand spatially tightly controlled, and they are usuallypresent in cells at low concentration. Furthermore,

Figure 6. Validation of direct TFL1targets. A, Quantification of the en-richment of TFL1-GR on the LFY, PIF5,and TPPH genes in 35Spro::TFL1-GRplants by ChIP. Fractions of input inmock-treated (dark gray) and DEX-treated (light gray) plants are shownfor one biological replicate. Data foradditional biological replicates areshown in Supplemental Figure S11. B,Expression of LFY, PIF5, and TPPH in35Spro::TFL1-GR plants in response toDEX treatment. Expression estimates(read counts) were extracted fromRNA-seq data. Reads are normalizedfor sequencing depth. Error bars showSD. C, Expression of LFY, PIF5, andTPPH in DEX- and mock-treated35Spro::TFL1-GR plants quantified byRT-qPCR 2 and 26 h after DEX appli-cation. Error bars show SD. Asterisksindicate significant differences be-tween mock- and DEX-treated plants(P , 0.05, Student’s t test).

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mobile signaling molecules are usually small moleculesto facilitate efficient translocation. Common signalmolecules include hormones, RNAs, small peptides,and transcription factors (Busch and Benfey, 2010; VanNorman et al., 2011). In plants, signaling molecules canmove either through the apoplastic space between cellsor symplastically from cell to cell through plasmodes-mata. A prominent example of long-distance signalingis the transport of the FT protein, a florigen that isproduced in leaf phloem companion cells and trans-ported through the sieve elements to induce floweringat the SAM (Srikanth and Schmid, 2011). Interestingly,TFL1, a protein that is closely related to FT but acts as arepressor of flowering, has been suggested to movewithin the SAM (Conti and Bradley, 2007). This notionis based on the observation that TFL1 mRNA isexpressed in the central part of the SAM, but the proteinhas awider distribution, reaching the outer layers of theapex (Conti and Bradley, 2007). However, the relevanceof TFL1 protein movement for its function in SAM de-velopment and flowering time regulation has not beenaddressed in detail.

Here, we first showed that the ability of TFL1 tomovewithin the SAM is essential for complementing theearly-flowering phenotype of the tfl1-1mutant (Fig. 1B).When tagged with a single copy of the Venus fluores-cent protein under the control of the genomic TFL1sequences (gTLF1-1xVenus), the fusion protein wasdetected most strongly in the center of the SAM, but italso reached the epidermal L1 layer of the SAM andlargely rescued the early-flowering phenotype of thetfl1-1 mutant (Fig. 2; Supplemental Fig. S6A). How-ever, rescue of tfl1-1 early flowering by gTFL1was notalways complete, possibly because our genomic con-structs lack the block V TFL1 cis-regulatory region,which has been reported to contain an enhancer ele-ment that contributes to the expression level of TFL1,without being required for correct temporal andspatial expression. Furthermore, when we expressedTFL1 under the control of the epidermis-specific ML1promoter, 44% of the T1 plants (ML1pro::TFL1) flow-ered similar to the wild type, with more than 14 totalleaves (Fig. 3A; Supplemental Fig. S7). Taken to-gether, these results suggest that TFL1 acts at least inpart in the apical cells of the tunica during the floraltransition and provide evidence that movementwithin the SAM contributes to TFL1 function.

It seems possible that TFL1 interacts in the tunicawith transcription factors, such as FD, to prevent theswitch from vegetative to reproductive development,thereby maintaining the inflorescence meristem in anundifferentiated state. In this context, it is also worth-while to note that ATC, a protein that is closely relatedto TFL1, has been shown to influence flowering timespecifically when expressed under the control of theML1 promoter under SD conditions (Huang et al.,2012).

TheML1 promoter is expressed in all epidermal cells,but its activity increases at the shoot apex during thetransition to flowering (Sessions et al., 1999). However,

even at the inflorescence meristem, activity of the ML1promoter is thought to be weaker than that of the 35Spromoter. Furthermore, the 35S promoter drives strongexpression in the vegetative SAM as well, and acrossthe entire apex, not only in the L1 cells. This could ex-plain why lines expressing TFL1 under the control ofthe ML1 promoter, while flowering later than the tfl1mutant, were nevertheless not as late flowering as the35Spro::TFL1 lines (Figs. 1C and 3A).

When expressed from the ML1 promoter, the TFL1-1xGFP signal accumulated in the L1 layer but could alsobe detected in L2 and to some extent even in L3 cells(Fig. 3B). The finding that the TFL1 protein can move ineither direction in the SAM suggests that movementoccurs via passive diffusion, following a concentrationgradient, rather than by active directional transport.This is not entirely surprising, as it has previously beenshown that small proteins such as GFP (27 kD), whichhas a similar size to TFL1 (20 kD), can move relativelyfreely at the apex (Crawford and Zambryski, 2000; Wuet al., 2003; Kim et al., 2005). However, this is in contrastto a previous report that TFL1movement was impairedin lfy mutants, in which the protein accumulates in thecentral region of the SAMwhere its mRNA is expressed(Conti and Bradley, 2007). One possible explanation forthis discrepancy is that LFY could regulate the sizeexclusion limit of plasmodesmata in the SAM, whichwould indirectly affect the movement of macromole-cules between cells. Alternatively, it seems possible thatLFY facilitates TFL1 movement through direct proteininteractions or by regulating the expression of an un-known factor that is required for TFL1 movement.

It should be noted that the homeodomain proteinWUSCHEL, which is expressed in the organizing centerof the SAM, has been shown to move upward towardthe stem cell population of the tip of the shoot meristem(Yadav et al., 2011; Daum et al., 2014), suggesting thatprotein movement within the SAM might be morecommon than originally thought.

TFL1 Movement Affects Meristem Determinacy

Another interesting outcome of our experiments re-lates to the regulation of meristem indeterminacy byTFL1. T1 plants expressing the mobile TFL1-1xGFP orimmobile TFL1-3xGFP variants under the control ofeither its own regulatory elements or the ML1 pro-moter, either in the presence or absence of NLS signal(Table 1), displayed substantial differences in shootdeterminacy. Indeterminate growth of the inflores-cences was restored in approximately 50% of T1 plantsexpressing the gTFL1-1xGFP protein. All the othercombinations showed a determinate growth of themain inflorescence, as in the tfl1-1mutant. These resultssuggest that TFL1 regulates shoot indeterminacy out-side the TFL1 expression domain, as TFL1 variants thatare prevented from moving by the large 3xGFP tagand/or nuclear localization (due to the addition of anNLS) are incapable of rescuing the determinate growth

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of the tfl1-1 mutant. An alternative explanation is that,to regulate the indeterminate growth of the shoot, TFL1is required at the same time both in the center of theSAM and in the tunica.In contrast to the effect of TFL1 on flowering time,

expression of TFL1 from the ML1 promoter was notsufficient to rescue the determinate growth of the tfl1-1mutant. This suggests that the function of TFL1 in theregulation of indeterminate growth is not limited to theL1 layer. Interestingly, the MADS domain transcriptionfactors AGAMOUS and SEPALLATA3, two importantregulators of floral development, have been reported toconvert the inflorescence meristem to a terminal flowerwhen expressed from a constitutive promoter, but theyhad no effect on the growth habit when expression wasrestricted to the L1 (Mizukami and Ma, 1992; Honmaand Goto, 2001; Urbanus et al., 2010). In summary, ourfindings indicate that TFL1 movement is crucial in theregulation of inflorescence meristem indeterminacy,although the precise region in which TFL1 activity isrequired remains to be determined.

TFL1 Is a Transcriptional Coregulator ofFlowering-Related Genes

The subcellular localization and molecular functionof TFL1 have been discussed controversially. However,it had been shown that a translational fusion betweenTFL1 and the strong transcription activation domainVP16 induced the expression of genes that are believedto be normally repressed by TFL1, suggesting that theprotein might function as a transcriptional corepressor(Hanano and Goto, 2011). Our ChIP-seq and RNA-seqresults support the idea that TFL1 acts as a transcrip-tional cofactor in the apex during the floral transition,identifying 115 putative direct targets that are bothbound and transcriptionally regulated by a TFL1-containing protein complex. TFL1 has been reportedto have transcriptional repressor activity based on thephenotype of SRDX-tagged plants (Hanano and Goto,2011). Consistent with this previous study, applicationof DEX to our inducible TFL1-GR lines also resulted inlate flowering, confirming the negative effect of TFL1on this process (Supplemental Fig. S10, A and B). It isthus surprising that our RNA-seq analysis identifiedmore up-regulated than down-regulated genes in the35Spro::TFL1-GR line in response to DEX treatment(Fig. 5B). However, as we sampled apices 24 h afterDEX application, it is likely that a substantial part of thedifferentially expressed genes are regulated by TFL1indirectly. Furthermore, in our RNA-seq analyses, DEXwas applied to 35Spro::TFL1-GR plants 6 d after theplants had been shifted from SD to LD. At this timepoint, the transition to flowering has started; however,expression of the early flower meristem and floralhomeotic genes LFY and AP1 can still be efficientlysuppressed byDEX application (Supplemental Fig. S10,C and D). Nevertheless, we cannot completely ruleout the possibility that other genes that are normallyrepressed by TFL1 were not responding to the DEX

treatment, which might explain the relatively largenumber of up-regulated transcripts in our analysis.However, given that the 35S::TFL1-SRDX lines closelyresemble 35S::TFL1 overexpression plants (Hanano andGoto, 2011), the most parsimonious explanation stillremains that TFL1 acts as a transcriptional corepressorof flowering time genes.As there is no evidence that TFL1 can directly interact

with DNA, binding is most likely facilitated throughtranscription factors. In this context, it is worthwhile tonote that among the TFL1-bound regions, G-box motifsare highly overrepresented. This suggests that TFL1interacts with DNA through transcription factors thatbind to G-box motifs, such as bZIP and bHLH proteins(Sibéril et al., 2001; Jones, 2004).One bZIP transcription factor that has been shown to

physically interact in complex with TFL1 is FD, an im-portant regulator of flowering time in Arabidopsis (Abeet al., 2005; Wigge et al., 2005). Interestingly, compari-son of TFL1-bound regions with direct FD targetsidentified 358 shared peaks, confirming that TFL1 is animportant FD interactor (Collani et al., 2019). Both TFL1and FD are involved in floral transition at the SAM andbind genes that regulate auxin-mediated signaling andresponse to stimuli. However, the two proteins clearlyhave partially separate roles in the regulation of de-velopment. For example, binding of FD was enrichedon genes involved in maintenance of the inflorescencemeristem and flower and carpel development, sug-gesting that FD but not TFL1 might have a yet unre-ported role in fruit and seed development. Similarly,TFL1 also appears to have FD-independent functions,as we identified 613 peaks unique to TFL1, suggestingthat it can form complexes with proteins other than FD.One likely candidate that might mediate TFL1 bindingto DNA is FD PARALOG (AtbZIP27), which has beenshown to interact with TFL1 (Wigge et al., 2005) andmutation of which enhances the late-flowering pheno-type of the fd-2 mutant (Jaeger et al., 2013). Amongthese TFL1-specific targets were many genes involvedin the regulation of carbohydrate metabolic processes,whose impact on SAM organization at the floral tran-sition has not yet been explored in detail, and responseto different wavelengths of light.Interestingly, LFY, which has long been known to be

genetically regulated by TFL1 (Ratcliffe et al., 1998), isamong the direct TFL1-specific target genes (Fig. 6).ChIP-seq identified a G-box in the second exon of LFY(Fig. 4D), which showed strong enrichment for TFL1but not for FD binding. This suggests that at the apexFD can induce LFY expression only indirectly, mostlikely by up-regulating the expression of SPL genes andSUPPRESSOR OF OVEREXPRESSION OF CO1 (Junget al., 2012). To the best of our knowledge, no positiveregulators that directly bind to the second exon of LFYhave been reported, indicating that the regulation byTFL1 is mediated by a yet unknown transcription fac-tor. Importantly, LFY is able to directly bind both FDand TFL1 cis-regulatory regions (Moyroud et al., 2011).This suggests the existence of a tight feedback loop between

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these genes that likely contributes to a fast and stable reg-ulation of the floral transition and meristem identity.

Another interesting case is the up-regulation of PIF5by TFL1, independently from FD (Figs. 4D and 6). Therole of PIF5, and even more so PIF4, as a positive reg-ulator of cell elongation in the hypocotyl has alreadybeen documented (Niwa et al., 2009; Kunihiro et al., 2011;Choi andOh, 2016). However, the role of PIFs at the SAMhas not yet been investigated in detail. Recently, it wasreported thatmisexpression ofPIFs from the FDpromoterhad only a minor effect on flowering time (Galvão et al.,2015). During the floral transition, the SAM first increasesin size due to a proliferation of cells in the meristem, fol-lowed by the elongation of internodes during bolting(Jacqmard et al., 2003). As the growth of the apical regionof the stem is in part due to an elongation of cells of the ribmeristem, we wonder whether PIFs could be involved inregulating these morphological changes during the floraltransition. In this context, it is worthmentioning that PIFsbelong to the bHLH class of transcription factors that bindG-box consensus sequences on the DNA (Leivar andQuail, 2011). These findings suggest that TFL1-PIFsmight interact in a protein complex; however, furtherexperiments will be required to test this hypothesis.

Finally, several genes related to trehalose 6-phosphatewere differentially expressed in response to TFL1 in-duction according to our RNA-seq experiments. As tre-halose 6-phosphate is a floral promoter, changes in theexpression of TPS and TPP genes at the apex under LDconditions could affect the floral transition (Wahl et al.,2013). Furthermore, in maize (Zea mays), the geneRAMOSA3, which encodes a TPP, regulates identityand determinacy of the axillary meristems (Satoh-Nagasawa et al., 2006). It would thus be extremely in-teresting to elucidate if a similar mechanism involvingTFL1 and some TPPs could be active in the regulation ofthe inflorescence architecture also in Arabidopsis.

CONCLUSION

In summary, we found that movement of TFL1 in theSAM is critical for its function in the regulation offlowering time and inflorescence development in Ara-bidopsis. Furthermore, TFL1 movement from the innerparts of the meristem to the L1 layer is probably not adirectional process. In the SAM, TFL1 is stronglyenriched at G-box motifs, suggesting that it is prefer-entially recruited to DNA through interaction withbZIP and possibly bHLH transcription factors. Our re-sults demonstrate that TFL1 acts as a transcription co-factor at the SAM during the floral transition, and weidentified a genome-wide list of putative TFL1 targets.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) accession Col-0 was used as the wild type.The tfl1-1 mutation and phenotype in the Col-0 background have been

described previously (Shannon and Meeks-Wagner, 1991). Plants were grownon soil at 65% relative humidity under wide-spectrum fluorescent lights with afluence rate of 125 to 175 mmol m22 s21. For flowering time studies and ChIPexperiments, seeds were stratified for 3 d in 0.1% (w/v) agar in the dark at 4°Cand directly planted on soil. Plants were grown on soil under LD (16 h of lightand 8 h of night) at 22°C during the day and 18°C during the night. Plantsintended for measuring flowering time were grown in a randomized design tominimize positional effects. Flowering timewas scored as rosette leaves, caulineleaves, and total leaf number, as well as days to flowering, which is defined asthe number of days from sowing until the emergence of the first visible flowerbuds. For RNA-seq experiments, plants were first grown under SD conditions(8 h of light and 16 h of night) at 21°C during the day and 19°C during the nightfor 3 weeks and then shifted to LD.

DNA Vectors and Plant Transformation

Sequences were amplified by PCR from cDNA or genomic DNA and clonedinto pGREEN vectors by using either a classical restriction enzyme cloningapproach or the GreenGate system (Lampropoulos et al., 2013). To generate the35Spro::TFL1-GR line, the CDS of TFL1was cloned into the pGreen0229 plasmidcontaining a 35Spro::GR construct (Yu et al., 2004). Final constructs weretransformed into Agrobacterium tumefaciens (strain GV3101 pMP90 pSoup) byelectroporation (Gene Pulser Xcell system). Arabidopsis plants of accession Col-0 and the tfl1-1 mutant were transformed by the floral dip method. BASTAselection (0.1%, v/v) was used for screening the transgenic lines on soil. Lists ofthe PCR primers used for cloning and the plant binary vectors generated in thisstudy can be found in Supplemental Tables S1 and S2.

DEX Treatment

To induce the translocation of TFL1-GR to the nucleus, 35Spro::TFL1-GRplants were treated after their shift to LD conditions with a solution containingeither 30 mM DEX, 0.03% (v/v) ethanol, and 0.01% (v/v) Silwet L-77 or a so-lutionwith an equal composition but without DEX (mock solution) by applyinga single drop directly to the shoot apex at ZT12.

Establishing and Verifying Experimental Conditionsfor RNA-Seq

To establish the experimental conditions and test the effect of DEX treatmenton LFY and AP1 expression, 12 manually dissected apices were collected forthree biological replicates each. Total RNA was extracted using the RNeasyMini Kit (Qiagen) and treated with DNaseI (Ambion) following the manufac-turer’s instructions. Then, 1 mg of RNA was reverse transcribed into single-stranded cDNA using SuperScriptII (Invitrogen) in a 20-mL reaction mixture.The resulting cDNA was diluted to a final volume of 90 mL and 5 mL of eachsample was used in qPCR. qPCR reactions were performed on a Fast II system(Applied Biosystems) using SYBR Green (Takara). The primers used for thequantitative analysis are described in Supplemental Table S1. qPCR was per-formed on three independent biological replicates, and TIP41 was used as areference gene. Relative expression was calculated based on the ddCt methodand normalized to the T0 control (mock) sample.

RNA-Seq and Validation

For RNA-seq experiments, 35Spro::TFL1-GR plants were grown for 3 weeksunder SD before being shifted to LD. DEX ormock solutionwas applied directlyto the shoot apex at ZT12 6 d after the shift to LD, and samples were collected24 h later. Approximately 60 manually dissected apices were collected for eachof three replicates per condition. Total RNA was extracted using the RNeasyMini Kit (Qiagen) and treated with DNaseI (Ambion) following the manufac-turer’s instructions. For RNA-seq analysis, the quality of the resulting RNAwaschecked on an Agilent 2100 Bioanalyzer instrument using the RNA6000 nanokit. Strand-specific RNA libraries were constructed using the TruSeq strandedmRNA kit (Illumina). Libraries were sequenced on a NextSeq500 platform(Illumina) using the High Output 75 cycles kit v2.0 (Illumina) to produce 75-nucleotide single-end reads. Libraries were sequenced at the genomics corefacility at Servicio Central de Soporte a la Investigación of the University ofValencia, Spain. All RNA-seq data are available from the European NucleotideArchive under accession number PRJEB29016.

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For validation of the RNA-seq results by RT-qPCR, 35Spro::TFL1-GRplants were grown 3 weeks under SD conditions and then shifted to LD. DEXor mock solution was applied at ZT0 6 d after the shift to LD, and sampleswere collected 2 and 26 h later. RNA extraction and RT-qPCRwere performedas described above.

ChIP-Seq and ChIP-PCR

For ChIP-seq experiments, approximately 300 mg of manually dissectedapices (gTFL1-1xVenus and Col-0) from 15-d-old plants grown on soil under LDwere harvested. Samples were first prefixed under vacuum for 30 min in 2 mM

ethyleneglycol-bis-succinimidyl-succinate solution and then postfixed for anadditional 30 min after the addition of formaldehyde to a final concentration of1% (v/v). ChIP was performed as previously described (Kaufmann et al., 2010).Anti-GFP fromAbcam (ab290) was used for immunoprecipitation of the Venus-tagged protein complex. ChIP-seq libraries were prepared by Novogene Bio-informatics Technology using the NEB Next Ultra II DNA Library Prep Kit,selecting fragments between 100 and 350 bp. Libraries were sequenced on anIlluminaHiSeq3000 systemusing the 50-bp single-end kit. All ChIP-seq data areavailable from the European Nucleotide Archive under accession numberPRJEB29016.

For the ChIP-PCR experiment using the anti-GR antibody (Abcam, ab3580),35Spro::TFL1-GR and Col-0 plants were grown for 3 weeks under SD conditionsand then transferred to LD. After 5 d in LD, plants were treatedwith one drop of30 mM DEX or mock directly applied to the shoot apex. Approximately 300 mgof apices were manually dissected 24 h after DEX application, and ChIP wasperformed as described above. For ChIP-PCR, a 1:3 dilution of immunopreci-pitated DNA was analyzed by qPCR, performed on a CFX96 Touch Real-TimePCR Detection System (Bio-Rad) using LightCycler 480 SYBR Green I Master(Roche). Oligonucleotides used as primers for qPCR are listed in SupplementalTable S1.

ChIP-Seq and RNA-Seq Analyses

ChIP-seq raw data were aligned to the Arabidopsis genome (TAIR10 re-lease) using bwa (Li and Durbin, 2010). Peaks were called with MACS2 (Zhanget al., 2008) using default parameters, and mapped reads from Col-0 were usedas a control. Overlapping peaks were retrieved using the R package DiffBindusing default parameters (Ross-Innes et al., 2012; Stark and Brown, 2013). Denovo motif analysis using MEME-ChIP (Machanick and Bailey, 2011) was usedfor identifying the binding site.

For RNA-seq analyses, ribosomal RNA sequences were filtered out usingSortMeRNA (Kopylova et al., 2012). Adapters were trimmed from theremaining reads using Trimmomatic (Bolger et al., 2014). The trimmed se-quences were then aligned against the Arabidopsis transcriptome (TAIR10)using STAR (Dobin et al., 2013) and reads were counted with HTSeqCount(Anders et al., 2015). DESeq2 with default parameters was used to performdifferential expression analysis (Love et al., 2014).

Confocal Laser Scanning Microscopy

To detect Venus- and GFP-tagged proteins, manually dissected apices werecollected from plants grown under LD conditions. Samples were fixed aspreviously described (Gregis et al., 2009). Sections between 70 and 90 mmthick were obtained using a Leica vibratome (VT1000S). Confocal laser scan-ning microscopy was performed using a Zeiss LSM 780 microscope (253magnification).

Statistical Analyses

Flowering time data were analyzed by one-way ANOVA with posthocTukey’s honestly significant difference based on Tukey-Kramer correction (P,0.05). Statistical significance calculations for the RT-qPCR data shown inFigure 6C were performed with two-tailed Student’s t test using three biological repli-cates with at least 12 plants per replicate. Differences in expression were deemed sig-nificant at P, 0.05. Normality of the data was determined with the Shapiro-Wilk test.

Accession Numbers

RNA-seq and ChIP-seq data were deposited at the European NucleotideArchive under project identifier PRJEB29016.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Flowering time of gTFL1 lines.

Supplemental Figure S2. gTFL1-1xGFP but not gTFL1-3xGFP comple-ments the flowering time and shoot meristem defects of the tfl1-1 mutant.

Supplemental Figure S3. Flowering time data for stable (T4)overexpressor lines.

Supplemental Figure S4. Flowering time data for stable (T4) tagged gTFL1 lines.

Supplemental Figure S5. Localization of gTFL1-1xVenus protein at theshoot apex.

Supplemental Figure S6. Localization of gTFL1-1xVenus and gTFL1-3xVenus protein in different T4 lines.

Supplemental Figure S7. Flowering time data for T1 lines expressingtagged and untagged TFL1 from the ML1 promoter.

Supplemental Figure S8. MEME output for the three most stronglyenriched motifs in TFL1-bound regions.

Supplemental Figure S9. GO analysis output for differentially bound(ChIP-seq) and expressed (RNA-seq) genes.

Supplemental Figure S10. Characterization of 35Spro::TFL1-GR lines.

Supplemental Figure S11. Validation of TFL1 target genes by ChIP-PCRusing 35Spro::TFL1-GR (#7.9).

Supplemental Figure S12. GO analysis output for genes bound by FD andTFL1 or specifically by TFL1.

Supplemental Table S1. List of primers used in this study.

Supplemental Table S2. List of plasmids used in this study.

Supplemental Data S1. List of the 971 TFL1-bound peaks identified inseedlings expressing gTFL1-1xVenus in tfl1-1.

Supplemental Data S2. List of genes regulated by TFL1-GR.

Supplemental Data S3. List of the 115 bound and differentiallyexpressed genes.

Supplemental Data S4. List of the 358 TFL1- and FD-bound peaks.

Supplemental Data S5. List of the 613 TFL1-bound peaks not sharedwith FD.

Supplemental Data S6. List of the 237 FD-bound peaks not sharedwith TFL1.

Received July 12, 2019; accepted January 17, 2020; published January 29, 2020.

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